EA044245B1 - COMPLEMENT C5 mRNA COMPOSITIONS AND METHODS OF THEIR APPLICATION - Google Patents

Abstract

The invention relates to mRNA, such as double-stranded ribonucleic acid (dsRNA), compositions targeting the gene of complement component C5, as well as methods of using such mRNA, such as dsRNA, compositions for inhibiting the expression of C5 and for treating subjects suffering from a disease associated with complement component C5, such as paroxysmal nocturnal hemoglobinuria.

Description

List of sequences

This application includes a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII file, created on March 10, 2014, is named 121301-00520 SL.txt and is 734,486 bytes in size.

State of the art

Complement was first discovered in the 1890s, when heat-stable antibodies present in normal serum were found to promote or induce bacterial killing (Walport, M.J. (2001) N Engl J Med. 344:1058). The complement system consists of more than 30 proteins that are either present as soluble proteins in the blood or as membrane-associated proteins. Complement activation leads to a cascade of enzymatic reactions known as the complement pathways, resulting in the formation of the potent anaphylotoxins C3a and C5a, which induce a variety of physiological responses ranging from chemoattraction to apoptosis. Complement was originally thought to play a critical role in innate immunity, resulting in a robust and rapid response against invading pathogens. However, it has become increasingly clear that complement also plays an important role in adaptive immunity involving T and B cells, which aid in the elimination of pathogens (Dunkelberger J.R. and Song W.C. (2010) Cell Res. 20:34; Molina H., et al. (1996) Proc Natl Acad Sci USA. 93:3357), in the maintenance of immunological memory that prevents re-invasion of pathogens, and is involved in numerous human pathological conditions (Qu, H., et al. (2009) Mol Immunol. 47:185; Wagner, E. and Frank M.M. (2010) Nat Rev Drug Discov. 9:43).

Complement activation is known to occur via three distinct pathways: alternative, classical, and lectin (Fig. 1), involving proteins that primarily exist as inactive zymogens that are then sequentially cleaved and activated. All complement activation pathways result in the cleavage of the C5 molecule to form the anaphylatoxin C5a and C5b, which subsequently forms the terminal complement complex (C5b-9). C5a exerts its predominant proinflammatory activity by interacting with the classical G protein receptor C5aR (CD88) and the non-G protein receptor C5L2 (GPR77), expressed on a variety of immune and non-immune cells. C5b-9 induces cytolysis through the formation of the membrane attack complex (MAC), with the lytic MAC subunits and soluble C5b-9 having multiple non-cytolytic immune functions. These two complement effectors, C5a and C5b-9, formed by cleavage from C5, are key components of the complement system responsible for the propagation and/or initiation of pathology in a variety of diseases, including paroxysmal nocturnal hemoglobinuria, rheumatoid arthritis, post-ischemic injury, and neurodegenerative diseases.

Currently, only one therapeutic agent targeting the C5-C5a components is available for the treatment of complement component C5-associated diseases: the anti-C5 antibody eculizumab (Soliris®). Although eculizumab has demonstrated efficacy in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) and is currently being evaluated in clinical trials for the treatment of additional complement component C5-associated diseases, eculizumab therapy requires high-dose weekly infusions, followed by biweekly infusions, at an annual cost of approximately $400,000. Accordingly, there is a need in the field for alternative therapies and combination therapies for individuals with complement component C5-associated diseases.

Brief description of the invention

The present invention relates to mRNA compositions that target RNA-induced silencing complex (RISC)-mediated cleavage of C5 gene RNA transcripts. The C5 gene may be within a cell, such as a cell within a subject, such as a human. The present invention also relates to methods and combination therapies for treating a subject with a disorder that can be beneficially affected by inhibiting or reducing C5 gene expression, such as a C5 complement component-associated disease such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) using mRNA compositions that target RNA-induced silencing complex (RISC)-mediated cleavage of C5 gene RNA transcripts to inhibit C5 gene expression.

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Accordingly, in one aspect, the present invention relates to an agent, which is a double-stranded ribonucleic acid (dsRNA), for inhibiting the expression of complement component C5, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5.

In another aspect, the present invention relates to an agent, which is a double-stranded ribonucleic acid (dsRNA), for inhibiting the expression of complement component C5, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity that comprises at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any of the antisense sequences listed in any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23.

In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of A-118320, A-118321, A-118316, A-118317, A-118332, A-118333, A-118396, A-118397, A-118386, A-118387, A-118312, A-118313, A-118324, A-118325, A-119324, A-119325, A-119332, A-119333, A-119328, A-119329, A-119322, A-119323, A-119324, A-119325, A-119334, A-119335, A-119330, A-119331, A-119326, A-119327, A-125167, A-125173, A-125647, A-125157, A-125173, and A125127. In another embodiment, the sense and antisense strands comprise sequences selected from the group consisting of sequences in any of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23. In one embodiment, the agent, which is a dsRNA, comprises at least one modified nucleotide.

In one aspect, the present invention relates to a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C5, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence AAGCAAGAUAUUUUUAUAAUA (SEQ ID NO:62) and wherein the antisense strand comprises the nucleotide sequence UAUUAUAAAAAUAUCUUGCUUUU (SEQ ID NO:113). In one embodiment, the dsRNA agent comprises at least one modified nucleotide, as described below.

In one aspect, the present invention relates to a double-stranded RNAi agent for inhibiting the expression of complement component C5, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides and wherein the sense strand is conjugated to a ligand attached at the 3' end.

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand contain modifications.

In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal deoxythymine (dT) nucleotide. In another embodiment, substantially all of the nucleotides of the antisense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal deoxythymine (dT) nucleotide. In another embodiment, the modified nucleotides are a short sequence of deoxythymine (dT) nucleotides. In another embodiment, the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end. In another embodiment, the sense strand is conjugated to one or more GalNAc derivatives attached via a branched divalent or trivalent linker at the 3' end.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a closed nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-naturally occurring base, a nucleotide containing a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesterol derivative or a bis-decylamide group of dodecanoic acid.

In another embodiment, the modified nucleotides comprise a short 3'-terminal deoxythymine (dT) nucleotide sequence.

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In one embodiment, the region of complementarity is at least 17 nucleotides in length. In another embodiment, the region of complementarity is between 19 and 21 nucleotides in length.

In one embodiment, the region of complementarity is 19 nucleotides in length.

In one embodiment, each strand is no more than 30 nucleotides in length.

In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

In one embodiment, the agent, which is a dsRNA, further comprises a ligand.

In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

In one embodiment, the ligand is a derivative of N-acetylgalactosamine (GalNAc).

In one embodiment, the ligand is but ^ ^{on n} _n

In one embodiment, the dsRNA agent is conjugated to a ligand as shown in the following scheme:

3'

AcHN £ H H where X is O or S.

In one embodiment, X is O.

In one embodiment, the region of complementarity consists of one of the antisense sequences in any of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23.

In one embodiment, the dsRNA agent is selected from the group consisting of AD-58123, AD-58111, AD-58121, AD-58116, AD-58133, AD-58099, AD-58088, AD-58642, AD58644, AD-58641, AD-58647, AD-58645, AD-58643, AD-58646, AD-62510, AD-62643, AD-62645, AD62646, AD-62650, and AD-62651.

In another aspect, the present invention relates to a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C5, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence AAGCAAGAUAUUUUUAUAAUA (SEQ ID NO:62) and wherein the antisense strand comprises the nucleotide sequence UAUUAUAAAAAUAUCUUGCUUUUdTdT (SEQ ID NO:2899).

In another aspect, the present invention relates to a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C5, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence asasGfcAfaGfaUfAfUfuUfuuAfuAfauaL96 (SEQ ID NO:2876) and wherein the antisense strand comprises the nucleotide sequence usAfsUfuAfuaAfaAfauaUfcUfuGfcuususudTdT (SEQ ID NO:2889).

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In one aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of complement component C5 in a cell, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the double-stranded RNAi agent is represented by formula (III):

sense: 5' n _p -N _a -(XXX) _± -N _b -Y Υ Υ —N _b — (Ζ Ζ Z)j -N _a - n _q 3 ' antisense: 3' n _p ' -N _a ' - (X 'X 'X ') _k -N _b '-Υ Ύ Ύ ' -N _b ' - (Ζ ' Ζ ' Z ') _b -N _a ' - n _q '5' (III), where each of i, j, k and l is independently equal to 0 or 1;

each of p, p', q and q' is independently equal to 0-6;

each of Na and Na' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of N _b and N _b ' independently represents an oligonucleotide sequence comprising 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of np, np', nq and nq', each of which may be present or absent, independently represents a protruding nucleotide;

each of XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides;

modifications N _b are different from modification Y, and modifications N _b ' are different from modification Y'; and where the sense strand is conjugated to at least one ligand.

In one embodiment, i equals 0; j equals 0; i equals 1; j equals 1; both i and j equal 0; or both i and j equal 1.

In one embodiment, k equals 0; l equals 0; k equals 1; l equals 1; both k and l equal 0; or both k and l equal 1.

In one embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

In one embodiment, the YYY motif is located at or near a cleavage site of the sense strand.

In one embodiment, the Y'Y'Y' motif is located at positions 11, 12, and 13 of the antisense strand from the 5' end.

In one embodiment, Y' is 2'-O-methyl.

In one embodiment, formula (III) is represented by formula (IIIa): sense: 5' n _p -N _a -YYY -N _a - n _q 3 ' antisense: 3' n _p ,-N _a ,- YΎΎ'- N _a >- n _q - 5' (Illa).

In another embodiment, formula (III) is represented by formula (IIIb): sense: 5' n _p -N _a -YYY -N _b -ZZZ -N _a - n _q 3' antisense: 3' n _p --N _a -- Y Ύ ¹ Y ' -N _b --Z ' Z ' Z ' - N _a -- n _q - 5' (IIIb), wherein each of N _b and N _b ' independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

In another embodiment, formula (III) is represented by formula (IIIc): sense: 5' n _p -N _a -XXX -N _b -YYY -N _a - n _q 3' antisense: 3' n _p --N _a -- X'X'X'-N _b -- Y'Y'Y'- N _a -- n _q - 5' (IIIc), wherein each of N _b and N _b ' independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

In another embodiment, formula (III) is represented by formula (IIId):

I sense: 5' n _p -N _a -XX X- N _b -Y Y Y -N _b -Z Z Z -N _a - n _q 3' antisense: 3' n _p --N _a -- X'X'X'- N _b --Y ' Y ' Y '-N _b --Z ' Z ' Z ' - N _a -- n _q - 5' (IIId), where each of N _b and N _b ' independently represents an oligonucleotide sequence containing 1-5 modified nucleotides, and each of Na and Na' independently represents an oligonucleotide sequence containing 2-10 modified nucleotides.

In one embodiment, the double-stranded region is 15-30 nucleotide pairs in length.

In one embodiment, the double-stranded region is 17-23 base pairs in length. In another embodiment, the double-stranded region is 17-25 base pairs in length. In another embodiment, the double-stranded region is 23-27 base pairs in length. In yet another embodiment, the double-stranded region is 19-21 base pairs in length. In another embodiment, the double-stranded region is 21-23 base pairs in length.

In one embodiment, each strand contains 15-30 nucleotides.

In one embodiment, the nucleotide modifications are selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'hydroxyl, and combinations thereof.

In another embodiment, the nucleotide modifications are 2'-O-methyl or 2'-fluoro modifications.

In one embodiment, the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. In one embodiment, the ligand is

In one embodiment, the ligand is attached to the 3' end of the sense strand.

In one embodiment, the RNAi agent is conjugated to a ligand as shown in the following scheme:

In one embodiment, the agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand.

In one embodiment, the strand is an antisense strand. In another embodiment, the strand is a sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand.

In one embodiment, the strand is an antisense strand. In another embodiment, the strand is a sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at both the 5' and 3' ends of one strand.

In one embodiment, the strand is an antisense strand.

In one embodiment, the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

In one embodiment, the Y nucleotides have a 2'-fluoro modification.

In one embodiment, the Y' nucleotides have a 2'-O-methyl modification.

In one embodiment, p’>0.

In one embodiment, p-2.

In one embodiment, q'=0, p=0, q=0, and the protruding nucleotides p' are complementary to the target mRNA.

In another embodiment, q'=0, p-0, q-0, and the protruding nucleotides p' are not complementary to the target mRNA.

In one embodiment, the sense strand comprises a total of 21 nucleotides and the antisense strand comprises a total of 23 nucleotides.

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In one embodiment, at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage.

In one embodiment, all np's are linked to adjacent nucleotides via phosphorothioate linkages.

In one embodiment, the RNAi agent is selected from the group of RNAi agents listed in Table 4, Table 18, Table 19, or Table 23. In another embodiment, the RNAi agent is selected from the group consisting of AD-58123, AD-58111, AD-58121, AD-58116, AD-58133, AD-58099, AD-58088, AD58642, AD-58644, AD-58641, AD-58647, AD-58645, AD-58643, AD-58646, AD-62510, AD-62643, AD62645, AD-62646, AD-62650, and AD-62651.

In one aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of complement component C5 in a cell, wherein said double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to a portion of an mRNA encoding complement component C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein said double-stranded RNAi agent is represented by formula (III):

sense: 5' n _p -N _a -(XXX) iN _b -YYY -N _b - (Ζ ZZ)j -N _a - n _q 3 ' antisense: 3' n _p '-N _a ' - (X'X'X ') _k -N _b '-Υ Ύ Ύ '-N _b ' - (Ζ ' Ζ ' Ζ ') _b -N _a ' - n _q '5' (III), where i, j, k and l are each independently 0 or 1;

each of p, p', q and q' is independently equal to 0-6;

each of Na and Na' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of N _b and Nb' independently represents an oligonucleotide sequence containing 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of np, np', n _q, and n _q ', each of which may be present or absent, independently represents a protruding nucleotide;

each of XXX, YYY, ZZZ, X'X'X', YY'Y' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides, and wherein the modifications are 2'-Omethyl- or 2'-fluoro-modifications;

modifications N _b are different from modification Y, and modifications N _b ' are different from modification Y'; and where the sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of complement component C5 in a cell, wherein said double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to a portion of an mRNA encoding complement component C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein said double-stranded RNAi agent is represented by formula (III):

sense: 5' n _p -N _a - (XXX) iN _b -YYY -N _b -(Ζ ZZ)j -N _a - n _q 3 ' antisense: 3' n _p ' -N _a ' - (X 'X 'X ') _k -N _b '-Y Ύ Ύ ' -N _b ' - (Ζ ' Ζ ' Z ') _b -N _a ' - n _q '5' (III), where each i, j, k and l are independently equal to 0 or 1;

each of n _p , n _q , and n _q ', each of which may be present or absent, independently represents a protruding nucleotide;

each of p, q and q' is independently equal to 0-6;

n _p '>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond;

each of Na and Na' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of N _b and N _b ' independently represents an oligonucleotide sequence comprising 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides, and wherein the modifications are 2'-Omethyl- or 2'-fluoro-modifications;

the Nb modifications are different from the Y modification, and the Nb' modifications are different from the Y' modification; and where the sense strand is conjugated to at least one ligand.

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In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of complement component C5 in a cell, wherein said double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to a portion of an mRNA encoding complement component C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein said double-stranded RNAi agent is represented by formula (III):

sense: 5' n _p -N _a - (XXX) iN _b -YYY -N _b -(ZZZ)j -N _a - n _q 3 ' antisense: 3' n _p '-N _a ' - (X'X'X ') _k -N _b '-Y ' Y ' Y '-N _b ' - (Z ' Z ' Z ') _b -N _a ' - n _q '5' (III), where each of i, j, k, and l is independently 0 or 1;

each of np, nq, and nq', each of which may be present or absent, independently represents a protruding nucleotide;

each of p, q and q' is independently equal to 0-6;

n _p '>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate bond;

each of Na and Na' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of N _b and Nb' independently represents an oligonucleotide sequence containing 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides, and wherein the modifications are 2'-Omethyl- or 2'-fluoro-modifications;

modifications N _b are different from modification Y, and modifications N _b ' are different from modification Y'; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of complement component C5 in a cell, wherein said double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to a portion of an mRNA encoding complement component C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein said double-stranded RNAi agent is represented by formula (III):

sense: 5' n _p -N _a - (XXX) iN _b -YYY -N _b -(ZZZ)j -N _a - n _q 3 ' antisense: 3' n _p ' -N _a ' - (X 'X 'X ') _k -N _b '-Y Ύ Ύ ' -N _b ' - (Z ' Z ' Z ') _b -N _a ' - n _q '5' (III), where each of i, j, k, and l is independently 0 or 1;

each of n _p , n _q , and n _q ', each of which may be present or absent, independently represents a protruding nucleotide;

each of p, q and q' is independently equal to 0-6;

n _p '>0, and at least one n _p ' is linked to an adjacent nucleotide via a phosphorothioate bond;

each of Na and Na' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of N _b and N _b ' independently represents an oligonucleotide sequence comprising 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides, and wherein the modifications are 2'-Omethyl- or 2'-fluoro-modifications;

modifications N _b differ from modification Y, and modifications N _b ' differ from modification Y';

wherein the sense strand comprises at least one phosphorothioate bond; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

In another aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of complement component C5 in a cell, wherein said double-stranded agent

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The RNAi comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region complementary to a portion of an mRNA encoding complement component C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein said double-stranded RNAi agent is represented by formula (III):

sense: 5' n _p -N _a -YYY - N _a - n _q 3' antisense: 3' n _p '-N _a '- ΥΎΎ'- N _a '- n _q '5' (Illa), where each of n _p , nq, and nq', each of which may be present or absent, independently represents a protruding nucleotide;

each of p, q and q' is independently equal to 0-6;

n _p '>0, and at least one n _p ' is linked to an adjacent nucleotide via a phosphorothioate bond;

each of N _a and N _a ' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of YYY and YY'Y' independently represents one motif of three identical modifications of three consecutive nucleotides, and wherein the modifications are 2'-O-methyl- or 2'-fluoro-modifications;

wherein the sense strand comprises at least one phosphorothioate bond; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

In one aspect, the present invention relates to a double-stranded RNAi agent for inhibiting the expression of complement component C5, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5, wherein substantially all of the nucleotides of the sense strand comprise modifications selected from the group consisting of a 2'-O-methyl modification and a 2'fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-end, wherein substantially all of the nucleotides of the antisense strand comprise modifications selected from the group consisting of a 2'-O-methyl modification and 2'-fluoro modifications, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached via a branched divalent or trivalent linker at the 3' end.

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides. In another embodiment, each strand contains 19-30 nucleotides.

In one aspect, the present invention relates to a cell comprising a dsRNA agent of the present invention.

In one aspect, the present invention relates to a vector encoding at least one chain of a dsRNA agent, wherein the dsRNA agent comprises a region of complementarity to at least a portion of an mRNA encoding complement component C5, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA agent targets the mRNA for cleavage.

In one embodiment, the region of complementarity is at least 15 nucleotides in length. In another embodiment, the region of complementarity is 19-21 nucleotides in length. In another embodiment, each strand comprises 19-30 nucleotides.

In one aspect, the present invention relates to a cell comprising a vector of the present invention.

In one aspect, the present invention relates to a pharmaceutical composition for inhibiting the expression of the complement component C5 gene, comprising the dsRNA agent of the present invention.

In one embodiment, the RNAi agent is administered in an unbuffered solution.

In one embodiment, the unbuffered solution is saline or water.

In one embodiment, the RNAi agent is administered in a buffer solution.

In one embodiment, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.

In another embodiment, the buffer solution is phosphate buffered saline (PBS).

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In another aspect, the present invention relates to a pharmaceutical composition comprising a double-stranded RNAi agent of the present invention and a lipid formulation.

In one embodiment, the lipid composition comprises LNP. In another embodiment, the lipid composition comprises MC3.

In one aspect, the present invention relates to a composition comprising an antisense polynucleotide agent selected from the group consisting of the sequences listed in any of Tables 3, 4, 5, 6, 19, 18, 20, 21 and 23.

In another aspect, the present invention relates to a composition comprising a sense polynucleotide agent selected from the group consisting of the sequences listed in any of Tables 3, 4, 5, 6, 19, 18, 20, 21 and 23.

In another aspect, the present invention relates to a modified antisense polynucleotide agent selected from the group consisting of the antisense sequences listed in any of Tables 4, 6, 18, 19, 21 and 23.

In a further aspect, the present invention relates to a modified sense polynucleotide agent selected from the group consisting of the sense sequences listed in any of Tables 4, 6, 18, 19, 21 and 23.

In one aspect, the present invention relates to methods for treating a subject suffering from a disease or disorder in which a reduction in the expression of complement component C5 would have a beneficial effect. The methods comprise administering to the subject a therapeutically effective amount of an agent that is a dsRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5, thereby treating the subject.

In another aspect, the present invention relates to methods for preventing at least one symptom in a subject suffering from a disease or disorder in which a reduction in the expression of the complement component C5 will have a beneficial effect. The methods comprise administering to the subject a therapeutically effective amount of an agent that is a dsRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5, thereby preventing at least one symptom in a subject suffering from a disease in which a reduction in the expression of C5 will have a beneficial effect.

In another aspect, the present invention relates to methods for treating a subject suffering from a disease or disorder in which a reduction in the expression of complement component C5 would have a beneficial effect. The methods comprise administering to the subject a therapeutically effective amount of an agent that is a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity that comprises at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any of the antisense sequences listed in any of Tables 3, 4, 5, 6, 18, 19, 20, 21, 23, thereby treating the subject.

In another aspect, the present invention relates to methods for preventing at least one symptom in a subject suffering from a disease or disorder in which a reduction in the expression of the complement component C5 will have a beneficial effect. The methods comprise administering to the subject a prophylactically effective amount of an agent that is a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity that comprises at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any of the antisense sequences listed in any of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23, thereby preventing at least one symptom in a subject suffering from a disease in which a reduction in the expression of C5 will have a beneficial effect.

In one aspect, the present invention relates to methods for treating a subject suffering from a disease or disorder in which a reduction in the expression of complement component C5 would have a beneficial effect, which comprises administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5, wherein substantially all of the nucleotides of the antisense strand and substantially all of the nucleotides of the sense strand are modified nucleotides, and wherein the sense strand is conjugated to one or more ligands at the 3'-end.

- 9 044245

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the administration is subcutaneous administration.

In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal dT nucleotide. In another embodiment, substantially all of the nucleotides of the antisense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal deoxythymine (dT) nucleotide. In another embodiment, the modified nucleotides are a short sequence of deoxythymine (dT) nucleotides. In another embodiment, the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end. In another embodiment, the sense strand is conjugated to one or more GalNAc derivatives attached via a branched divalent or trivalent linker at the 3' end.

In another aspect, the present invention relates to methods for preventing at least one symptom in a subject suffering from a disease or disorder in which a reduction in the expression of complement component C5 would have a beneficial effect, which comprises administering to the subject a prophylactically effective amount of a double-stranded RNAi agent, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5, wherein substantially all of the nucleotides of the antisense strand and substantially all of the nucleotides of the sense strand are modified nucleotides, and wherein the sense strand is conjugated to a single ligand at the 3' end.

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the administration is subcutaneous administration.

In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal dT nucleotide. In another embodiment, substantially all of the nucleotides of the antisense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal deoxythymine (dT) nucleotide. In another embodiment, the modified nucleotides are a short sequence of deoxythymine (dT) nucleotides. In another embodiment, the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end. In another embodiment, the sense strand is conjugated to one or more GalNAc derivatives attached via a branched divalent or trivalent linker at the 3' end.

In one aspect, the present invention relates to methods for treating a subject suffering from a disease or disorder in which a reduction in the expression of complement component C5 would have a beneficial effect. The methods comprise administering to the subject a therapeutically effective amount of an agent that is a dsRNA comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the double-stranded RNAi agent is represented by formula (III):

semantic: 5' p _r -N _a - (XXX) iN _b -YYY -N _b - (Ζ Ζ Z)j -N _a - n _q 3 ' antisense: 3' p _r ' -N _a ' - (X 'X 'X ') _k -N _b '-Υ Ύ Ύ ' -N _b ' - (Ζ ' Ζ ' Ζ ' ) _b -N _a ' - n _q '5' (III), where each of i, j, k and l is independently equal to 0 or 1;

each of p, p', q and q' is independently equal to 0-6;

each of N _a and N _a ' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of N _b and N _b ' independently represents an oligonucleotide sequence comprising 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of np, np', n _q and nq', each of which may be present or absent, independently represents a protruding nucleotide;

- 10 044245 each of XXX, YYY, ZZZ, X'X'X', YY'Y' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides;

modifications N _b differ from modification Y, and modifications N _b ' differ from modification

Y'; and wherein the sense strand is conjugated to at least one ligand; wherein a subject is treated, wherein a subject suffering from a disease or disorder is treated, wherein a reduction in the expression of complement component C5 will have a beneficial effect.

In another aspect, the present invention relates to methods for preventing at least one symptom in a subject suffering from a disease or disorder in which a reduction in the expression of complement component C5 would have a beneficial effect. The methods comprise administering to the subject a prophylactically effective amount of an agent that is a dsRNA comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the double-stranded RNAi agent is represented by formula (III):

sense: 5' n _p -N _a - (XXX) iN _b -YYY -N _b -(ZZZ)j -N _a - n _q 3 ' antisense: 3' n _p ' -N _a ' - (X 'X 'X ') _k -N _b '-Y Ύ Ύ ' -N _b ' - (Z ' Z ' Z ') _b -N _a ' - n _q '5' (III), where each of i, j, k, and l is independently 0 or 1;

each of p, p', q and q' is independently equal to 0-6;

each of Na and Na' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of N _b and N _b independently represents an oligonucleotide sequence containing 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of np, np', n _q, and n _q ', each of which may be present or absent, independently represents a protruding nucleotide;

each of XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides;

the Nb modifications are different from the Y modification, and the Nb' modifications are different from the Y' modification; and wherein the sense strand is conjugated to at least one ligand; whereby at least one symptom in a subject suffering from a disease in which a reduction in the expression of C5 would have a beneficial effect is prevented, thereby preventing at least one symptom in a subject suffering from a disease or disorder in which a reduction in the expression of the complement component C5 would have a beneficial effect.

In one embodiment, administration of dsRNA to a subject results in a reduction in intravascular hemolysis, stabilization of hemoglobin levels, and/or a reduction in C5 protein accumulation.

In one embodiment, the disease is a disease associated with complement component C5. In one embodiment, the disease associated with complement component C5 is selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA), antiphospholipid antibody syndrome, lupus nephritis, ischemia-reperfusion injury, typical or infectious hemolytic uremic syndrome (tHUS), dense deposit disease (DDD), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); syndrome involving hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, bullous pemphigoid associated with Shiga-like toxin of E. coli, hemolytic uremic syndrome, C3 nephropathy, vasculitis associated with antibodies to neutrophil cytoplasm; graft rejection reactions caused by humoral and vascular mechanisms, graft dysfunction, myocardial infarction, allogeneic transplantation, sepsis, coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, sepsis associated with the systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type 1 diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disorder, peripheral vascular disorders, renovascular hypertension, disorders mesenteric/intestinal vessels, vasculitis, Henoch-Schonlein nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, vasculitis associated with immune complexes, Takayasu disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barré syndrome and percutaneous transluminal coronary angioplasty (PTCA). In another embodiment, the disease associated with complement component C5 is paroxysmal nocturnal hemoglobinuria (PNH). In yet another embodiment, the disease associated with complement component C5 is atypical hemolytic uremic syndrome (aHUS).

In one embodiment, the subject is a human.

In another embodiment, the methods of the present invention further comprise administering to the subject an antibody to complement component C5 or an antigen-binding fragment thereof.

In one embodiment, the antibody or antigen-binding fragment thereof inhibits the cleavage of complement component C5 into fragments C5a and C5b. In another embodiment, the antibody to complement component C5 is eculizumab.

In another embodiment, the methods of the present invention further comprise administering to the subject a meningococcal vaccine.

In one embodiment, eculizumab is administered to the subject once weekly at a dose of less than about 600 mg for 4 weeks, followed by a fifth dose about one week later at less than about 900 mg, and then at a dose of less than about 900 mg about every two weeks thereafter.

In another embodiment, eculizumab is administered to the subject once weekly at a dose of less than about 900 mg for 4 weeks, followed by a fifth dose after about one week at less than about 1200 mg, and then at a dose of less than about 1200 mg about every two weeks thereafter.

In one embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 900 mg for 4 weeks, followed by a fifth dose after about one week at less than about 1200 mg, and then at a dose of less than about 1200 mg approximately every two weeks thereafter.

In another embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 600 mg for 2 weeks, followed by a third dose about one week later at less than about 900 mg, and then at a dose of less than about 900 mg about every two weeks thereafter.

In another embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 600 mg for 2 weeks, followed by a third dose about one week later at less than about 600 mg, and then at a dose of less than about 600 mg about every two weeks thereafter.

In another embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 600 mg for 1 week, followed by a second dose about one week later at less than about 300 mg, and then at a dose of less than about 300 mg about every two weeks thereafter.

In one embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 300 mg for 1 week, followed by a second dose about one week later at less than about 300 mg, and then at a dose of less than about 300 mg about every two weeks thereafter.

In another embodiment, the methods of the present invention further comprise plasmapheresis or plasma exchange in the subject. In one such embodiment, eculizumab is administered to the subject at a dose of less than about 600 mg or at a dose of less than about 300 mg.

In another embodiment, the methods of the present invention further comprise infusing the plasma into the subject. In one such embodiment, eculizumab is administered to the subject at a dose of less than about 300 mg.

In one embodiment, eculizumab is administered to the subject at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 15 mg/kg. In another embodiment, eculizumab is administered to the subject at a dose of about 5 mg/kg to about 15 mg/kg.

In one embodiment, eculizumab is administered to the subject at a dose selected from the group consisting of 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, and 15 mg/kg.

In one embodiment, eculizumab is administered to a subject by intravenous infusion.

In another embodiment, eculizumab is administered to the subject subcutaneously.

In one embodiment, the dsRNA agent is administered at a dose of from about 0.01 mg/kg to about 10 mg/kg or from about 0.5 mg/kg to about 50 mg/kg.

- 12 044245

In another embodiment, the dsRNA agent is administered at a dose of from about 10 mg/kg to about 30 mg/kg.

In one embodiment, the dsRNA agent is administered at a dose selected from the group consisting of 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg.

In one embodiment, the dsRNA agent is administered to the subject once per week. In another embodiment, the dsRNA agent is administered to the subject twice per week. In another embodiment, the dsRNA agent is administered to the subject twice per month.

In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

In one embodiment, the dsRNA agent and eculizumab are administered to the subject subcutaneously. In another embodiment, the dsRNA agent and eculizumab are administered to the subject simultaneously.

In one embodiment, the dsRNA agent is first administered to the subject for a period of time sufficient to reduce the level of complement component C5 in the subject, and then eculizumab is administered at a dose of less than about 600 mg.

In one embodiment, the level of complement component C5 in the subject is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

In one embodiment, eculizumab is administered at a dose of about 100-500 mg.

In one embodiment, the methods of the present invention further comprise measuring hemoglobin and/or LDH levels in the subject.

In one embodiment, the dsRNA is conjugated to a ligand.

In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA.

In one embodiment, the ligand is a derivative of N-acetylgalactosamine (GalNAc).

In one aspect, the present invention relates to methods for inhibiting the expression of complement component C5 in a cell. The methods comprise contacting the cell with an agent that is a dsRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5; and maintaining the cell obtained in step (a) for a time sufficient to degrade the mRNA transcript of the C5 gene, thereby inhibiting the expression of the C5 gene in the cell.

In another aspect, the present invention relates to methods for inhibiting the expression of complement component C5 in a cell. The methods comprise contacting the cell with an agent that is a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity that comprises at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any of the antisense sequences listed in any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23; and maintaining the cell obtained in step (a) for a time sufficient to degrade the mRNA transcript of the C5 gene, thereby inhibiting the expression of the C5 gene in the cell.

In another aspect, the present invention relates to methods for inhibiting the expression of a complement component C5 in a cell, comprising contacting the cell with an agent that is a dsRNA comprising a sense strand and an antisense strand comprising a region of complementarity, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5, wherein substantially all of the nucleotides of the antisense strand and substantially all of the nucleotides of the sense strand are modified nucleotides and wherein the sense strand is conjugated to one or more ligands at the 3'-end; and maintaining the cell obtained in the first step for a time sufficient to destroy the mRNA transcript of the C5 gene, thereby inhibiting the expression of the C5 gene in the cell.

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal dT nucleotide. In another embodiment, substantially all of the nucleotides of the antisense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal deoxy-thymine (dT) nucleotide. In another embodiment, the modified nucleotides are a short sequence of deoxy-thymine (dT) nucleotides. In another embodiment, the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end. In another embodiment, the sense strand is conjugated to one or more GalNAc derivatives attached via a branched divalent or trivalent linker at the 3' end.

In another aspect, the present invention relates to methods for inhibiting the expression of complement component C5 in a cell. The methods comprise contacting the cell with an agent that is a dsRNA comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the double-stranded RNAi agent is represented by formula (III):

semantic: 5' n _p -N _a - (XXX) iN _b -YYY -N _b - (Ζ Ζ Z)j -N _a - n _q 3 ' antisense: 3' n _p ' -N _a ' - (X 'X 'X ') _k -N _b '-Y Ύ Ύ ' -N _b ' - (Ζ ' Ζ ' Z ') _b -N _a ' - n _q '5' (III), where each of i, j, k and l is independently equal to 0 or 1;

each of p, p', q and q' is independently equal to 0-6;

each of N _a and N _a ' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of N _b and N _b ' independently represents an oligonucleotide sequence comprising 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of np, np', n _q, and n _q ', each of which may be present or absent, independently represents a protruding nucleotide;

each of XXX, YYY, ZZZ, X'X'X', YYY and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides;

the N _b modifications are different from the Y modification, and the Nb' modifications are different from the Y'modification; and wherein the sense strand is conjugated to at least one ligand; and maintaining the cell obtained in step (a) for a time sufficient to degrade the C5 gene mRNA transcript, thereby inhibiting expression of the C5 gene in the cell.

In one embodiment, the cell is in a subject.

In one embodiment, the subject is a human.

In one embodiment, the human subject suffers from a disease associated with complement component C5.

In one embodiment, the disease associated with the complement component C5 is selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA), antiphospholipid antibody syndrome, lupus nephritis, ischemia-reperfusion injury, typical or infectious hemolytic uremic syndrome (tHUS), dense deposit disease (DDD), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, high liver enzymes, and low platelet syndrome (HELLP syndrome); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; pauci-immune vasculitis; bullous epidermolysis; recurrent fetal loss; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis bullous pemphigoid associated with Shiga-like toxin of E. coli hemolytic uremic syndrome, C3 nephropathy, vasculitis associated with antibodies to neutrophil cytoplasm; Allograft rejection reactions mediated by humoral and vascular mechanisms, graft dysfunction, myocardial infarction, allogeneic transplantation, sepsis, coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, sepsis associated with the systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type 1 diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular accident, peripheral vascular disorders, renovascular hypertension, mesenteric/intestinal vascular disorders, vasculitis, Henoch-Schonlein nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex-associated vasculitis, Takayasu disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barré syndrome and percutaneous transluminal coronary angioplasty (PTCA). In another embodiment, the disease associated with complement component C5 is

- 14 044245 paroxysmal nocturnal hemoglobinuria (PNH). In another embodiment, the disease associated with complement component C5 is atypical hemolytic uremic syndrome (aHUS).

In one embodiment, the methods further comprise contacting the cell with an antibody to complement component C5 or an antigen-binding fragment thereof.

In one embodiment, the antibody or antigen-binding fragment thereof inhibits the cleavage of complement component C5 into C5a and C5b fragments.

In one embodiment, the antibody to complement component C5 or antigen-binding fragment thereof is eculizumab.

In one embodiment, the methods further comprise contacting the cell with a meningococcal vaccine.

In one embodiment, the cell is contacted with eculizumab once a week at a dose of less than about 600 mg for 4 weeks, followed by a fifth dose after about one week at less than about 900 mg, and then at a dose of less than about 900 mg about every two weeks thereafter.

In another embodiment, the cell is contacted with eculizumab once a week at a dose of less than about 900 mg for 4 weeks, followed by a fifth dose about one week later at less than about 1200 mg, and then at a dose of less than about 1200 mg about every two weeks thereafter.

In another embodiment, the cell is contacted with eculizumab once a week at a dose of less than about 900 mg for 4 weeks, followed by a fifth dose about one week later at less than about 1200 mg, and then at a dose of less than about 1200 mg about every two weeks thereafter.

In another embodiment, the cell is contacted with eculizumab once a week at a dose of less than about 600 mg for 2 weeks, followed by a third dose about one week later at less than about 900 mg, and then at a dose of less than about 900 mg about every two weeks thereafter.

In one embodiment, the cell is contacted with eculizumab once per week at a dose of less than about 600 mg for 2 weeks, followed by a third dose after about one week at less than about 600 mg, and then at a dose of less than about 600 mg about every two weeks thereafter.

In another embodiment, the cell is contacted with eculizumab once a week at a dose of less than about 600 mg for 1 week, followed by a second dose about one week later at less than about 300 mg, and then at a dose of less than about 300 mg about every two weeks thereafter.

In one embodiment, the cell is contacted with eculizumab once per week at a dose of less than about 300 mg for 1 week, followed by a second dose about one week later at less than about 300 mg, and then at a dose of less than about 300 mg about every two weeks thereafter.

In one embodiment, the cell is in a subject.

In one embodiment, the methods of the present invention further comprise plasmapheresis or plasma exchange in the subject. In one embodiment, eculizumab is administered to the subject at a dose of less than about 600 mg. In another embodiment, eculizumab is administered to the subject at a dose of less than about 300 mg.

In one embodiment, the methods of the present invention further comprise infusing the plasma into the subject. In one embodiment, eculizumab is administered to the subject at a dose of less than about 300 mg.

In one embodiment, the cell is contacted with eculizumab at a dose of from about 0.01 mg/kg to about 10 mg/kg or from about 0.5 mg/kg to about 15 mg/kg.

In another embodiment, the cell is contacted with eculizumab at a dose of from about 5 mg/kg to about 15 mg/kg.

In one embodiment, the cell is contacted with eculizumab at a dose selected from the group consisting of 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, and 15 mg/kg.

In one embodiment, eculizumab is administered to a subject by intravenous infusion. In another embodiment, eculizumab is administered to a subject subcutaneously.

In one embodiment, the cell is contacted with the dsRNA agent at a dose of from about 0.01 mg/kg to about 10 mg/kg or from about 0.5 mg/kg to about 50 mg/kg.

In another embodiment, the cell is contacted with the dsRNA agent at a dose of from about 10 mg/kg to about 30 mg/kg.

In one embodiment, the cell is contacted with an agent that is dsRNA at a dose selected from the group consisting of 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg.

- 15 044245

In one embodiment, the cell is contacted with the dsRNA agent once per week. In another embodiment, the dsRNA agent is administered to the subject twice per week. In another embodiment, the cell is contacted with the dsRNA agent twice per month.

In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

In one embodiment, the dsRNA agent and eculizumab are administered to the subject subcutaneously. In another embodiment, the dsRNA agent and eculizumab are administered to the subject simultaneously.

In one embodiment, the cell is contacted with the dsRNA agent and eculizumab simultaneously.

In one embodiment, the dsRNA agent is administered to the subject initially for a period of time sufficient to reduce the level of complement component C5 in the subject, and eculizumab is then administered at a dose of less than about 600 mg.

In one embodiment, the level of complement component C5 in the subject is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

In one embodiment, eculizumab is administered at a dose of about 100-500 mg.

In one embodiment, the cell is first contacted with a dsRNA agent for a period of time sufficient to reduce the level of complement component C5 in the cell, and then the cell is contacted with eculizumab at a dose of less than about 600 mg.

In one embodiment, the level of complement component C5 in the cell is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

In one embodiment, the cell is contacted with eculizumab at a dose of about 100-500 mg/kg.

In one aspect, the present invention provides methods for inhibiting the expression of C5 in a subject. The methods comprise administering to the subject a therapeutically effective amount of an agent comprising a dsRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5, thereby inhibiting the expression of C5 in the subject.

In another aspect, the present invention provides methods for inhibiting the expression of C5 in a subject. The methods comprise administering to the subject a therapeutically effective amount of an agent that is a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity that comprises at least 15 contiguous nucleotides that differs by no more than 3 nucleotides from any of the antisense sequences listed in any of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23, thereby inhibiting the expression of C5 in the subject.

In another aspect, the present invention relates to methods for inhibiting the expression of complement component C5 in a subject, comprising administering to the subject a therapeutically effective amount of an agent that is a dsRNA comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 5, wherein substantially all of the nucleotides of the antisense strand and substantially all of the nucleotides of the sense strand are modified nucleotides and wherein the sense strand is conjugated to one or more ligands at the 3' end, thereby inhibiting the expression of the C5 gene in the subject.

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the administration is subcutaneous administration.

In one embodiment, substantially all of the nucleotides of the sense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal dT nucleotide. In another embodiment, substantially all of the nucleotides of the antisense strand are modified nucleotides selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 3'-terminal deoxythymine (dT) nucleotide. In another embodiment, the modified nucleotides are a short sequence of deoxythymine (dT) nucleotides. In another embodiment, the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end. In another embodiment, the sense strand is conjugated to one or more GalNAc derivatives attached via a branched divalent or trivalent linker at the 3' end.

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In another aspect, the present invention provides methods for inhibiting C5 expression in a subject. The methods comprise administering to the subject a therapeutically effective amount of an agent that is a dsRNA comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding C5, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the double-stranded RNAi agent is represented by formula (III):

semantic: 5' n _p -N _a -(XXX) iN _b -YYY -N _b - (Ζ Ζ Z)j -N _a - n _q 3 ' antisense: 3' n _p ' -N _a ' - (X 'X 'X ') _k -N _b '-Y Ύ Ύ ' -N _b ' - (Ζ ' Ζ ' Z ') _b -N _a ' - n _q '5' (III), where each of i, j, k and l is independently equal to 0 or 1;

each of p, p', q and q' is independently equal to 0-6;

each of Na and Na' independently represents an oligonucleotide sequence comprising 0-25 nucleotides that are either modified or unmodified, or combinations thereof, wherein each sequence comprises at least two nucleotides modified in different ways;

each of Nb and Nb' independently represents an oligonucleotide sequence containing 0-10 nucleotides that are either modified or unmodified, or combinations thereof;

each of n _p , n _p ', n _{q ,} and n _q ', each of which may be present or absent, independently represents a protruding nucleotide;

each of XXX, YYY, ZZZ, X'X'X', ΥΎΎ and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides;

the Nb modifications are different from the Y modification, and the Nb' modifications are different from the Y' modification; and wherein the sense strand is conjugated to at least one ligand, thereby inhibiting C5 expression in the subject.

In one embodiment, the methods further comprise administering to the subject an antibody to complement component C5 or an antigen-binding fragment thereof.

In one embodiment, the antibody to complement component C5 or antigen-binding fragment thereof is eculizumab.

In one embodiment, the antibody or antigen-binding fragment thereof inhibits the cleavage of complement component C5 into C5a and C5b fragments.

In one embodiment, the methods of the present invention further comprise administering to the subject a meningococcal vaccine.

In one embodiment, eculizumab is administered to the subject once weekly at a dose of less than about 600 mg for 4 weeks, followed by a fifth dose about one week later at less than about 900 mg, and then at a dose of less than about 900 mg about every two weeks thereafter.

In another embodiment, eculizumab is administered to the subject once weekly at a dose of less than about 900 mg for 4 weeks, followed by a fifth dose after about one week at less than about 1200 mg, and then at a dose of less than about 1200 mg about every two weeks thereafter.

In one embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 900 mg for 4 weeks, followed by a fifth dose after about one week at less than about 1200 mg, and then at a dose of less than about 1200 mg approximately every two weeks thereafter.

In another embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 600 mg for 2 weeks, followed by a third dose about one week later at less than about 900 mg, and then at a dose of less than about 900 mg about every two weeks thereafter.

In one embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 600 mg for 2 weeks, followed by a third dose about one week later at less than about 600 mg, and then at a dose of less than about 600 mg about every two weeks thereafter.

In another embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 600 mg for 1 week, followed by a second dose about one week later at less than about 300 mg, and then at a dose of less than about 300 mg about every two weeks thereafter.

In another embodiment, the subject is less than 18 years of age and eculizumab is administered to the subject once weekly at a dose of less than about 300 mg for 1 week, followed by a second dose about one week later at less than about 300 mg, and then at a dose of less than about 300 mg about every two weeks thereafter.

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In one embodiment, the methods further include plasmapheresis or plasma exchange in the subject. In one embodiment, eculizumab is administered to the subject at a dose of less than about 600 mg. In another embodiment, eculizumab is administered to the subject at a dose of less than about 300 mg.

In one embodiment, the methods further comprise infusing the plasma into the subject. In one embodiment, eculizumab is administered to the subject at a dose of less than about 300 mg.

In one embodiment, eculizumab is administered to the subject at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 15 mg/kg. In another embodiment, eculizumab is administered to the subject at a dose of about 5 mg/kg to about 15 mg/kg.

In another embodiment, eculizumab is administered to the subject at a dose selected from the group consisting of 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, and 30 mg/kg.

In one embodiment, eculizumab is administered to a subject by intravenous infusion. In another embodiment, eculizumab is administered to a subject subcutaneously.

In one embodiment, the dsRNA agent is administered at a dose of from about 0.01 mg/kg to about 10 mg/kg or from about 0.5 mg/kg to about 15 mg/kg.

In one embodiment, the dsRNA agent is administered at a dose of from about 10 mg/kg to about 30 mg/kg. In another embodiment, the dsRNA agent is administered at a dose selected from the group consisting of 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, and 30 mg/kg.

In one embodiment, the dsRNA agent is administered to the subject once per week. In another embodiment, the dsRNA agent is administered to the subject twice per week. In another embodiment, the dsRNA agent is administered to the subject twice per month.

In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

In one embodiment, the dsRNA agent and eculizumab are administered to the subject subcutaneously. In another embodiment, the dsRNA agent and eculizumab are administered to the subject simultaneously.

In one embodiment, the dsRNA agent is first administered to the subject for a period of time sufficient to reduce the level of complement component C5 in the subject, and then eculizumab is administered at a dose of less than about 600 mg.

In one embodiment, the level of complement component C5 in the subject is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

In one embodiment, eculizumab is administered at a dose of about 100-500 mg.

In one embodiment, the dsRNA agent is conjugated to a ligand.

In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

In one embodiment, the ligand is a derivative of N-acetylgalactosamine (GalNAc).

Brief description of graphic materials

Fig. 1 is a schematic representation of the three complement pathways: alternative, classical, and lectin.

Fig. 2 is a graph showing the percentage of complement component C5 remaining in C57BL/6 mice after a single 10 mg/kg dose of the indicated mRNAs.

Fig. 3 is a graph showing the percentage of complement component C5 remaining in C57BL/6 mice after a single 10 mg/kg dose of the indicated mRNAs.

Fig. 4 is a graph showing the percentage of complement component C5 remaining in C57BL/6 mice 48 h after a single 10 mg/kg dose of the indicated mRNAs.

Fig. 5A is a graph showing the percentage of hemolysis remaining on days 4 and 7 in rats following a single subcutaneous administration of AD-58642 at a dose of 2.5 mg/kg, 10 mg/kg, or 25 mg/kg.

Figure 5B shows Western blot data showing the amount of complement component C5 remaining at day 7 in rats following a single subcutaneous administration of AD-58642 at a dose of 2.5 mg/kg, 10 mg/kg, or 25 mg/kg.

Fig. 6A and 6B are graphs showing the percentage of complement component C5 remaining in C57BL/6 mice 5 days after a single dose of AD-58642 at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, or 25 mg/kg.

Fig. 7A and 7B are graphs showing the percentage of hemolysis remaining at day 5 in C57BL/6 mice following a single dose of AD-58642 at 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, or 25 mg/kg.

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Figure 8 shows Western blot data showing the amount of complement component C5 remaining at day 5 in C57BL/6 mice after a single dose of 1.25 mg/kg AD-58642,

2.5 mg/kg, 5 mg/kg, 10 mg/kg, or 25 mg/kg.

Fig. 9 is a graph showing the amount of complement component C5 protein remaining in the serum of mice on days 5 and 9 after a single dose of AD-58641 at 0.625 mg/kg, 1.25 mg/kg, 2.5 mg/kg, 5.0 mg/kg, or 10 mg/kg. The lower limit of quantification (LLOQ) of the assay is shown as a dotted line.

Fig. 10 is a graph showing the amount of complement component C5 protein remaining in the serum of mice at day 8 following administration of AD-58641 on days 0, 1, 2, and 3 at a dose of 0.625 mg/kg, 1.25 mg/kg, or 2.5 mg/kg. The lower limit of quantification (LLOQ) of the assay is shown as a dotted line.

Figures 11A and 11B show the efficacy and cumulative effect of repeated administration of AD-58641 in rats. Figure 11A is a graph showing the hemolytic activity retained in rat serum on days 0, 4, 7, 11, 14, 18, 25, and 32 following repeated administration at 2.5 mg/kg/dose or 5.0 mg/kg/dose, q2w x3 (twice a week for 3 weeks). Figure 11B is a Western blot showing the amount of complement component C5 protein remaining in the serum of the animals.

Fig. 12 is a graph showing the amount of complement component C5 protein in the serum of cynomolgus macaques at various time points before, during, and after two cycles of subcutaneous administration of AD-58641 at a dose of 2.5 mg/kg or 5 mg/kg every third day for eight doses. C5 protein levels were normalized to the average of three pre-treatment samples.

Fig. 13 is a graph showing the percentage of hemolysis remaining in the serum of cynomolgus macaques at various time points before, during, and after two cycles of subcutaneous administration of AD-58641 at a dose of 2.5 mg/kg or 5 mg/kg every third day for eight doses. The percentage of hemolysis was calculated as the ratio of maximum hemolysis to background hemolysis in control samples.

Fig. 14 is a graph showing the percentage of complement component C5 protein remaining at day 5 in the serum of C57BL/6 mice after a single 1 mg/kg dose of the indicated mRNAs.

Fig. 15 is a graph showing the percentage of complement component C5 protein remaining at day 5 in the serum of C57BL/6 mice after a single dose of 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg of the indicated mRNAs.

Fig. 16 is a graph showing the percentage of complement component C5 protein remaining in the serum of C57BL/6 mice on days 6, 13, 20, 27, and 34 after a single 1 mg/kg dose of the indicated mRNAs.

Fig. 17 is a graph showing the percentage of hemolysis remaining in the serum of rats at various time points after administration of a 5 mg/kg dose of the indicated compounds on days 0, 4, and 7.

Fig. 18A shows the nucleotide sequence of complement component 5 (C5) of Homo sapiens (SEQ ID NO:1); Fig. 18B shows the nucleotide sequence of complement component 5 (C5) of Macaca mulatta (SEQ ID NO:2); Fig. 18C shows the nucleotide sequence of complement component 5 (C5) of Mus musculus (SEQ ID NO:3); Fig. 18D shows the nucleotide sequence of complement component 5 (C5) of Rattus norvegicus (SEQ ID NO:4); Fig. 18E shows the reverse complement of SEQ ID NO:1 (SEQ ID NO:5); Fig. 18E shows the reverse complement of SEQ ID NO:2 (SEQ ID NO:6); Fig. 18C shows the reverse complement of SEQ ID NO:3 (SEQ ID NO:7); and Fig. 18H shows the reverse complement of SEQ ID NO:4 (SEQ ID NO:8).

Detailed description of the invention

The present invention relates to mRNA agents that target RNA-induced silencing complex (RISC)-mediated cleavage of complement component C5 gene RNA transcripts.

The mRNAs of the present invention comprise an RNA strand (antisense strand) comprising a region of about 30 nucleotides or less in length, such as 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 nucleotides in length, wherein the region is substantially complementary to at least a portion of the C5 gene mRNA transcript. The use of these mRNAs provides for the targeted destruction of the C5 gene mRNA in mammals. Very low doses of C5 mRNA, in particular, can specifically and effectively mediate RNA interference (RNAi), resulting in significant inhibition of C5 gene expression. The present inventors have demonstrated that mRNAs targeting C5 can mediate RNAi in vitro and in vivo, resulting in significant inhibition of C5 gene expression. Thus, methods and compositions incorporating these mRNAs are useful in treating a subject,

- 19 044245 in whom a reduction in the level and/or activity of the C5 protein would have a beneficial effect, such as a subject suffering from a disease associated with the complement component C5, such as paroxysmal nocturnal hemoglobinuria (PNH).

The present invention also relates to methods and combination therapies for treating a subject with a disorder that can be beneficially affected by inhibiting or reducing expression of the C5 gene, such as a disease associated with the complement component C5, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) using mRNA compositions that target RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the complement component C5 gene.

The present invention also relates to methods for preventing at least one symptom, such as hemolysis, in a subject suffering from a disorder in which inhibition or reduction of C5 gene expression would have a beneficial effect, such as a disease associated with complement component C5, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). The present invention further relates to mRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the complement component C5 gene. The C5 gene may be within a cell, such as a cell within a subject, such as a human.

The combination therapy of the present invention comprises administering to a subject suffering from a disease associated with complement component C5, an RNAi agent of the present invention and an additional therapeutic agent, such as an antibody or antigen-binding fragment thereof to complement component C5, such as eculizumab.

The combination therapies of the present invention reduce the level of C5 in a subject (e.g., by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 99%) by targeting C5 mRNA with the iRNA agent of the present invention and, accordingly, allow for a reduction in the therapeutically (or prophylactically) effective amount of eculizumab required in treating a subject, thereby reducing treatment costs and allowing for easier and more convenient methods of administering eculizumab, such as subcutaneous administration.

The following detailed description discloses methods for producing and using compositions containing mRNA for inhibiting and expressing the C5 gene, as well as compositions, uses and methods for treating subjects suffering from diseases or disorders in which inhibition and/or reduction of expression of this gene will have a beneficial effect.

I. Definitions.

To facilitate the understanding of the present invention, definitions of relevant terms are first provided. Furthermore, it should be noted that where a value or range of values for a variable is listed in this document, values and ranges intermediate to the listed values are also intended to be part of the present invention.

The singular form is used in this document to denote one or more (i.e., at least one) grammatical entities of an entry. For example, "element" refers to a single element or multiple elements, such as a plurality of elements.

The expression including is used in this document to mean the phrase including without limitation and is used interchangeably with it.

The expression or is used in this document to mean the expression and/or and is used interchangeably with it unless the context clearly indicates otherwise.

The complement component C5 used herein, used interchangeably with the expression C5, refers to a well-known gene and polypeptide also known in the art as CPAMD4, a C3- and PZP-like protein containing an alpha-2-macroglobulin domain, an analog of the anaphylotoxin C5a, a hemolytic complement (Hc), and a unit of complement C5. The sequence of the human C5 mRNA transcript can be found, for example, in GenBank, No. GI: 38016946 (NM_001735.2; SEQ ID NO: 1). The sequence of the macaque C5 mRNA can be found, for example, in GenBank, No. GI: 297270262 (XM_001095750.2; SEQ ID NO: 2). The mouse C5 mRNA sequence can be found, for example, in GenBank, GI No. 291575171 (NM_010406.2; SEQ ID NO:3). The rat C5 mRNA sequence can be found, for example, in GenBank, GI No. 392346248 (XM_345342.4; SEQ ID NO:4). Additional examples of C5 mRNA sequences are readily available in public databases, such as GenBank.

The term C5, as used herein, also refers to naturally occurring variations in the DNA sequences of the C5 gene, such as single nucleotide polymorphisms in the C5 gene. Numerous SNPs in the C5 gene have been identified and can be found, for example, in the NCBI dbSNP (see, for example, ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs in the C5 gene can be found in the NCBI dbSNP, accession numbers rs121909588 and rs121909587.

As used herein, the target sequence refers to a contiguous portion of the nucleotide sequence of the mRNA molecule produced during transcription of the C5 gene, in

- 20 044245 including to mRNA, which is the product of RNA processing of the primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a support for mRNA-directed cleavage at or near this portion of the nucleotide sequence of the mRNA molecule formed during transcription of the C5 gene.

The target sequence may be approximately 9-36 nucleotides in length, such as approximately 15-30 nucleotides in length. For example, the target sequence may be approximately 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the ranges and lengths listed above are also considered to be part of the present invention.

As used herein, the term "chain containing a sequence" refers to an oligonucleotide containing a chain of nucleotides that is characterized by a sequence designated using standard nucleotide nomenclature.

Each of G, C, A, T, and U typically refers to a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, which is described in more detail below, or a nucleotide-mimicking replacement moiety (see, for example, Table 2). It is well known to those skilled in the art that guanine, cytosine, adenine, and uracil can be replaced by other moieties without significantly altering the base pairing properties of an oligonucleotide containing a nucleotide bearing such a replacement moiety. For example, without limitation, a nucleotide containing inosine as a base can form base pairs with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine can be substituted in the dsRNA nucleotide sequences described in the present invention with nucleotides containing, for example, inosine. In another example, adenine and cytosine at any point in the oligonucleotide can be substituted with guanine and uracil, respectively, to form a G-U wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods described in the present invention.

The terms "mRNA," "RNAi agent," "agent comprising mRNA," and "RNA interference agent," when used interchangeably herein, refer to an agent that contains RNA as defined herein and that mediates the targeted cleavage of an RNA transcript via the RNA-induced silencing complex (RISC) pathway. The mRNA directs sequence-specific destruction of mRNA through a process known as RNA interference (RNAi). The mRNA modulates, for example, inhibits, the expression of C5 in a cell, such as a cell of a subject, such as a mammalian subject.

In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, such as a C5 mRNA target sequence, to direct cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is cleaved into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a type III ribonuclease-like enzyme, is involved in the processing of dsRNA into short interfering RNAs of 19-23 base pairs in length with characteristic two-base overhangs at the 3' end (Bernstein, et al., (2001) Nature 409:363). siRNAs are then incorporated into the RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to direct target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present invention relates to a single-stranded RNA (siRNA) formed intracellularly that promotes the formation of the RISC complex to effect silencing of a target gene, i.e., the C5 gene. Accordingly, the term siRNA is also used herein to refer to RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded siRNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to Argonaute 2, an endonuclease in the RISC complex, which then cleaves the target mRNA. Single-stranded siRNAs are typically 15-30 nucleotides in length and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated by reference. Any antisense nucleotide sequences described herein can be used as single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150: 883-894.

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In another embodiment, the mRNA for use in the compositions, uses, and methods of the present invention is double-stranded RNA and is referred to herein as a double-stranded RNAi agent, a double-stranded RNA (dsRNA) molecule, an agent that is dsRNA, or dsRNA. The term dsRNA refers to a complex of ribonucleic acid molecules with a duplex structure, comprising two antiparallel and substantially complementary nucleic acid strands considered to have a sense and antisense orientation with respect to a target RNA, i.e., the C5 gene. In some embodiments of the present invention, double-stranded RNA (dsRNA) triggers the cleavage of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but, as described in detail herein, each or both strands may also include one or more nucleotides that are not ribonucleotides, such as a deoxyribonucleotide and/or a modified nucleotide. Furthermore, the RNAi agent used herein may include ribonucleotides with chemical modifications; the RNAi agent may include significant modifications of a plurality of nucleotides. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications that are used in a siRNA-type molecule are encompassed by the term "RNAi agent" within the context of this description and claims.

The duplex region may be of any length that allows specific degradation of the desired target RNA via the RISC pathway, and may be from about 9 to 36 base pairs in length, such as about 15-30 base pairs in length, such as about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 2024, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the ranges and lengths listed above are also considered to be part of the present invention.

The two strands that form a duplex structure may be different parts of a single larger RNA molecule, or they may be separate RNA molecules. In cases where the two strands are part of a single larger molecule and are therefore connected by an uninterrupted chain of nucleotides from the 3' end of one strand to the 5' end of the corresponding other strand, the duplex structure connecting the RNA strands is called a hairpin loop. A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides.

When two essentially complementary strands of dsRNA contain separate RNA molecules, these molecules may not necessarily be covalently linked. When two strands are covalently linked by a method other than an uninterrupted chain of nucleotides from the 3' end of one strand to the 5' end of the corresponding other strand, forming a duplex structure, the connecting structure is called a linker. The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the RNAi agent may contain one or more nucleotide overhangs.

In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as a C5 mRNA target sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is cleaved into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a type III ribonuclease-like enzyme, is involved in processing dsRNA into short interfering RNAs of 19-23 base pairs in length with characteristic two-base overhangs at the 3' end (Bernstein, et al., (2001) Nature 409:363). siRNAs are then incorporated into the RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to direct target recognition (Nykanen, et al., (2001) Cell 107:309). After binding to the corresponding target mRNA, one or more endonucleases in RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an mRNA, such as a dsRNA. For example, when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa, a nucleotide overhang exists. A dsRNA may contain an overhang of at least one nucleotide.

- 22 044245 nucleotides; alternatively, the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five or more nucleotides. The nucleotide overhang may comprise or consist of a nucleotide/nucleoside analogue, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of the overhang may be at the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

In one embodiment, the antisense strand of the dsRNA comprises 1-10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or nucleotides, protruding at the 3'-end and/or 5'-end. In one embodiment, the sense strand of the dsRNA comprises 1-10 nucleotides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or nucleotides protruding at the 3'-end and/or 5'-end. In another embodiment, one or more nucleotides of the overhang are replaced with a nucleoside thiophosphate.

A blunt end or blunt end means that there are no unpaired nucleotides at the end of a double-stranded RNAi agent, i.e., there is no nucleotide overhang. A blunt-ended RNAi agent is a dsRNA that is double-stranded along its entire length, i.e., it does not have a nucleotide overhang at either end of the molecule. RNAi agents of the present invention include RNAi agents with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends.

The term "antisense strand" or "guide strand" refers to a strand of mRNA, such as dsRNA, that includes a region that is substantially complementary to a target sequence, such as C5 mRNA. As used herein, the term "region of complementarity" refers to a region of the antisense strand that is substantially complementary to a sequence, such as a target sequence, such as the nucleotide sequence C5, as defined herein. In cases where the region of complementarity is not completely complementary to the target sequence, mismatches may be present in internal or terminal regions of the molecule. Typically, the most favorable mismatches are located in terminal regions, such as within 5, 4, 3, or 2 nucleotides of the 5' and/or 3' end of the mRNA.

The term "sense strand" or "passenger strand" as used herein means a strand of mRNA that includes a region that is substantially complementary to a region of the antisense strand, as that term is described herein.

As used herein, the term "cleavage site" refers to a region adjacent to a cleavage site. A cleavage site is the target site at which cleavage occurs. In some embodiments, the cleavage site comprises three bases at either end of the cleavage site and adjacent to it. In some embodiments, the cleavage site comprises two bases at either end of the cleavage site and adjacent to it. In some embodiments, the cleavage site is primarily located at a site flanking nucleotides 10 and 11 of the antisense strand, and the cleavage site comprises nucleotides 11, 12, and 13.

As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence with respect to a second nucleotide sequence means the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising a second nucleotide sequence, as will be understood by one of skill in the art. Such conditions, for example, can be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing (see, for example, Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered in the body, can be used. A person skilled in the art will be able to determine the set of conditions most suitable for analyzing the complementarity of two sequences in accordance with the final use of the hybridized nucleotides.

Complementary sequences within mRNA, such as within dsRNA, described herein include base pairs of an oligonucleotide or polynucleotide comprising a first nucleotide sequence of the oligonucleotide or polynucleotide comprising a second nucleotide sequence along the entire length of one or both nucleotide sequences. Such sequences may be referred to as being fully complementary to each other herein. However, when a first sequence is characterized herein as being substantially complementary to a second sequence, then the two sequences may be fully complementary or they may form one or more, but typically no more than 5, 4, 3, or 2, mismatched base pairs when hybridized to form a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to their end use, such as inhibition of gene expression via the RISC pathway. However, when two oligonucleotides are intended to form one or more single nuclei upon hybridization,

- 23,044,245 stranded overhangs, such overhangs will not be considered mismatches for the purposes of determining complementarity. For example, a dsRNA containing one 21-nucleotide-long oligonucleotide and another 23-nucleotide-long oligonucleotide, where the longer oligonucleotide contains a 21-nucleotide sequence that is completely complementary to the shorter oligonucleotide, may still be considered completely complementary for the purposes described herein.

The complementary sequences used herein may also include or be formed entirely from base pairs not constructed according to the Watson-Crick model and/or base pairs formed from unnatural and modified nucleotides, to the extent that the above requirements regarding their ability to hybridize are met. Such base pairs not constructed according to the Watson-Crick model include, but are not limited to, wobble or Hoogsteen G:U base pairing.

The expressions "complementary," "fully complementary," and "inherently complementary" may be used herein to refer to a base match between the sense strand and the antisense strand of a dsRNA or between the antisense strand of an mRNA agent and a target sequence, as will be apparent from the context of their use.

As used herein, a polynucleotide that is substantially complementary to at least a portion of messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., mRNA encoding C5). For example, a polynucleotide is complementary to at least a portion of C5 mRNA if the sequence is substantially complementary to a continuous portion of the mRNA encoding C5.

In general, the majority of the nucleotides in each strand are ribonucleotides, but, as described in detail herein, each or both strands may also include one or more nucleotides that are not ribonucleotides, such as a deoxyribonucleotide and/or a modified nucleotide. Furthermore, mRNA may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications used in an mRNA molecule are encompassed by the term "mRNA" within the context of this description and claims.

In one aspect of the present invention, an agent for use in the methods and compositions of the present invention is a single-stranded antisense RNA molecule that inhibits a target mRNA via the inhibitory mechanism of antisense RNA. The single-stranded antisense RNA molecule is complementary to a sequence in the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation stoichiometrically by base pairing with mRNA and by physically interfering with the translation mechanism, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule can be from about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, a single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20 or more contiguous nucleotides of any of the antisense sequences described herein.

The term "lipid nanoparticle" or "LNP" refers to a vesicle containing a lipid layer enclosing a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., mRNA, or a plasmid from which mRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

A subject as used herein is an animal, such as a mammal, including primates (such as a human, a non-human primate such as a monkey and a chimpanzee), non-primates (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or birds (such as a duck or goose). In one embodiment, the subject is a human, such as a human being treated or diagnosed with a disease, disorder, or condition that would benefit from reducing C5 expression; a human at risk of developing a disease, disorder, or condition that would benefit from reducing C5 expression; a human suffering from a disease, disorder, or condition that would benefit from reducing C5 expression; and/or a human being treated for a disease, disorder, or condition that would benefit from reducing C5 expression, as described herein.

As used herein, the terms "treat" or "treat" refer to a beneficial or desired result, including, but not limited to, alleviating or ameliorating one or more symptoms associated with adverse complement pathway activation (e.g., hemolysis and/or chronic inflammation); reducing the degree of adverse complement pathway activation; stabilizing (i.e., not worsening) the state of chronic inflammation and/or hemolysis; or alleviating or temporarily alleviating adverse complement pathway activation (e.g., chronic inflammation and/or hemolysis), whether established or not established. Treatment may also mean prolonging life compared to expected survival in the absence of treatment.

The expression below in the context of the level of complement component C5 in a subject, or a disease marker, or a symptom, refers to a statistically significant reduction in such level. The reduction can be by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, and, preferably, up to a level accepted within the normal range for a person without such a disorder.

As used herein, preventing or providing prophylaxis, when used in relation to a disease, disorder, or condition thereof, in which a reduction in C5 expression would have a beneficial effect, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, disorder, or condition, for example, a symptom of adverse complement activation, such as chronic inflammation, hemolysis, and/or thrombosis. The likelihood of developing thrombosis is reduced, for example, when an individual with one or more risk factors for developing thrombosis either does not develop thrombosis or develops thrombosis to a lesser degree of severity relative to a population with the same risk factors and not receiving treatment as described herein. Failure to develop the disease, disorder, or condition, or a reduction in the development of a symptom associated with such disease, disorder, or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the demonstration of delayed symptoms with a delay (e.g., days, weeks, months, or years) is considered effective prevention.

As used herein, the term complement component C5-associated disease is a disease or disorder that is caused by or associated with complement activation. Such diseases are typically associated with inflammation and/or activation of the immune system, such as membrane attack complex-mediated lysis, anaphylaxis, and/or hemolysis. Non-limiting examples of complement component C5-associated diseases include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA), antiphospholipid antibody syndrome, lupus nephritis, ischemia-reperfusion injury, typical or infectious hemolytic uremic syndrome (tHUS), dense deposit disease (DDD), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), multiple sclerosis (MS); Macular degeneration (eg, age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelet count syndrome (HELLP syndrome); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, bullous pemphigoid, Shiga-like toxin-associated E. coli, hemolytic uremic syndrome, C3 nephropathy, anti-neutrophil cytoplasmic antibody-associated vasculitis (eg, granulomatosis with polyangiitis (formerly known as Wegener granulomatosis), Churg-Strauss syndrome, and microscopic polyangiitis); Allograft rejection reactions mediated by humoral and vascular mechanisms, graft dysfunction, myocardial infarction (e.g., tissue injury and ischemia in myocardial infarction), allogeneic transplantation, sepsis (e.g., adverse outcome of sepsis), coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, sepsis associated with the systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type 1 diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disorders, peripheral (e.g., musculoskeletal) vascular disorders, renovascular hypertension, mesenteric/intestinal vascular disorders, vasculitis, Henoch-Schonlein nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex-related vasculitis, Takayasu disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barré syndrome, and percutaneous transluminal coronary angioplasty (PTCA) (see, e.g., Holers (2008) Immunological Reviews 223:300–316; Holers and Thurman (2004) Molecular Immunology 41:147-152; U.S. Patent Publication No. 20070172483).

In one embodiment, the disease associated with complement component C5 is paroxysmal nocturnal hemoglobinuria (PNH). PNH can be classic PNH or PNH in the setting of other bone marrow failure syndrome and/or myelodysplastic syndrome (MDS), such as cytopenias. In another embodiment, the disease associated with complement component C5 is atypical hemolytic uremic syndrome (aHUS).

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II. mRNA according to the invention.

The present invention relates to mRNAs that inhibit expression of the complement component C5 gene. In one embodiment, the mRNA agent comprises double-stranded ribonucleic acid (dsRNA) molecules for inhibiting expression of the C5 gene in a cell, e.g., a cell of a subject, e.g., a mammal, such as a human, suffering from a disease associated with the complement component C5, e.g., PNH. The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed upon expression of the C5 gene. The region of complementarity is approximately 30 nucleotides or less in length (e.g., approximately 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). When contacted with a cell expressing a C5 gene, the mRNA inhibits expression of the C5 gene (e.g., a human, non-primate, or avian C5 gene) by at least about 10%, as assessed by, for example, PCR or a branched DNA (bDNA)-based assay, or a protein-based assay, for example, by immunofluorescence assay using, for example, Western blotting or flow cytometry techniques.

dsRNA comprises two RNA strands that are complementary and hybridize to form a duplex structure under the conditions under which the dsRNA will be used. One strand of dsRNA (the antisense strand) contains a region of complementarity that is essentially complementary, and typically completely complementary, to a target sequence. The target sequence may be derived from an mRNA sequence produced by expression of the C5 gene. The other strand (the sense strand) contains a region that is complementary to the antisense strand, and the two strands hybridize to form a duplex structure when combined under appropriate conditions. As described herein and as is known in the art, dsRNA complementary sequences may also be contained as self-complementary regions of a single nucleic acid molecule, rather than being located on separate oligonucleotides.

Typically, the duplex structure is 15 to 30 base pairs in length, such as 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,

18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24,

19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29,

21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the ranges and lengths listed above are also considered part of the present invention.

Similarly, the region of complementarity of the target sequence is between 15 and 30 nucleotides in length, for example, within 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the ranges and lengths listed above are also considered to be part of the present invention.

In some embodiments, the dsRNA is approximately 15 to approximately 20 nucleotides in length, or approximately 25 to approximately 30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, as is well known in the art, dsRNAs longer than approximately 21-23 nucleotides can serve as substrates for Dicer. As will also be understood by those of ordinary skill in the art, the region of RNA that represents the target for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where appropriate, the portion of the target mRNA is a contiguous sequence of the target mRNA of sufficient length to allow it to serve as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

It will also be understood by one skilled in the art that the duplex region is the main functional part of the dsRNA, for example, a duplex region of from about 9 to 36 base pairs, such as about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 932, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-

26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, until the region is processed into a functional duplex of, for example, 15-30 base pairs that directs the desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, one skilled in the art will appreciate that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is a non-naturally occurring miRNA. In another embodiment, the agent that is an mRNA is used

- 26 044245 used to target C5 expression is not formed in the target cell by cleavage of the larger dsRNA.

The dsRNA described herein may further include one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang may exhibit unexpectedly high inhibitory properties relative to their blunt-ended counterparts. The nucleotide overhang may comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of the overhang may be on the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

dsRNA can be synthesized using standard methods known in the art, as described below, for example, using an automated DNA synthesizer such as those commercially available from, for example, Biosearch, Applied Biosystems, Inc.

The mRNA compounds of the present invention can be prepared using a two-step procedure. First, individual strands of a double-stranded RNA molecule are prepared separately. The component strands are then annealed. Individual strands of the siRNA compound can be prepared using liquid-phase synthesis, solid-phase organic synthesis, or both. Organic synthesis has the advantage of readily producing oligonucleotide strands containing unnatural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using liquid-phase synthesis, solid-phase organic synthesis, or both.

In one aspect, a dsRNA of the present invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the group of sequences presented in any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23, and the antisense strand corresponding to the sense strand is selected from the group of sequences from any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23. In this aspect, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to a sequence of mRNA formed upon expression of the C5 gene. Thus, in this aspect, the dsRNA will comprise two oligonucleotides, wherein one oligonucleotide is described as the sense strand in any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23, and the second oligonucleotide is described as an antisense strand corresponding to the sense strand in any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23. In one embodiment, the substantially complementary dsRNA sequences are contained in separate oligonucleotides. In another embodiment, the substantially complementary dsRNA sequences are contained in a single oligonucleotide.

It should be understood that although some of the sequences in Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23 are described as modified and/or conjugated sequences, an RNA from among the mRNAs of the present invention, for example, a dsRNA of the present invention, may comprise any of the sequences indicated in Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23 that are not modified or conjugated, and/or modified and/or conjugated otherwise than described herein.

It is well known to those skilled in the art that dsRNA with a duplex structure of approximately 20 and 23 base pairs, such as 21 base pairs, have been found to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, it has been found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, due to the nature of the oligonucleotide sequences shown in any of Table 3, 4, 5, 6, 18, 19, 20, 21, and 23, the dsRNAs described herein may contain at least one strand of at least 21 nucleotides in length. It is reasonable to expect that shorter duplexes having one of the sequences of any of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23, minus only a few nucleotides at one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23, and differing in their ability to inhibit C5 gene expression by no more than about 5, 10, 15, 20, 25 or 30% inhibition compared to dsRNA containing the entire sequence, are considered within the scope of the present invention.

Furthermore, the RNAs presented in any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23 identify site(s) in the C5 transcript that are susceptible to RISC-mediated cleavage. Accordingly, the present invention further describes mRNAs that target one of these sites. For the purposes of this document, an mRNA is said to target a particular site of an RNA transcript if the mRNA promotes cleavage of the transcript anywhere within that particular site. Such an mRNA will typically comprise at least about 15 contiguous nucleotides from one of the sequences presented in any of Tables 3, 4, 5, 6, 18, 19, 20, 21 and 23, coupled with additional nucleotide sequences taken from a region adjacent to the selected sequence in the C5 gene.

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While the target sequence is typically approximately 15-30 nucleotides in length, there is considerable variability in the suitability of specific sequences within this range for directing the cleavage of any given target RNA. Various software packages and the principles outlined in this document provide guidance for identifying optimal target sequences for any given target gene, but an empirical approach can also be adopted, in which a window or mask of a given size (as a non-limiting example, 21 nucleotides) is placed literally or figuratively (including, for example, in silico) on the target RNA sequence to identify sequences within a range of sizes that can serve as target sequences. By gradually moving the sequence window one nucleotide above or below the initial position of the target sequence, the next potential target sequence can be identified until a complete set of possible sequences is determined for any given target size. This method, combined with the systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally, can identify those RNA sequences that, when targeted with an mRNA agent, mediate the best inhibition of target gene expression. Thus, while sequences identified, for example, in any of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23, are effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by gradually moving a single nucleotide window above or below the given sequences to identify sequences with the same or improved inhibition characteristics.

Furthermore, it is contemplated that for any identified sequence, for example, in any of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23, further optimization can be achieved by systematically either adding or removing nucleotides to form longer or shorter sequences and testing those sequences generated by moving a longer or shorter window up or down the target RNA from that point. Again, combining this approach to generating new targets in candidates with testing the efficiency of mRNA based on those target sequences in inhibition assays known in the art and/or as described herein can lead to further improvements in inhibition efficiency. Further, such optimized sequences can be adjusted by, for example, introducing modified nucleotides as described herein or as known in the art, adding or altering an overhang, or other modifications known in the art and/or described herein, to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, improving transmembrane delivery, targeting a specific location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

The mRNA described herein may contain one or more mismatches to the target sequence. In one embodiment, the mRNA described herein contains no more than 3 mismatches. If the antisense strand of the mRNA contains mismatches to the target sequence, it is preferred that the region of mismatch is not in the center of the region of complementarity. If the antisense strand of the mRNA contains mismatches to the target sequence, it is preferred that the mismatch be limited to the last 5 nucleotides from either the 5' or 3' end of the region of complementarity. For example, for an mRNA agent consisting of 23 nucleotides, the strand complementary to the C5 gene region typically does not contain any mismatches within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether mRNA containing a mismatch to a target sequence is effective in inhibiting C5 gene expression. Considering the effectiveness of mRNA with a mismatch in inhibiting C5 gene expression is important, especially if a particular region of complementarity in the C5 gene is known to have a polymorphic sequence variation in the population.

III. Modified mRNAs according to the invention.

In one embodiment, an RNA from among the mRNAs of the present invention, e.g., a dsRNA, is unmodified and does not contain, for example, chemical modifications and/or conjugations known in the art and described herein. In another embodiment, an RNA from among the mRNAs of the present invention, e.g., a dsRNA, is chemically modified to improve stability or other beneficial characteristics. In some embodiments, substantially all of the nucleotides of the mRNAs of the present invention are modified. In other embodiments, all of the nucleotides of the mRNAs of the present invention are modified. The mRNAs of the present invention in which substantially all of the nucleotides are modified are substantially, but not completely, modified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

The nucleic acids described in the present invention can be synthesized and/or modified by methods well known in the art, such as those described in Current protocols in nucleic acid chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, end modifications, such as 5'-terminal modifications (phosphorylation, conjugation, inverted linkages) or 3'-terminal modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that pair with an extended set of partners, deletion of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2'-position or the 4'-position) or sugar substitution; and/or backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of mRNA compounds used in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or internucleoside linkages that are not naturally occurring. RNAs containing modified backbones include, among others, those that do not contain a phosphorus atom in the backbone. In the context of this disclosure, and as is sometimes referred to in the art, modified RNAs that do not contain a phosphorus atom in their internucleoside backbone may also be considered oligonucleosides. In some embodiments, a modified mRNA will contain a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidate and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates with normal 3'-5'-linkages, their 2'-5'-linked analogs, as well as those whose polarity is inverted, where adjacent pairs of nucleoside units are linked via 3'-5'- to 5'3'- or 2'-5'- to 5'-2'-. Also includes various salts, mixed salts, and free acid forms.

Illustrative U.S. patents that disclose the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;

5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361;

5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614;

6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816;

7,273,933; 7,321,029 and U.S. Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom are those formed by internucleoside linkages of short alkyl or cycloalkyl chains, mixed internucleoside linkages of heteroatoms and alkyl or cycloalkyl chains, or internucleoside linkages of one or more shorter heteroatomic or heterocyclic chains. They include those with morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methylene-formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methylene-imino and methylene-hydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and others, including mixed constituents of N, O, S and CH ₂ .

Illustrative U.S. patents that describe the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437 and 5,677,439, the entire contents of each of which are incorporated herein by reference.

Other embodiments include suitable RNA mimetics for use in mRNA, in which both the sugar linkage and the internucleoside linkage, i.e., the backbone of the nucleotide units, are replaced with new moieties. The base units are maintained during hybridization with the target compound of the corresponding nucleic acid. One such oligomeric compound, an RNA mimetic that has demonstrated excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nitrogenous bases are retained and bind directly or indirectly to the nitrogen atoms of the aza group of the amide portion of the backbone. Illustrative U.S. patents that describe the preparation of PNA compounds include, but are not limited to, U.S. patents. Nos. 5,539,082, 5,714,331, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the mRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments described in the present invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular --CH ₂ --NH--CH ₂ -, --CH ₂ --N(CH ₃ )--O--CH ₂ -- [known as the methylene (methylimino) or MMI backbone], --CH ₂ --O--N(CH ₃ )--CH ₂ --, --CH ₂ --N(CH ₃ )--N(CH ₃ )--CH ₂ -- and --N(CH ₃ )--CH ₂ --CH ₂ -- [where the native phosphodiester backbone is represented as --O--P--O--CH2--] from the aforementioned U.S. patent. No. 5,489,677 and the amide backbones of the aforementioned U.S. Pat. No. 5,602,240. In some embodiments, the RNAs described herein have a morpholino backbone structure as in the aforementioned U.S. Pat. No. 5,034,506.

Modified RNAs may also contain one or more substituted sugar moieties. mRNAs, such as dsRNAs, described herein may include one of the following substituents at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl may be substituted or unsubstituted with Cl- _Cl0- alkyl or _C2 - _Cl0- alkenyl and alkynyl. Illustrative suitable modifications include O[(CH2) _n O] _m CH3, O(CH2) _n OCH3, O(CH2) _n NH2, O(CH2) _n CH3, O(CH2) _n ONH2, and O( _CH2 ) _n ON[( _CH2 ) _{n CH3} ] ₂ , wherein n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following substituents at the 2' position: C1- _C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, _SCH3 , OCN, Cl, Br, CN, _CF3 , _OCF3 , SOCH3, _SO2CH3 , ONO2, NO2, _N3 , NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino-, poly alkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of mRNA, or group for improving the pharmacodynamic properties of mRNA, and other substituents having similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., the O( _CH2 ) _2ON ( _CH3 ) ₂ group, also known as 2'-DMAOE, as described in the examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e. 2'-O-- _CH2 --O-- _CH2 --N( _CH2 ) ₂ .

Other modifications include 2'-methoxy (2'-OCH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ), and 2'-fluoro (2'-F). Similar modifications can be made at other positions in the RNA of mRNAs, particularly at the 3' position of the sugar at the 3' end of the nucleotide, or at the 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide. mRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Illustrative U.S. patents that describe the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633 and 5,700,920, some of which generally recognize the present application. The entire contents of each of the above-mentioned applications are incorporated herein by reference.

mRNA may also include modifications or substitutions of the nitrogenous base (often referred to in the art simply as a base). As used herein, unmodified or naturally occurring nitrogenous bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nitrogenous bases include other synthetic and natural nitrogenous bases such as deoxythymine (dT), 5-methylcytosine (5-MeC), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted compounds of uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Additional nitrogenous bases include those described in U.S. Pat. No. 3,687,808, which are described in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; which are described in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J.L., ed. John. Wiley & Sons, 1990, which are described by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and which are described by Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993. Some of these nitrogenous bases are particularly useful for increasing the binding affinity of the oligomeric compounds described in the present invention. They include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. Substitutions of 5-methylcytosine have been shown to increase nucleic acid duplex stability at 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are illustrative base substitutions, even more so in combination with the 2'-O-methoxyethyl sugar modification.

Illustrative U.S. patents that describe the preparation of some of the above-mentioned modified nitrogenous bases, as well as other modified nitrogenous bases, include, but are not limited to, the above-mentioned U.S. Patent Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066;

- 30 044245

5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;

5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;

6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672 and 7,495,088, the entire contents of each of which are incorporated herein by reference.

RNAs from mRNAs can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide containing a modified ribose moiety, where the ribose moiety contains an additional bridge connecting the 2' and 4' carbon atoms. This structure effectively locks the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439–447; Mook, OR et al., (2007) Mol Canc Ther 6(3):833–843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185–3193).

Illustrative U.S. patents that describe the production of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490, 6,670,461, 6,794,499, 6,998,484, 7,053,207, 7,084,125, and 7,399,845, the entire contents of each of which are incorporated herein by reference.

Potential stabilizing modifications at the ends of RNA molecules may include N(acetylaminocaproyl)-4-hydroxyprolinol (Nur-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Nur-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ester), N-(aminocaproyl) 4-hydroxyprolinol (Nur-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT(idT), etc. A disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

A. Modified mRNAs containing motifs according to the invention.

In some aspects of the present invention, the double-stranded RNAi agents of the present invention include those with chemical modifications as disclosed, for example, in U.S. Provisional Patent Application No. 61/561,710, filed November 18, 2011, or in PCT/US2012/065691, filed November 16, 2012, the entire contents of each of which are incorporated herein by reference.

As shown herein and in Provisional Application No. 61/561,710 or PCT Application No. PCT/US2012/065691, superior results can be obtained by introducing one or more motifs of three identical modifications of three consecutive nucleotides into the sense strand and/or antisense strand of an RNAi agent, in particular at or near a cleavage site. In some embodiments, the sense strand and antisense strand of an RNAi agent may be completely modified in a different manner. The introduction of such motifs disrupts the pattern of modifications, if any, of the sense and/or antisense strand. The RNAi agent, for example the sense strand, may be optionally conjugated to a ligand that is a GalNAc derivative. The resulting RNAi agents are characterized by superior gene silencing activity.

More specifically, it was unexpectedly found that when the sense strand and the antisense strand of a double-stranded RNAi agent were fully modified to have one or more motifs of three identical modifications of three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent, then the gene silencing activity of the RNAi agent was best enhanced.

Accordingly, the present invention provides double-stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., the complement component C5 (C5) gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can vary in length from 12 to 30 nucleotides. For example, each strand may be 14 to 30 nucleotides long, 17 to 30 nucleotides long, 25 to 30 nucleotides long, 27 to 30 nucleotides long, 17 to 23 nucleotides long, 17 to 21 nucleotides long, 17 to 19 nucleotides long, 19 to 25 nucleotides long, 19 to 23 nucleotides long, 19 to 21 nucleotides long, 21 to 25 nucleotides long, or 21 to 23 nucleotides long.

The sense strand and antisense strand typically form a double-stranded RNA duplex (dsRNA), also referred to herein as the RNAi agent. The duplex region of the RNAi agent can be 12-30 base pairs in length. For example, the duplex region can be 14-30 base pairs in length, 17-30 base pairs in length, 27-30 base pairs in length, 17-23 base pairs in length, 17-21 base pairs in length, 17-19 base pairs in length, 19-25 base pairs in length, 19-23 base pairs in length, 19-21 base pairs in length, 21-25 base pairs in length, or 21-23 base pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may comprise one or more overhangs and/or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhang may be 1-6 nucleotides in length, such as 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs may be a result of

- 31 044245 one thread is longer than the other, or that two threads of the same length are arranged in a checkerboard pattern.

The overhang may form a mismatch with the target mRNA, or it may be complementary to the gene sequences with which the target interaction occurs, or it may have a different sequence. The first and second strands may also be connected, for example, by additional bases to form a hairpin or by other non-base linkers.

In one embodiment, each of the nucleotides in the overhang region of the RNAi agent can independently be a modified or unmodified nucleotide, including, but not limited to, a sugar with a 2'-modification, such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA, or it can be complementary to the gene sequences with which the target interaction occurs, or it can have a different sequence.

The 5' or 3' overhangs of the sense strand, the antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang(s) comprise two nucleotides with a phosphorothioate between the two nucleotides, wherein the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3' end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3' overhang is present on the antisense strand. In one embodiment, the 3' overhang is present on the sense strand.

An RNAi agent may contain only a single overhang, which can enhance the RNAi's interfering activity without affecting its overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand or, alternatively, at the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Typically, the antisense strand of an RNAi has a nucleotide overhang at the 3' end, and the 5' end is blunt. Without wishing to be bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the overhang at the 3' end of the antisense strand facilitate the inclusion of the guide strand in the RISC process.

In one embodiment, the RNAi agent is a blunt-ended oligomer 19 nucleotides in length, wherein the sense strand comprises at least one motif of three 2'-F modifications of three consecutive nucleotides at positions 7, 8, 9 from the 5' end. The antisense strand comprises at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

In another embodiment, the RNAi agent is a 20 nucleotide long blunt-ended oligomer, wherein the sense strand comprises at least one motif of three 2'-F modifications of three consecutive nucleotides at positions 8, 9, 10 from the 5' end. The antisense strand comprises at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

In another embodiment, the RNAi agent is a 21 nucleotide long blunt-ended oligomer, wherein the sense strand comprises at least one motif of three 2'-P modifications of three consecutive nucleotides at positions 9, 10, 11 from the 5' end. The antisense strand comprises at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-P modifications of three consecutive nucleotides at positions 9, 10, 11 from the 5'end; the antisense strand comprises at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at positions 11, 12, 13 from the 5' end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is at the 3' end of the antisense strand. In cases where the 2-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are overhanging nucleotides and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide in the sense strand and the antisense strand of the RNAi agent, including nucleotides that are part of motifs, are modified nucleotides. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc ₃ ).

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein, starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired from the sense strand and up to 6 consecutive 3'-terminal nucleotides are unpaired from the sense strand, thereby forming a 3'-single-stranded overhang of 1-6 nucleotides; wherein the 5'-end of the antisense strand comprises from 10-30 consecutive nucleotides unpaired from the sense strand, thereby forming a 5'-single-stranded overhang of 10-30 nucleotides; wherein at least the 5'-terminal and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand, wherein the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA over a length of at least 19 ribonucleotides of the antisense strand to reduce expression of the target gene upon introduction of the double-stranded nucleic acid into a mammalian cell; and wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one of the motifs occurs at or near the cleavage site. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the RNAi agent comprises a first strand with a length that is at least 25 and at most 29 nucleotides, and a second strand with a length that is at most 30 nucleotides, with at least one motif of three 2'-O-methyl modifications of three consecutive nucleotides at position 11, 12, 13 from the 5'-end; wherein the 3'-end of the first strand and the 5'-end of the second strand form a blunt end, and the second strand is 1-4 nucleotides longer at the 3'-end than the first strand, wherein the duplex region is at least 25 nucleotides in length, and the second strand is sufficiently complementary to the target mRNA over at least 19 nucleotides of the length of the second strand, to reduce the expression of the target gene, wherein the RNAi agent is introduced into mammalian cells, and wherein cleavage of the RNAi agent with a dicer preferably results in siRNA containing the 3'-end of the second strand, thereby reducing the expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent comprises at least one motif of three identical modifications of three consecutive nucleotides, wherein one of the motifs is at a cleavage site in the sense strand.

In one embodiment, the antisense strand of the RNAi agent may also comprise at least one motif of three identical modifications of three consecutive nucleotides, wherein one of the motifs is at or near a cleavage site in the sense strand.

For an RNAi agent with a duplex region of 17-23 nucleotides in length, the cleavage site of the antisense strand is typically located at approximately the 10th, 11th, and 12th positions from the 5' end. Thus, motifs of three identical modifications can be located at positions 9, 10, 11; positions 10, 11, 12; positions 11, 12, 13; positions 12, 13, 14, or positions 13, 14, 15 of the antisense strand, with the counting starting from ¹ nucleotide from the 5' end of the antisense strand or the counting starting from ¹ paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary according to the length of the RNAi duplex region from the 5' end.

The sense strand of the RNAi agent may comprise at least one motif of three identical modifications of three consecutive nucleotides at the strand cleavage site; and the antisense strand may be characterized by at least one motif of three identical modifications of three consecutive nucleotides at or near the strand cleavage site. In cases where the sense strand and antisense strand form a dsRNA duplex, the sense strand and antisense strand may be aligned such that one three-nucleotide motif in the sense strand and one three-nucleotide motif in the antisense strand overlap by at least one nucleotide, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may comprise multiple motifs of three identical modifications of three consecutive nucleotides. The first motif may be at or near the cleavage site of the strand, and the other motifs may be flanking modifications. The term "flanking modification" as used herein means a motif occurring in another part of the strand that is separated from the motif at or near the cleavage site of the same strand. The flanking modification is either adjacent to the first motif or separated by at least one or more nucleotides. In cases where the motifs are directly adjacent to each other, then the chemical structure of the motifs is different from each other, and when the motifs are separated by one or more nucleotides, then the chemical structures may be the same or different. They may

- 33 044245 two or more flanking modifications are present. For example, when two flanking modifications are present, each flanking modification can be located at one end relative to the first motif, which is located at or near the cleavage site, or on both sides of the leading motif.

Like the sense strand, the antisense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs located at or near the strand cleavage site. This antisense strand may also contain one or more flanking modifications, if aligned similarly to the flanking modifications that may be present in the sense strand.

In one embodiment, the flanking modification in the sense strand or antisense strand of the RNAi agent typically does not include the first one or first two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

In another embodiment, the flanking modification in the sense strand or antisense strand of the RNAi agent typically does not include the first one or first two paired nucleotides in the duplex region at the 3' end, 5' end, or both ends of the strand.

In cases where each of the sense strand and the antisense strand of the RNAi agent contains at least one flanking modification, the flanking modifications may fall at the same end of the duplex region and have an overlap of one, two, or three nucleotides.

In cases where each of the sense strand and the antisense strand of the RNAi agent contains at least two flanking modifications, the sense strand and the antisense strand may be aligned such that two modifications, each from one strand, fall at one end of the duplex region with an overlap of one, two, or three nucleotides; two modifications, each from one strand, fall at the other end of the duplex region with an overlap of one, two, or three nucleotides; two modifications of one strand fall on either side of the leading motif with an overlap of one, two, or three nucleotides in the duplex region.

In one embodiment, each nucleotide in the sense strand and antisense strand of the RNAi agent, including nucleotides that are part of motifs, may be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more changes in one or both unlinked phosphate oxygen atoms and/or one or more linked phosphate oxygen atoms; a change in a ribose sugar component, such as the 2'-hydroxyl in the ribose sugar; complete replacement of the phosphate moiety with dephosphorized linkers; modification or substitution of a naturally occurring base; and substitution or modification of the ribose phosphate backbone.

Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated throughout the nucleic acid, such as modifications of a base, a phosphate moiety, or an unlinked O-phosphate moiety. In some cases, a modification will occur at all positions in the nucleic acid, but in many cases it will not. For example, a modification may occur only at the 3' or 5' end, or it may occur only at a terminal region, such as the terminal nucleotide or the last 2, 3, 4, 5, or 10 nucleotides of the chain. A modification may occur in a double-stranded region, in a single-stranded region, or in both. A modification may occur only in a double-stranded region of RNA, or it may occur only in a single-stranded region of RNA. For example, a phosphorothioate modification at the unbound O position may occur at only one or both ends, may occur only in a terminal region, such as at the terminal nucleotide position or in the last 2, 3, 4, 5, or 10 nucleotides of the chain, or may occur in both double-stranded and single-stranded regions, particularly at the end. The 5' end or ends may be phosphorylated.

This may be possible, for example, to improve stability, to include specific bases in the overhangs, or to include modified nucleotides or nucleotide substituents in the single-stranded overhangs, such as in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhangs. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, such as by the modifications described herein. Modifications may include, for example, the use of modifications at the 2'-position of the ribose sugar using modifications known in the art, such as the use of deoxyribonucleotides modified with 2'-deoxy-2'-fluoro-(2'T) or 2'-O-methyl in place of the ribose sugar of the nitrogenous base, and modifications of the phosphate group, such as phosphorothioate modifications. The overhangs may not be homologous to the target sequence.

In one embodiment, each residue of the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. The strands may contain multiple modifications. In one embodiment, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

At least two different modifications are typically present in the sense strand and the antisense strand. These two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

In one embodiment, N _a and/or N _b have alternating pattern modifications. The term "alternating motif" as used herein means a motif with one or more modifications, wherein each modification occurs on alternating nucleotides of a single strand. The term "alternating nucleotide" may mean one for every two nucleotides, or one for every three nucleotides, or a similar pattern. For example, if each of A, B, and C represents one type of nucleotide modification, then the alternating motif may be ABABABABABA..., AABBAABBAABB..., AAABAABAAAB..., AAABAAABAAAB..., AAABBBAAAABBB... or ABCABCABCABC..., etc.

The types of modifications contained in the alternating motif may be the same or different. For example, if each of A, B, C, and D represents one type of nucleotide modification, then the alternating pattern, i.e., the modifications of every other nucleotide, may be the same, but each of the sense strand and antisense strand may be selected from several possible modifications in the alternating motif, such as ABABAB..., ACACAC..., BDBDBD..., or CDCDCD..., etc.

In one embodiment, the RNAi agent of the present invention comprises a modification pattern for the alternating motif of the sense strand that is shifted relative to the modification pattern for the alternating motif of the antisense strand. The shift may be such that a modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand, and vice versa. For example, when pairing the sense strand with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand may begin with ABABAB from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with BABABA from the 5'-3' end of the strand in the duplex region. As another example, the alternating motif in the sense strand may start with AABBAABB from the 5'-3' end of the strand, and the alternating motif in the antisense strand may start with BBAABBAA from the 5'-3' end of the strand in the duplex region, so that there is a complete or partial shift in modification patterns between the sense strand and the antisense strand.

In one embodiment, the RNAi agent initially comprises a pattern of alternating 2'-O-methyl modification and 2'-P modification motifs in the sense strand and initially has a shift in relation to the pattern of alternating 2'-O-methyl modification and 2'-F modification motifs in the antisense strand, i.e., a 2'-O-methyl modified nucleotide in the sense strand base pairs with a 2'-F modified nucleotide in the antisense strand and vice versa. 1 position in the sense strand may begin with a 2'-F modification, and 1 position in the antisense strand may begin with a 2'-O-methyl modification.

The introduction of one or more motifs consisting of three identical modifications of three consecutive nucleotides into the sense strand and/or antisense strand disrupts the original pattern of modifications present in the sense strand and/or antisense strand. This disruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs consisting of three identical modifications of three consecutive nucleotides into the sense and/or antisense strand unexpectedly enhances gene silencing activity against the target gene.

In one embodiment, when a motif of three identical modifications of three consecutive nucleotides is introduced into either strand, the modification of the nucleotide following the motif is a modification different from the modification of the motif. For example, a portion of the sequence containing the motif is ... _NaYYYNb ..., where Y is a modification of the motif of three identical modifications of three consecutive nucleotides, and _Na and _Nb are a modification of the nucleotide following the YYY motif that is different from the modification _of Y, and where Na and Nb may be the same or different modifications. Alternatively, Na and/or _Nb may be present or absent when the flanking modification is present.

The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur at any nucleotide of the sense strand, the antisense strand, or both strands at any position within the strand. For example, an internucleotide linkage modification may occur at every nucleotide of the sense strand and/or the antisense strand; each internucleotide linkage modification may occur in an alternating pattern in the sense strand and/or the antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern.

The alternating pattern of internucleotide linkage modification of the sense strand may be the same or different from that of the antisense strand, and the alternating pattern of internucleotide linkage modification of the sense strand may be characterized by a shift relative to the alternating pattern of internucleotide linkage modification of the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

In one embodiment, RNAi has a modification of the phosphorothioate or methylphosphonate internucleotide linkage in the overhang region. For example, the overhang region may comprise two nucleotides with a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Modifications to the internucleotide linkage can also be performed to connect the overhanging nucleotides to the terminal paired nucleotides in the duplex region. For example, at least 2, 3, 4, or all of the overhanging nucleotides can be linked via a phosphorothioate or methylphosphonate internucleotide linkage, and, optionally, additional phosphorothioate or methylphosphonate internucleotide linkages can be present connecting the overhanging nucleotide to the paired nucleotide that follows the overhanging nucleotide. For example, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides and the third is a paired nucleotide adjacent to the overhanging nucleotide. These terminal three nucleotides may be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and/or the 5' end of the antisense strand.

In one embodiment, a two-nucleotide overhang is located at the 3' end of the antisense strand, and two phosphorothioate internucleotide linkages are present between the terminal three nucleotides, wherein two of the three nucleotides are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

In one embodiment, the RNAi agent comprises a mismatch(s) to a target in a duplex or combination thereof. The mismatch may occur in an overhang region or a duplex region. Base pairs can be arranged based on their propensity to promote dissociation or melting (e.g., by the free energy of association or dissociation of a particular pairing; the simplest approach is to examine the pairs on an individual base pair basis, although next-neighbor analysis or the like can also be performed). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and TrC is preferred over G:C (1=inosine). Mismatches, such as non-canonical or non-canonical pairing types (which are described elsewhere in this document), are preferred over canonical pairing types (A:T, A:U, G:C); and mating types that include universal bases are preferred over canonical mating types.

In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs in the duplex regions from the 5' end of the antisense strand, independently selected from the group consisting of: A:U, G:U, I:C, and mismatched pairs, such as non-canonical or non-canonical pairing types or pairing types that include universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

In one embodiment, the nucleotide at position 1 in the duplex region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs in the duplex region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region from the 5' end of the antisense strand is an AU base pair.

In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, a short sequence of deoxythymine nucleotides is present, for example, two dT nucleotides at the 3' end of the sense and/or antisense strand.

In one embodiment, the sequence of the sense chain may be represented by formula (I):

5' n _p -N _a -(XXX ) iN _b -YYY -N _b -(Z Ζ Ζ ) jN _a -n _q 3' (I) where each of i and j is independently equal to 0 or 1;

each of p and q is independently equal to 0-6;

each of Na independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, wherein each sequence contains at least two nucleotides modified in different ways;

each of N _b independently represents an oligonucleotide sequence containing 0-10 modified nucleotides;

each of np and n _q independently represents a protruding nucleotide;

where N _b and Y have different modifications; and each of XXX, YYY, and ZZZ independently represents one motif of three identical modifications of three consecutive nucleotides. Preferably, in YYY, all nucleotides are 2'-F modified.

In one embodiment, Na and/or N _b have alternating pattern modifications.

- 36 044245

In one embodiment, the YYY motif is at or near a cleavage site of the sense strand. For example, if the RNAi agent comprises a duplex region that is 17-23 nucleotides in length, the YYY motif may be at or near the cleavage site (e.g., may be at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13) in the sense strand, wherein the counting starts from ¹ nucleotide from the 5' end or, optionally, the counting starts from ¹ paired nucleotide in the duplex region from the 5' end.

In one embodiment, i equals 1 and j equals 0, or i equals 0 and j equals 1, or both i and j equal 1. The sense chain may thus be represented by the following formulas:

5' n _p -N _a -YYY-N _b -ZZZ-N _a -n _q 3'(lb);

5' n _p -N _a -XXX-N _b -YYY-N _a -n _q 3'(Ic); or

5' n _p -N _a -XXX-N _b -YYY-N _b -ZZZ-N _a -n _q 3' (Id) .

In cases where the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N _a can independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In cases where the sense strand is represented by formula (Ic), Nb is an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N _a can independently be an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In cases where the sense strand is represented by formula (Id), each Nb independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5, or 6. Each N _a may independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from the others.

In other embodiments, i is 0 and j is 0, and the sense chain may be represented by the formula

5' n _p -N _a -YYY- N _a -n _q 3' (la).

In cases where the sense strand is represented by formula (Ia), each N _a may independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the sequence of the antisense strand of RNAi may be represented by formula (II)

5' n _q ,-N _a ^r -(Z'Z'Z') _k -N _b '-Y'Y'Y'-N _b '-(X'X'X ' ) _Σ -N' _a -n _p '3' (II) where each of k and l is independently equal to 0 or 1;

each of p' and q' is independently equal to 0-6;

each of N _a ' independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, wherein each sequence contains at least two nucleotides modified in different ways;

each Nb' independently represents an oligonucleotide sequence containing 0-10 modified nucleotides;

each of np' and nq' independently represents a protruding nucleotide;

where Nb' and Y' have different modifications; and each of X'X'X', Y'Y'Y' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides.

In one embodiment, N _a ' and/or Nb' have alternating pattern modifications.

The Y'Y'Y' motif is located at or near the cleavage site of the antisense strand. For example, if the RNAi agent comprises a duplex region that is 17-23 nucleotides in length, the Y'Y'Y' motif may be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14, or 13, 14, 15 of the antisense strand, with counting starting from ¹ nucleotide from the 5' end or, optionally, starting from ¹ paired nucleotide in the duplex region from the 5' end. Preferably, the Y'Y'Y' motif is at positions 11, 12, 13.

In one embodiment, in the Y'Y'Y' motif, all nucleotides are 2'-OMe modified.

In one embodiment, k equals 1 and l equals 0, or k equals 0 and l equals 1, or both k and l equals 1.

- 37 044245

The antisense strand can thus be represented by the following formulas:

5' n _q --N _a '-Z 'Z 'Z '-N _b '-Y'Y'Y'-N _a '-n _pl З'(Пь);

5' n _q '-N _a '-Υ Ύ Ύ '-N _b ^g -X'X'X '-Pr- 3'(Is); or

5' n _q .-N _a '- Z 'Z 'Z '-Nb'-Y ^r Y ^r Y ^r -N _b '- X'X'X '-N _a '-n _p . 3' (Hd).

In cases where the antisense strand is represented by formula (IIb), N _b ' is an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na' independently is an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In cases where the antisense strand is represented by formula (IIc), N _b ' is an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N _a ' independently is an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In cases where the antisense strand is represented by formula (IId), each N _b ' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 02, or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N _b is 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the formula

5' n _p .-N _a .-Y'Y'Y'- N _a --n _q >3' (la).

In cases where the antisense strand is represented by formula (IIa), each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

Each of X', Y' and Z' may be the same or different from the others.

Each nucleotide of the sense strand and antisense strand can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z', in particular, can represent a 2'-Omethyl modification or a 2'-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may comprise a YYY motif located at positions 9, 10, and 11 of the strand, where the duplex region is 21 nucleotides long, counting starting from the ^1st nucleotide from the 5' end or, optionally, counting starting from the ^1st paired nucleotide in the duplex region from the 5'end; and Y is a 2'-F modification. The sense strand may further comprise an XXX motif or ZZZ motifs as flanking modifications at the opposite end of the duplex region; and each of XXX and ZZZ independently represents a 2'-OMe modification or a 2'-B modification.

In one embodiment, the antisense strand may comprise a Y'Y'Y' motif located at positions 11, 12, 13 of the strand, wherein the counting starts from the ^1st nucleotide from the 5' end or, optionally, the counting starts from the ^1st paired nucleotide in the duplex region from the 5'end; and Y' is a 2'-O-methyl modification. The antisense strand may further comprise an X'X'X' motif or Z'Z'Z' motifs as flanking modifications at the opposite end of the duplex region; and each of X'X'X' and Z'Z'Z' independently represents a 2'-OMe modification or a 2'-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, RNAi agents for use in the methods of the present invention may comprise a sense strand and an antisense strand, each strand comprising from 14 to 30 nucleotides, the RNAi duplex being represented by formula (III):

sense: 5' n _p -N _a - (XXX) iN _b -YYY -N _b -(ZZZ)j -N _a - n _q 3 ' antisense: 3' n _p ' -N _a ' - (X 'X 'X ') _k -N _b '-Y Ύ Ύ ' -N _b ' - (Z ' Z ' Z ') _b -N _a ' - n _q '5' (III), where each of i, j, k, and l is independently 0 or 1;

each of p, p', q and q' is independently equal to 0-6;

each of N _a and N _a independently represents an oligonucleotide sequence containing 0-25 modified nucleotides, wherein each sequence contains at least two nucleotides modified in different ways;

each of N _b and N _b ' independently represents an oligonucleotide sequence containing 0-10 modified nucleotides;

where each of np', np, n _q ' and n _q , each of which may be present or absent, independently represents a protruding nucleotide; and

- 38 044245 each of XXX, YYY, ZZZ, X'X'X', ΥΎΎ' and Z'Z'Z' independently represents one motif of three identical modifications of three consecutive nucleotides.

In one embodiment, i equals 0 and j equals 0; or i equals 1 and j equals 0; or i equals 0 and j equals 1; or both i and j equals 0; or both i and j equals 1. In another embodiment, k equals 0 and l equals 0; or k equals 1 and l equals 0; k equals 0 and l equals 1; or both k and l equals 0; or both k and l equals 1.

Illustrative combinations of sense strand and antisense strand that form an RNAi duplex include the formulas below:

' n _p - N _a -YYY -N _a -n _q 3 '

3' n _p '-N _a '-Y ' Y ' Υ ' -N _a 'n _q '5' (Sha)

5' η _ρ -N _a -YYY -¾ -ZZZ -N _a -n _q 3'

3' n _p '-N _a '-Y'Y'Y'-Nb'-Z 'Z 'Z '-N _a 'n _q '5' (Illb) ' n _p -N _a - XXX -N _b -YYY - N _a -n _q 3 '

3' n _p '-N _a '-X'X'X'-Nb'-Y'Y'Y'-N _a '-n _q '5' (IIIc) ' n _p -N _a -XXX -N _b -YYY -N _b - ZZZ -N _a -n _q 3 '3' n _p '-N _a '-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-N _a -n _q '5' (Hid)

In cases where the RNAi agent is represented by formula (IIIa), each of the N _a independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In cases where the RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each N _a independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In cases where the RNAi agent is represented by formula (IIIc), each of Nb, Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 02, or 0 modified nucleotides. Each of N _a independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

In cases where the RNAi agent is represented by formula (IIId), each of Nb, Nb' independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 02, or 0 modified nucleotides. Each of _Na , _Na ' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Each of _Na , _Na ', Nb, and Nb' independently contains alternating pattern modifications.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) may be the same or different from the others.

In cases where the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the nucleotides of Y may form a base pair with one of the nucleotides of Y'. Alternatively, at least two of the nucleotides of Y form base pairs with the corresponding nucleotides of Y'; or all three of the nucleotides of Y form base pairs with the corresponding nucleotides of Y'.

In cases where the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the nucleotides of Z may form a base pair with one of the nucleotides of Z'. Alternatively, at least two of the nucleotides of Z form base pairs with the corresponding nucleotides of Z'; or all three of the nucleotides of Z form base pairs with the corresponding nucleotides of Z'.

In cases where the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the nucleotides of X may form a base pair with one of the nucleotides of X'. Alternatively, at least two of the nucleotides of X form base pairs with the corresponding nucleotides of X'; or all three of the nucleotides of X form base pairs with the corresponding nucleotides of X'.

In one embodiment, the modification of nucleotide Y is different from the modification of nucleotide Y', the modification of nucleotide Z is different from the modification of nucleotide Z', and/or the modification of nucleotide X is different from the modification of nucleotide X'.

In one embodiment, when the RNAi agent is represented by formula (IIId), the modifications of N _a are 2'-O-methyl or 2'-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the modifications of N _a are 2'-O-methyl or 2'-fluoro modifications, and n _p '>0, and at least one n p' is linked to an adjacent nucleotide

- 39 044245 via a phosphorothioate linkage. In yet another embodiment, where the RNAi agent is represented by formula (IIId), the modifications of N _a are 2'-O-methyl or 2'-fluoro modifications, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker (described below). In another embodiment, where the RNAi agent is represented by formula (IIId), the N _a modifications are 2'-O-methyl or 2'-fluoro modifications, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

In one embodiment, where the RNAi agent is represented by formula (IIIa), the N _a modifications are 2'-O-methyl or 2'-fluoro modifications, np'>0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes may target the same gene or two different genes, or each of the duplexes may target the same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer comprising three, four, five, six, or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes may target the same gene or two different genes, or each of the duplexes may target the same gene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5' end, and one or both 3' ends are optionally conjugated to a ligand. Each agent may target the same gene or two different genes, or each agent may target the same gene at two different target sites.

Various publications have described multimeric RNAi agents that can be used in the methods of the present invention. Such publications include WO 2007/091269, U.S. Patent No. 7,858,769, WO 2010/141511, WO 2007/117686, WO 2009/014887, and WO 2011/031520, the entire contents of which are hereby incorporated by reference.

As described in more detail below, an RNAi agent containing one or more carbohydrate moieties conjugated to the RNAi agent may enhance one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with other moieties, such as a non-carbohydrate (preferably cyclic) carrier to which the carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been replaced in this manner is referred to herein as a ribose-replacement modification subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all atoms in the ring are carbon atoms, or a heterocyclic ring system, i.e., One or more atoms in the ring may be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier may be a monocyclic ring system or may contain two or more rings, such as fused rings. The cyclic carrier may be a fully saturated ring system or may contain one or more double bonds.

The ligand can be attached to the polynucleotide via a carrier. Carriers comprise: (i) at least one point of attachment to the backbone, preferably two points of attachment to the backbone, and (ii) at least one binding point of attachment. The term "point of attachment to the backbone" as used herein means a functional group, for example, a hydroxyl group, or, more typically, a bond accessible for introduction of the carrier into the backbone and which is suitable for this, for example, a phosphate or a modified phosphate, for example, a sulfur-containing backbone of ribonucleic acid. The term "binding point of attachment" (TAP) in some embodiments means a ring atom of the cyclic carrier, for example, a carbon atom or a heteroatom (other than the atom that provides the point of attachment to the backbone), to which the selected moiety is bound. The moiety may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is linked via an intermediate linkage to a cyclic support. Thus, the cyclic support will often include a functional group, such as an amino group, or, more typically, provide a linkage that is suitable for introducing or linking another chemical building block, such as a ligand, to the constituent ring.

RNAi agents can be conjugated to a ligand via a carrier, wherein the carrier can be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]-dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methods of the present invention is an agent selected from the group consisting of agents listed in any of Tables 3, 4, 5, 6, 18, 19, 20, 21, and 23. Such agents may further comprise a ligand.

IV. Ligand-conjugated mRNA.

Another modification of the RNA from among the mRNAs of the present invention includes chemical linkage to one or more RNA ligands, fragments or conjugates that enhance the activity, cellular distribution or cellular uptake of the mRNA. Such moieties include, but are not limited to, lipid groups such as the cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioester such as beryll-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, such as dodecanediol or undecyl residues (SaisonBehmoaras et al., EMbO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:36513654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In one embodiment, the ligand alters the distribution, targeting, or lifetime of the mRNA agent into which it is incorporated. In preferred embodiments, the ligand provides increased affinity for a selected target, e.g., a molecule, cell, or cell type, compartment, e.g., a cellular compartment, or part of an organ, tissue, organ, or body region, e.g., compared to species lacking such a ligand. Preferred ligands will not participate in duplex pairing in a duplex nucleic acid.

Ligands may include a naturally occurring substance such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid which is polylysine (PLL), poly-L-aspartic acid, poly-κ-glutamic acid, styrene-maleic anhydride copolymer, L-lactide-glycolide copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) meacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide polyamine, polyamine peptidomimetic, polyamine dendrimer, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or alpha-helical peptide.

Ligands also include targeting groups, such as a cell or tissue targeting agent such as a lectin, glycoprotein, lipid, or protein such as an antibody that binds to a specific cell type such as a kidney cell. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surface protein A, carbohydrate mucin, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or RGD peptide, or an RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g., acridines), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphirin),

- 41 044245 polycyclic aromatic hydrocarbons (e.g. phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bisO(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine and peptide conjugates (e.g. antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/uptake aids (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleotides (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ tetraazamacrocycle complex), dinitrophenyl, HRP, or AP.

Ligands can be proteins, such as glycoproteins, or peptides, such as molecules with specific affinity for a co-ligand, or antibodies, such as an antibody that binds to a specific cell type, such as a liver cell. Ligands can also include hormones and hormone receptors. They can also include species other than peptides, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent glucose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose, or polyvalent fucose. A ligand, for example, can be lipopolysaccharide, a p38 MAP kinase activator, or an NF-kB activator.

A ligand may be a substance, such as a drug, that can increase the uptake of an mRNA agent by a cell, for example, by disrupting the cell's cytoskeleton, such as by disrupting microtubules, microfilaments, and/or intermediate filaments. The drug may, for example, be taxon, vincristine, vinblastine, cytochalasin, nocodazole, yaplakinolide, latrunculin A, phalloidin, svingolide A, indanocin, or myoservin.

In some embodiments, the ligand is attached to mRNA as described herein and acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binding agents, PEG, vitamins, etc. Illustrative PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. Oligonucleotides that contain a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, such as oligonucleotides of approximately 5 bases, 10 bases, 15 bases, or 20 bases, containing multiple phosphorothioate linkages in the backbone, are also useful as ligands in the present invention (e.g., as PK-modulating ligands). Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

The ligand-conjugated oligonucleotides of the present invention can be synthesized using an oligonucleotide that bears a reactive side chain, such as those obtained by attaching a binding molecule to the oligonucleotide (described below). This reactive oligonucleotide can be directly reacted with commercially available ligands, ligands synthesized with any of a variety of protecting groups, or ligands that have a binding moiety attached to it.

The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by well-known solid-phase synthesis. Equipment for such synthesis is commercially available from several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Additionally or alternatively, any other means for such synthesis known in the art can be used. Similar methods are also known for preparing other oligonucleotides, such as phosphorothioates and alkylated derivatives.

Ligand-conjugated oligonucleotides and a ligand-molecule carrying sequence-specific linked nucleosides of the present invention, oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide- or nucleoside-conjugate precursors that already carry a binding moiety, ligand-nucleotide or nucleoside-conjugate precursors that already carry a ligand molecule, or building blocks carrying a ligand other than a nucleoside.

When using nucleotide-conjugate precursors that already carry a binding moiety, the synthesis of sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the binding moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized using an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard phosphoramidites and non-standard phosphoramidites that are commercially available and commonly used in oligonucleotide synthesis.

- 42 044245

A. Lipid conjugates.

In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid molecules preferably bind to a serum protein, such as human serum albumin (HSA). The HSA-binding ligand allows for distribution of the conjugate to a target tissue, such as a target tissue other than kidney tissue. For example, the target tissue may be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid ligand can (a) increase resistance to conjugate degradation, (b) enhance targeting or transport to a target cell or cell membrane, and/or (c) be used to adjust binding to a serum protein, such as HSA.

A lipid ligand can be used to inhibit, for example, by regulating, the binding of a conjugate to the target tissue. For example, a lipid or lipid ligand that binds more strongly to HSA is less likely to target the kidneys and, therefore, is less likely to be eliminated from the body. A lipid or lipid ligand that binds less strongly to HSA can be used to target the conjugate to the kidneys.

In a preferred embodiment, the lipid ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity such that the conjugate will be preferentially distributed to tissue other than kidney tissue. However, it is preferred that the affinity not be so strong that HSA-ligand binding is irreversible.

In another preferred embodiment, the lipid ligand binds weakly or not at all to HSA, so the conjugate will be preferentially distributed to the kidney. Other renal cell-targeting moieties can also be used instead of or in addition to lipid ligands.

In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by a target cell, such as a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, such as malignant or benign cell proliferation, such as cancer cells.

Illustrative vitamins include vitamins A, E, and K. Other illustrative vitamins include B vitamins, such as folate, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients absorbed by target cells, such as liver cells. Also included are HAS and low-density lipoprotein (LDL).

B. Means that ensure penetration into the cell.

In another aspect, the ligand is a cell penetration agent, preferably a helical cell penetration agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidyl mimetic, non-peptide invertomers or pseudopeptide linkages, and the use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and lipophobic phase.

The ligand can be a peptide or a peptidomimetic.

A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. Attaching peptides and peptidomimetics to mRNA agents can influence the pharmacokinetic distribution of the mRNA, for example, by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be approximately 5-50 amino acids in length, such as approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

The peptide or peptide mimetic may, for example, be a cell penetration peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety may be a peptide dendrimer, a sterically hindered peptide, or a cross-linked peptide. Alternatively, the peptide moiety may include a hydrophobic membrane trafficking sequence (MTS). An exemplary peptide containing a hydrophobic MTS is RFGF, with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:9). An RFGF analog (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO:10)) containing a hydrophobic MTS may also be a targeting moiety. The peptide moiety may be a delivery peptide that can transport large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:11) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:12)) have been found to function as delivery peptides. The peptide or peptide mimetic may be encoded by random sequences

- 43 044245 DNA sequences, such as a peptide identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic linked to a dsRNA agent via an introduced monomeric unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD) peptide or RGD mimetic. The peptide moiety can be characterized by a length ranging from about 5 amino acids to about 40 amino acids. The peptide moieties can be characterized by a structural modification, such as to improve stability or control conformational properties. Any of the structural modifications described below can be used.

The RGD peptide for use in the compositions and methods of the present invention can be linear or cyclic and can be modified, such as glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics can include D-amino acids, as well as synthetic RGD mimetics. In addition to RGD, other moieties that target the integrin ligand can be used. Preferred conjugates with such a ligand target PECAM-1 or VEGF.

A cell-penetrating peptide is capable of penetrating a cell, such as a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A cell-penetrating peptide, for example, can be an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide-linked peptide (e.g., α-defensin, β-defensin, or bactenecin), or a peptide containing only one or two predominant amino acids (e.g., PR-39 or indolicidin). A cell-penetrating peptide can also include a cellular nuclear localization signal (NLS). For example, a cell entry peptide could be a two-component amphipathic peptide such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS from the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate conjugates.

In some embodiments of the compositions and methods of the present invention, an mRNA oligonucleotide is provided that further comprises a carbohydrate. Carbohydrate-conjugated mRNA is preferred for in vivo delivery of nucleic acids, as well as in compositions suitable for in vivo therapeutic use, as described herein. As used herein, a carbohydrate refers to a compound that is either a carbohydrate per se, consisting of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom attached to each carbon atom; or a compound having a carbohydrate moiety as a portion thereof, consisting of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Typical carbohydrates include sugars (mono-, di-, tri-, and oligosaccharides containing approximately 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starches, cellulose, glycogen, and polysaccharide gums. Specific monosaccharides include sugars of C5 and higher (e.g., C5, C6, C7, or C8); di- and trisaccharides include sugars composed of two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is N-acetylgalactosamine, such as

Formula II

- 44 044245

In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is selected from the group consisting of:

- 45 044245

- 46 044245

- 47 044245

HO

AcHN

- 48 044245

Another illustrative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

(Formula XXIII), where one of X and Y is an oligonucleotide, the other is hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell penetration peptide.

D. Linkers.

In some embodiments, the conjugate or ligand described herein may be attached to the mRNA oligonucleotide by various linkers, which may be cleavable or non-cleavable.

The term linker or linking group means an organic group that connects two parts of a compound, such as covalently attaching two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR ⁸ , C(O), C(O)NH, SO, SO2, SO2NH, or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkynyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylene groups may be interrupted or removed by O, S, S(O), SO2, N( ^R8 ), C(O), substituted or unsubstituted 8 aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; wherein R is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is between about 1-24 carbon atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

The cleavable linking group is sufficiently stable outside the cell, but upon entering the target cell, it cleaves, releasing the two portions that the linker holds together. In a preferred embodiment, the cleavable linking group cleaves at least about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more, or at least about 100 times faster in the target cell or under a first-pass condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second-pass condition (which may, for example, be selected to mimic or represent conditions found in blood or serum).

Cleavable linking groups are sensitive to the cleavage agents, such as pH, redox potential, or the presence of degenerative molecules. Typically, cleavage agents are more abundant or found at higher levels or activity inside cells than in serum or blood. Examples of such degenerative agents include: reducing agents that are selected for specific substrates or that have no substrate specificity, including, for example, oxidative or reducing enzymes or reducing agents such as mercaptans present in cells that can degrade a redox cleavable linking group by reduction; esterases; endosomes; or agents capable of creating an acidic environment, such as those that result in a pH of five or less; enzymes that can hydrolyze or destroy the acid-cleavable linking group by acting as a normal acid, peptidases (which may be substrate-specific) and phosphatases.

A cleavable linking group, such as a disulfide bond, can be pH-sensitive. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes are characterized by a more acidic environment, with a pH of 5.5 to 6.0, and lysosomes are characterized by an even more acidic environment, with a pH of 5.0. Some linkers will have a cleavable linking group that cleaves under favorable pH conditions, thereby releasing the cationic lipid from the ligand into the cell or into a desired cellular compartment.

The linker may contain a cleavable linking group that is cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a liver-targeting ligand may be linked to a cationic lipid via a linker containing an ester group. Liver cells are rich in esterases, and therefore, the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other esterase-rich cell types include lung, renal cortex, and testicular cells.

Linkers containing peptide bonds can be used to target peptidase-rich cell types, such as liver cells and synoviocytes.

Typically, the suitability of a candidate cleavable binding group can be assessed by evaluating the ability of a disrupting agent (or condition) to cleave the candidate binding group. It would also be desirable to test the candidate cleavable binding group for its ability to resist cleavage in blood or when brought into contact with other non-target tissues. In this way, the relative sensitivity to cleavage can be determined between the first and second conditions, where the first is chosen as an indicator of cleavage in the target cell and the second is chosen as an indicator of cleavage in other tissues or biological fluids, such as blood or serum. Evaluations can be performed in free-cell systems, in cells, in cell culture, in organ or tissue culture, or in animal models. It may be useful to conduct initial evaluations in free-cell or culture conditions and confirm them with further evaluations in animal models. In preferred embodiments, the beneficial candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in a cell (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox-cleavable linking groups.

In one embodiment, the cleavable linking group is a redox cleavable linking group that cleaves upon reduction or oxidation. An example of a reductive cleavable linking group is a disulfide linking group (-S-S-). The methods described herein can be used to determine the suitability of a candidate cleavable linking group to be a reductive cleavable linking group or, for example, to be suitable for use in a particular mRNA fragment and a particular targeting agent. For example, the candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the rate of cleavage that can be observed in a cell, such as a target cell. The candidate can also be evaluated under conditions selected to mimic conditions in blood or serum. In one embodiment, the candidate compounds are cleaved by no more than about 10% in blood. In other embodiments, the beneficial candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in a cell (or under in vitro conditions selected to simulate intracellular conditions) than in blood (or under in vitro conditions selected to simulate extracellular conditions). The rate of degradation of the candidate compounds can be determined using standard enzymatic kinetics assays under conditions selected to simulate the intracellular environment and compared to conditions selected to simulate the extracellular environment.

ii. Phosphate-based cleavable linking groups.

In another embodiment, the cleavable linker comprises a cleavable phosphate-based linking group. The cleavable phosphate-based linking group is cleavable by agents that break down or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as cellular phosphatases. Examples of phosphate-based linking groups are -OF ^, (O)(OR ^k )-O-, -OP(S)(OR ^k )-O-, -OP(S)(SR ^k )-O-, -SP(O)(OR ^k )-O-, -OP(O)(OR ^k )-S-, -SP(O)(OR ^k )-S-, -OP(S)(OR ^k )-S-, -SP(S)(OR ^k )-O-, -OP(O)(R ^k )-O-, -OP(S)(R ^k )-O-, -SP(O)(R ^k )-O-, -SP(S)(R ^k )-O-, -SP(O)(R ^k )-S-, -OP(S)(R ^k )-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-,

- 50 044245

-S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

iii. Acid-cleavable linking groups.

In another embodiment, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved in the presence of an acid. In preferred embodiments, acid-cleavable linking groups are cleaved under acidic conditions at a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by means such as enzymes that can act as a general acid. Specific organelles in the cell whose contents have a low pH, such as endosomes and lysosomes, can provide conditions for the cleavage of acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleaving groups may have the general formula C=NN-, C(O)O, or -OC(O). In a preferred embodiment, when the carbon is attached to the ester oxygen (alkoxy group), it is an aryl group, a substituted alkyl group, or a tertiary alkyl such as dimethylpentyl or tert-butyl. These candidates can be evaluated using methods similar to those described above.

iv. Ester-based linking groups.

In another embodiment, the cleavable linker comprises an ester-based linking group. The cleavable ester-based linking group is cleavable by enzymes such as esterases and amidases in cells. Examples of cleavable ester-based linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Cleavable ester-based linking groups are represented by the general formula C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

v. Peptide-based cleavable groups.

In another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleavable by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to form oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to form peptides and proteins. A peptide-based cleavable group is typically limited to the peptide bond (i.e., the amide bond) formed between amino acids to form peptides and proteins, and does not include the entire amide functional group. Peptide-based cleavable linking groups are represented by the general formula NHCHRAC(O)NHCHRBC(O)-, where RA and RB represent the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

- 51 044245

In one embodiment, the mRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of carbohydrate conjugates of mRNA with linkers from the compositions and methods of the present invention include, but are not limited to

(Formulas XXVI)

(Formulas XXVII),

- 52 044245

(formula XXX), where one of Y or X is an oligonucleotide, the other is hydrogen.

In some embodiments of the compositions and methods of the present invention, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branched linker.

In one embodiment, the dsRNA of the present invention is conjugated to a divalent or trivalent branched linker selected from the group of structures shown in any of Formulas (XXXI) to (XXXIV): Formulas XXXI

Formulas XXXII

₍ 2A_ _R 2A , _T 2B_|_2B

uZAZA

UZV RV

Formulas XXXIII ₅ or Formulas XXXIV ;

where q ^2A , q ^2B , q ^3A , q ^3B , q ^4A , q ^4B , q ^5A , q ^5B and q ^5C independently represent for each occurrence 0-20 and where the repeating units may be the same or different;

each of ^P2A , ^P2B , P3A, ^P3B , ^P4A , ^P4B , ^P5A , ^P5B , P5C, ^T2A , ^T2B , ^T3A , ^T3B , ^T4A , ^T4B , ^T4A , ^T5B , ^{T5C ,} independently for each case is absent, replaces CO, NH, O, S, OC(O), NHC(O), _CH2 , ^CH2NH or CH2O;

Q ^2A , Q ^2b , Q ^3A , Q ^3b , Q ^4A , Q ^4b , Q ^5A , Q ^5B , Q ^5C independently for each occurrence is absent, is alkylene, substituted alkylene, wherein one or more methylenes may be interrupted or terminated by one or more of O, S, S(O), SO ₂ , N(R ⁿ ), C(R')=C(R''), C=C or C(O);

each of R ^2A , R ^2B , R ^3A , R ^3B , R ^4A , R ^4B , R ^5A , R ^5B , R ^5C independently for each case is absent, represents NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R ^a )C(O), -C(O)-CH(R ^a )-NH-, CO, CH=NO,

- 53 044245 or heterocyclyl;

L ^2A , L ^2B , L ^3A , L ^3B , L ^4A , L ^4B , L ^5A , L ^5B and L ^5C represent a ligand; i.e., each independently for each instance is a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide; and R ^a is H or an amino acid side chain. Trivalent conjugated derivatives of GalNac are particularly useful for use with RNAi agents to inhibit target gene expression, such as those represented by formula (XXXV):

Formula XXXV

where L ^5A , L ^5B and L ^5C represent a monosaccharide such as a GalNAc derivative.

Examples of suitable divalent and trivalent branched linker groups conjugated to GalNac derivatives include, but are not limited to, the structures identified above as formulas II, VII, XI, X, and XIII.

Illustrative U.S. patents that describe the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, 8,106,022, the entire contents of each of which are incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact, more than one of the above modifications can be included in a single compound or even in a single nucleoside within the mRNA. The present invention also includes mRNA compounds that are chimeric.

Chimeric mRNA compounds or chimeras in the context of the present invention are mRNA compounds, preferably dsRNA, that comprise two or more chemically distinct regions, each of which consists of at least one monomer, i.e., a nucleotide in the case of a dsRNA compound. Such mRNAs typically comprise at least one region wherein the RNA is modified to provide the mRNA with increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Additionally, the mRNA region can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand in an RNA:DNA duplex. Activation of RNase H therefore leads to cleavage of the target RNA, thereby significantly increasing the efficiency of mRNA inhibition during gene expression. Consequently, comparable results can often be obtained with shorter mRNAs using chimeric dsRNAs compared to phosphorothioate-deoxy hybridizations of dsRNAs of the same target region. Cleavage of the target RNA can typically be detected by gel electrophoresis and, if necessary, by hybridization methods known in the art for linked nucleic acids.

In some cases, mRNA can be modified with a non-ligand group. A number of non-ligand molecules have been conjugated to mRNA to enhance mRNA activity, cellular distribution, or cellular uptake. Methods for performing such conjugations are available in the scientific literature. Such non-ligand moieties included lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioester such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahe

- 54 044245 dron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Illustrative U.S. patents that describe the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with RNA still bound to the solid support or after RNA cleavage in the solution phase.

Purification of the RNA conjugate by HPLC typically results in pure conjugate.

IV. Delivery of mRNA according to the invention.

Delivery of the mRNA of the present invention to a cell, such as a cell of a subject, such as a human subject (e.g., a subject in need thereof suffering from a disease associated with complement component C5), can be accomplished in various ways. For example, delivery can be accomplished by contacting the cell with the mRNA of the present invention either in vitro or in vivo. In vivo delivery can also be accomplished directly by administering a composition containing the mRNA, such as dsRNA, to the subject. Alternatively, in vivo delivery can be accomplished indirectly by administering one or more vectors that encode and direct the expression of the mRNA. Such alternatives are described further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with the mRNA of the present invention (see, for example, Akhtar S. and Julian R.L. (1992) Trends Cell. Biol. 2(5):139-144 and WO 94/02595, which are incorporated herein by reference in their entireties). With respect to in vivo delivery, factors that are considered in the context of delivering an mRNA molecule include, for example, the biological stability of the delivered molecule, the prevention of non-specific effects, and the accumulation of the delivered molecule in the target tissue. Non-specific effects of mRNA can be minimized by local administration, such as by direct injection or implantation into the tissue, or by local administration of the drug. Local administration at the treatment site maximizes the local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be damaged by the agent or that may destroy the agent, and allows for the administration of a lower total dose of the mRNA molecule. Several studies have demonstrated effective knockdown of gene products by local administration of mRNA. For example, intraocular delivery of dsRNA to VEGF, either by intravitreal injection in cynomolgus macaques (Tolentino, M.J., et al. (2004) Retina 24:132–138) or subretinal injection in mice (Reich, S.J., et al. (2003) Mol. Vis. 9:210–216), was shown to prevent new vessel formation in an experimental model of age-related macular degeneration. Furthermore, direct intratumoral injection of dsRNA into mice reduces tumor size (Pille, J., et al. (2005) Mol. Ther. 11:267-274) and can increase the survival of tumor-bearing mice (Kim, W.J., et al. (2006) Mol. Ther. 14:343-350; Li, S., et al. (2007) Mol. Ther. 15:515-523). RNA interference has also been successful in local delivery to the CNS by direct delivery (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P.H., et al. (2005) Gene Ther. 12:59–66; Makimura, H., et al. (2002) BMC Neurosci. 3:18; Shishkina, G.T., et al. (2004) Neuroscience 129:521–528; Thakker, E.R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270–17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594–602) and to the lungs by intranasal administration (Howard, K.A., et al. (2006) Mol. Ther. 14:476-484; Zhang, X., et al. (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al. (2005) Nat. Med. 11:50-55). Regarding the administration of mRNA systemically for the treatment of disease, the RNA can be modified or, alternatively, delivered using a drug delivery system; both methods act to prevent the rapid degradation of dsRNA by endo- and exonucleases in vivo. RNA modification or a pharmaceutical carrier can also allow targeting of the mRNA composition to the target tissue and can avoid unwanted off-target effects. mRNA molecules can be modified by chemical conjugation with lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, mRNA directed against ApoB and conjugated to a lipophilic cholesterol moiety was administered systemically to mice and resulted in knockdown of apoB mRNA in both the liver and small intestine (Soutschek, J., et al. (2004) Nature 432:173-178). Conjugation of mRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, J.O., et al. (2006) Nat. Biotechnol. 24:1005-1015). Alternatively, mRNA can be delivered using drug delivery systems such as a nanoparticle, dendrimer, polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems promote the binding of the mRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to ensure efficient uptake of mRNA by the cell. Cationic lipids, dendrimers, or polymers can be either bound to mRNA or acted upon to form a vesicle or micelle (see, e.g., Kim S.H., et al. (2008) Journal of Controlled Release 129(2):107-116) that enclose the mRNA. Formation

- 55 044245 vesicles or micelles also prevent degradation of mRNA when administered systemically. Methods for preparing and administering cationic complexes with mRNA are within the skill of the art (see, e.g., Sorensen, D.R., et al. (2003) J. Mol. Biol 327:761–766; Verma, U.N., et al. (2003) Clin. Cancer Res. 9:1291–1300; Arnold, A.S. et al. (2007) J. Hypertens. 25:197–205, which are incorporated herein by reference in their entireties). Some non-limiting examples of drug delivery systems suitable for systemic delivery of mRNA include DOTAP (Sorensen, D.R., et al. (2003), supra; Verma, U.N., et al. (2003), supra), oligofectamine, solid nucleic acid-lipid particles (Zimmermann, T.S., et al. (2006) Nature 441:111–114), cardiolipin (Chien, P.Y., et al. (2005) Cancer Gene Ther. 12:321–328; Pal, A., et al. (2005) Int J. Oncol. 26:1087–1091), polyethyleneimine (Bonnet M.E., et al. (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)-containing peptides (Liu, S. (2006) Mol. Pharm. 3:472487) and polyamidoamines (Tomalia, DA., et al. (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al. (1999) Pharm. Res. 16:1799-1804). In some embodiments, mRNA is complexed with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of mRNA and cyclodextrins can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety.

A. Vector-encoded mRNAs according to the invention.

mRNAs targeting the C5 gene can be expressed by transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A., et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113; Conrad, International PCT Publication No. WO 00/22114; and Conrad, U.S. Patent No. 6,054,299). Expression can be transient (approximately hours to weeks) or long-term (weeks to months or longer) depending on the particular construct used and the target tissue or cell type. Such transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which may be an integrating or non-integrating vector. Transgenes can also be engineered to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

Individual strands or strands of mRNA can be transcribed from an expression vector promoter. Two separate expression vectors can be co-introduced into a target cell (e.g., by transfection or infection) to express the two individual strands, producing, for example, dsRNA. Alternatively, each individual strand of dsRNA can be transcribed from promoters located on the same expression plasmid. In one embodiment, dsRNA is expressed as inverted repeat polynucleotides connected by a linker polynucleotide sequence such that the dsRNA has a stem-loop structure.

Expression vectors for mRNA are typically DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably compatible with vertebrate cells, can be used to generate recombinant constructs for mRNA expression, as described herein. Expression vectors for eukaryotic cells are well known in the art and are available from a number of commercial sources. Vectors are typically provided that contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of mRNA expression vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells explanted from a patient followed by reintroduction into the patient, or by any other method that allows delivery to the desired target cell.

Target cells can be transfected with mRNA expression plasmids complexed with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). The present invention also contemplates multiple lipid-mediated transfections for mRNA-mediated knockdown patterns targeting different regions of the target RNA over a period of a week or more. Successful introduction of vectors into host cells can be monitored using a variety of known methods. For example, transient transfection can be detected using a reporter, such as a fluorescent marker, such as green fluorescent protein (GFP). Stable transfection of cells ex vivo can be confirmed using markers that confer resistance to certain environmental factors (e.g., antibiotics and drugs), such as resistance to hygromycin B, on the transfected cell.

Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retroviral vectors, including, but not limited to, lentivirus vectors, Moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canaripox or fowlpox; and (j) hel

- 56 044245 per-dependent or weak adenovirus. Viruses with impaired replication may also be advantageous. Different vectors will or will not integrate into the cell genome. If necessary, constructs can include viral sequences for transfection. Alternatively, the construct can be inserted into vectors capable of episomal replication, such as EPV- and EBV-based vectors. To ensure mRNA expression in target cells, constructs for recombinant mRNA expression typically require regulatory elements, such as promoters, enhancers, etc. Other aspects to consider regarding vectors and constructs are described further below.

Vectors suitable for mRNA delivery will include regulatory elements (promoter, enhancer, etc.) sufficient to express the mRNA in the desired target cell or tissue. Regulatory elements can be selected to achieve either constitutive or regulated/inducible expression.

mRNA expression can be precisely regulated, for example, by using an inducible regulatory sequence that is responsive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems suitable for directing dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical dimerization inducers, and isopropyl-beta-Dthiogalactopyranoside (IPTG). One skilled in the art will be able to select the appropriate regulatory/promoter sequence based on the intended use of the mRNA transgene.

Viral vectors that contain nucleic acid sequences encoding mRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581–599 (1993)). Such retroviral vectors contain the components necessary for proper packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding mRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid to the patient. A more detailed description of retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291–302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells to render the stem cells more resistant to chemotherapy. Other sources illustrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV-based vectors described in U.S. Patent Nos. 6,143,520; and 5,981,276, which are incorporated herein by reference.

Adenoviruses are also contemplated for use in the delivery of mRNA according to the present invention. Adenoviruses are particularly promising vehicles, for example, for delivering genes to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium, where they cause mild disease. Other targets for adenovirus-based delivery systems include the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), provide a review article on adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenoviral vectors to transfer genes into the respiratory epithelium of rhesus macaques. Additional examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT publications WO 94/12649 and Wang et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing the mRNA described in the present invention, a method for constructing a recombinant AV vector, and a method for delivering the vector to target cells are described in Xia H et al. (2002) Nat. Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors can also be used to deliver the mRNA of the present invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146). In one embodiment, the mRNA can be expressed as two separate, complementary single-stranded RNA molecules using a recombinant AAV vector, for example, with either the U6 or H1 RNA promoters or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA described in the present invention, methods for constructing a recombinant AV vector, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987) J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are incorporated herein by reference.

Another viral vector suitable for delivery of mRNA according to the present invention is a poxvirus, such as vaccinia virus, for example, attenuated vaccinia virus,

- 57 044245 such as modified virus Ankara (MVA) or NYVAC, avipox such as fowlpox or canaripox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses or by substituting different viral capsid proteins, as needed. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies virus, Ebola virus, Mokola virus, and the like. AAV vectors can be designed to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J.E. et al. (2002) J Virol 76:791–801, the entire disclosure of which is incorporated herein by reference.

A vector pharmaceutical may comprise the vector in a suitable diluent or may comprise a sustained-release matrix incorporating a gene delivery vehicle. Alternatively, when the whole gene delivery vector can be produced by native recombinant cells, such as retroviral vectors, the pharmaceutical may comprise one or more cells that produce the gene delivery system.

V. Pharmaceutical compositions according to the invention.

The present invention also includes pharmaceutical compositions and formulations that include the mRNA of the present invention. In one embodiment, provided herein are pharmaceutical compositions comprising the mRNA described herein and a pharmaceutically acceptable carrier.

The phrase pharmaceutically acceptable is used herein to mean compounds, substances, compositions, and/or dosage forms that, within the margin of medical judgment, are suitable for use in contact with the tissues of a human subject and an animal subject without undue toxicity, irritation, allergic reaction, or other problem or complication commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, excipient (e.g., lubricant, talc, magnesium, calcium or zinc stearate, or stearic acid), or solubilizing encapsulating substance, that is involved in the transport and transportation of the reference compound from one organ or body part to another organ or body part. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents such as magnesium stearate, sodium lauryl sulfate, and talc; (8) fillers such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) fillers such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances used in pharmaceutical formulations.

Pharmaceutical compositions containing mRNA are useful for treating a disease or disorder associated with the expression or activity of the C5 gene, such as a disease associated with the complement component C5. Such pharmaceutical compositions are formulated depending on the delivery route. One example is compositions that are formulated for systemic administration via the parenteral route, such as subcutaneous (SC) or intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery to the brain parenchyma, such as by infusion into the brain, such as continuous infusion using a pump. The pharmaceutical compositions of the present invention can be administered in doses sufficient to inhibit the expression of the C5 gene. Typically, a suitable dose of the mRNA of the present invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, typically in the range of about 1 to 50 mg per kilogram of body weight per day. For example, dsRNA can be administered in an amount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.

For example, dsRNA can be administered at a dose of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1,

- 58 044245

1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,

3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4,

6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1,

9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or approximately 10 mg/kg. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

In another embodiment, the dsRNA is administered at a dose of from about 0.1 to about 50 mg/kg, from about 0.25 to about 50 mg/kg, from about 0.5 to about 50 mg/kg, from about 0.75 to about 50 mg/kg, from about 1 to about 50 mg/kg, from about 1.5 to about 50 mg/kg, from about 2 to about 50 mg/kg, from about 2.5 to about 50 mg/kg, from about 3 to about 50 mg/kg, from about 3.5 to about 50 mg/kg, from about 4 to about 50 mg/kg, from about 4.5 to about 50 mg/kg, from about 5 to about 50 mg/kg, from about 7.5 to about 50 mg/kg, from about 10 to about 50 mg/kg, from about 15 to about 50 mg/kg, from about 20 to about 50 mg/kg, from about 20 to about 50 mg/kg, from about 25 to about 50 mg/kg, from about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, from about 35 to about 50 mg/kg, from about 40 to about 50 mg/kg, from about 45 to about 50 mg/kg, from about 0.1 to about 45 mg/kg, from about 0.25 to about 45 mg/kg, from about 0.5 to about 45 mg/kg, from about 0.75 to about 45 mg/kg, from about 1 to about 45 mg/kg, from about 1.5 to about 45 mg/kg, from about 2 to about 45 mg/kg, from about 2.5 to about 45 mg/kg, from about 3 to about 45 mg/kg, from about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, from about 10 to about 20 mg/kg, or from about 15 to about 20 mg/kg. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

For example, dsRNA can be administered at a dose of about 0.1, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or approximately 10 mg/kg. Values and ranges intermediate to the listed values are also considered to be part of the present invention.

In another embodiment, the dsRNA is administered at a dose of from about 0.5 to about 50 mg/kg, from about 0.75 to about 50 mg/kg, from about 1 to about 50 mg/kg, from about 1.5 to about 50 mg/kg, from about 2 to about 50 mg/kg, from about 2.5 to about 50 mg/kg, from about 3 to about 50 mg/kg, from about 3.5 to about 50 mg/kg, from about 4 to about 50 mg/kg, from about 4.5 to about 50 mg/kg, from about 5 to about 50 mg/kg, from about 7.5 to about 50 mg/kg, from about 10 to about 50 mg/kg, from about 15 to about 50 mg/kg, from about 20 to about 50 mg/kg, from about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, from about 10 to about 45 mg/kg, from about 15 to about 45 mg/kg, from about 20 to about 45 mg/kg, from about 20 to about 45 mg/kg, from about 25 to about 45 mg/kg, from about 25 to about 45 mg/kg, from about 30 to about 45 mg/kg, from about 35 to about 45 mg/kg, from about 40 to about 45 mg/kg, from about 0.5 to about 40 mg/kg, from about 0.75 to about 40 mg/kg, from about 1 to about 40 mg/kg, from about 1.5 to about 40 mg/kg, from about 2 to about 40 mg/kg, from about 2.5 to about 40 mg/kg, from about 3 to about 40 mg/kg, from about 3.5 to about 40 mg/kg, from about 4 to about 40 mg/kg, from about 4.5 to about 40 mg/kg, from about 5 to about 40 mg/kg, from about 7.5 to about 40 mg/kg, from about 10 to about 40 mg/kg, from about 15 to about 40 mg/kg, from about 20 to about 40 mg/kg, from about 20 to about 40 mg/kg, from about 25 to about 40 mg/kg, from about 25 to about 40 mg/kg, from about 30 to about 40 mg/kg, from about 35 to about 40 mg/kg, from about 0.5 to about 30 mg/kg, from about 0.75 to about 30 mg/kg, from about 1 to about 30 mg/kg, from about 1.5 to about 30 mg/kg, from about 2 to about 30 mg/kg, from about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, from about 3 to about 20 mg/kg, from about 3.5 to about 20 mg/kg, from about 4 to about 20 mg/kg, from about 4.5 to about 20 mg/kg, from about 5 to about 20 mg/kg, from about 7.5 to about 20 mg/kg, from about 10 to about 20 mg/kg, or from about 15 to about 20 mg/kg. In one embodiment, the dsRNA is administered at a dose of from about 10 mg/kg to about 30 mg/kg. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

For example, a single therapeutic amount of mRNA of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or approximately 50 mg/kg. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

In some embodiments, the subject is administered, e.g., subcutaneously or intravenously, multiple doses of a therapeutic amount of mRNA, e.g., at a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65,

- 60 044245

0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,

1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,

4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,

7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,

9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5,

21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or approximately 50 mg/kg. A multiple-dose regimen may include administering a therapeutic amount of mRNA daily, such as for two days, three days, four days, five days, six days, seven days, or longer.

In other embodiments, the subject is administered, e.g., subcutaneously or intravenously, a repeated dose of a therapeutic amount of mRNA, e.g., at a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or approximately 50 mg/kg. A repeat dose regimen may involve administering a therapeutic amount of mRNA on a regular basis, such as every second day, every third day, every fourth day, twice a week, once a week, every other week, or once a month.

The pharmaceutical composition can be administered by intravenous infusion over a period of time, such as a 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, or approximately 25-minute period. Administration can be repeated, for example, regularly, such as once a week, once every two weeks (i.e., every two weeks) for one month, two months, three months, four months, or longer. After the initial treatment regimen, treatment can be administered less frequently. For example, after administration once a week or once every two weeks for three months, administration can be repeated once a month for six months, or a year, or longer.

The pharmaceutical composition can be administered once daily, or the mRNA can be administered as two, three, or more sub-doses at intervals throughout the day, or even by continuous infusion or delivery via a controlled-release formulation. In such a case, the amount of mRNA contained in each sub-dose should be correspondingly smaller to provide the total daily dose. The dosage unit can also be formulated for delivery over several days, for example, using a traditional sustained-release formulation that provides for the delayed release of mRNA over a period of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering agents to a specific site, such as those that can be used with the agents of the present invention. In such an embodiment, the dosage unit contains an appropriate multiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositions may be long-acting, such that subsequent doses are administered at intervals of no more than 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of the present invention, a single dose of the pharmaceutical compositions of the present invention is administered once a week. In other embodiments of the present invention, a single dose of the pharmaceutical compositions of the present invention is administered every two months.

One skilled in the art will appreciate that certain factors may influence the dose and timing necessary to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, types of previous treatments, the general health and/or age of the subject, and other existing medical conditions. Furthermore, treatment of a subject with a therapeutically effective amount of a composition may comprise a single treatment period or a series of treatment periods. Estimates of effective doses and in vivo half-lives for individual mRNAs encompassed by the present invention can be obtained using conventional methodologies or by conducting in vivo studies using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have provided a range of mouse models for studying various human diseases, such as disorders that can be beneficially targeted by reducing C5 expression. These models can be used for in vivo mRNA studies and for determining therapeutically effective doses. Suitable mouse models are known in the art and include, for example, a mouse model of collagen-induced arthritis (Courtenay, JS, et al. (1980) Nature 283, 666-668), myocardial ischemia (Homeister JW and Lucchesi BR (1994) Annu Rev Pharmacol Toxicol 34:17-40), a mouse model of egg white-induced asthma (e.g., Tomkinson A., et al. (2001). J. Immunol. 166, 5792-5800), (NZBxNZW)Fl, MRL/Fas ^lpr (MRL/lpr), and the BXSB mouse model (Theofilopoulos, AN and Kono, DH 1999. Murine lupus models: gene-specific and genome-wide studies. In Lahita R.G., ed., Systemic Lupus Erythematosus, 3rd edn, p. 145. Academic Press, San

Diego, CA), a mouse model of aHUS (Goicoechea de Jorge et al. (2011) Development of atypical hemolytic uremic syndrome is dependent on complement C5, J Am Soc Nephrol 22:137–145.

The pharmaceutical compositions of the present invention can be administered by various routes depending on whether local or systemic treatment is required and the area to be treated. Administration can be local (e.g., by transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by means of an inhaler; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., by means of an implanted device; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular administration.

mRNA can be delivered in such a way that it targets a specific tissue, such as the liver (e.g., liver hepatocytes).

Pharmaceutical compositions and formulations for topical use may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Traditional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be used. Suitable formulations for topical use include those in which the mRNAs described herein are mixed with a topical delivery vehicle, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine), negative (e.g., dimyristoyl phosphatidylglycerol (DMPG)) and cationic (e.g., dioleoyl tetramethylaminopropyl (DOTAP) and dioleoyl phosphatidylethanolamine (DOTMA)). The mRNAs described in the present invention can be encapsulated in liposomes or can form complexes with them, particularly with cationic liposomes. Alternatively, the mRNAs can form complexes with lipids, particularly with cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl-1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or _{C1.20 alkyl} esters (e.g., isopropyl myristate (IPM)), monoglyceride, diglyceride, or a pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

A. Compositions with mRNA containing membrane molecular assemblies.

mRNA for use in the compositions and methods of the present invention can be formulated for delivery into a membrane molecular assembly, such as a liposome or a micelle.

As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, such as a single bilayer or multiple bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed of a lipophilic material and an aqueous interior. The aqueous interior contains the mRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain the mRNA composition, although in some instances it may. Liposomes are suitable for transporting and delivering active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissue, the liposome bilayer fuses with the bilayer of cellular membranes. As the liposome and cell fuse, the internal aqueous content, which includes mRNA, is delivered into the cell, where the mRNA can specifically bind to the target RNA and mediate RNAi. In some cases, liposomes are also specifically targeted, for example, to direct mRNA to specific cell types.

Liposomes containing an RNAi agent can be prepared by a number of methods. In one example, the lipid component of the liposome is dissolved in a detergent to form a micelle with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Illustrative detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that contain the lipid component. The cationic groups of the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, for example, by dialysis, to yield a liposomal preparation of the RNAi agent.

If necessary, a carrier compound that promotes condensation can be added to the

- 62 044245 condensation reaction time, for example, by controlled addition. For example, the carrier compound could be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to promote condensation.

Methods for producing stable polynucleotide delivery vehicles that include a polynucleotide/cationic lipid complex as structural components of the delivery vehicles are further described, for example, in WO 96/37194, the entire contents of which are incorporated herein by reference. Formation of liposomes can also include one or more aspects of the exemplary methods described in Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for producing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used in cases where consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Such methods are easily adapted for packaging RNAi agents into liposomes.

Liposomes are divided into two broad classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized into an endosome. The liposomes rupture, releasing their contents into the cell cytoplasm, due to the acidic pH within the endosome (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes that are pH-sensitive or negatively charged entrap nucleic acids rather than forming a complex with them. Because both the nucleic acid and lipid have the same charge, repulsion occurs instead of complex formation. However, some nucleic acids are trapped in the aqueous internal environment of these liposomes. pH-sensitive liposomes were used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoylphosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods for in vitro and in vivo introduction of liposomes into cells include U.S. Patent No. 5,283,185; U.S. Patent No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems were also investigated to determine their suitability for drug delivery to the skin, specifically systems containing a non-ionic surfactant and cholesterol. Non-ionic liposomal formulations containing Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A to the dermal layer of mouse skin. The results showed that these non-ionic liposomal systems were effective in promoting cyclosporin A deposition in various skin layers (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6) 466).

Liposomes also include sterically stabilized liposomes, which, as used herein, mean liposomes containing one or more specialized lipids that, when incorporated into the liposomes, result in an increased circulating lifetime compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which the vesicle-forming portion of the liposome (A) contains one or more glycolipids, such as monosialoganglioside G _M1 , or (B) is derived from one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Without wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derived lipids, the increased circulating half-life of these sterically stabilized liposomes is a consequence of decreased uptake by reticuloendothelial system (RES) cells (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

A variety of liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) described the ability of monosialoganglioside _GM1 , galactocerebroside sulfate, and phosphatidylinositol to increase the half-life of liposomes in blood. These findings were commented on by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes containing (1) sphingomyelin and (2) ganglioside GM1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes, although they cannot fuse with the plasma membrane as effectively, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

Additional advantages of liposomes include the following: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs; and liposomes can protect encapsulated RNAi agents in internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, volume 1, p. 245). Important aspects in preparing liposomal formulations include the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acid to form lipid-nucleic acid complexes that are capable of fusing with negatively charged lipids in the cell membranes of tissue culture cells, resulting in delivery of the RNAi agent (see, e.g., Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analog, 1,2-bis-(oleoyloxy)-3-(trimethylammonium)propane (DOTAP), can be used in combination with a phospholipid to form vesicles that complex with DNA. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, MD) is an effective delivery system for highly anionic nucleic acids into tissue culture cells. It contains positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. The net charge of the resulting complexes is also positive when liposomes with a sufficient positive charge are used. The positively charged complexes obtained in this manner spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids, for example, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis-(oleoyloxy)-3,3(trimethylammonium)propane (DOTAP) (Boehringer Mannheim, Indianapolis, IN), differs from DOTMA in that the oleyl moieties are linked by ester rather than ether linkages.

Other published cationic lipid compounds include those that have been conjugated to a number of moieties, including, for example, carboxyspermine, which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycinedioctaolylamide (DOGS) (Transfectam™, Promega, Madison, WI) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES) (see, for example, U.S. Patent No. 5,171,678).

Another cationic lipid conjugate includes lipid-derivatized cholesterol (DC-Chol) that have been formulated as liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, prepared by conjugating polylysine to DOPE, has been reported to be effective in transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, such liposomes containing conjugated cationic lipids are known to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing formulations. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, CA) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, MD). Other cationic lipids suitable for oligonucleotide delivery are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suitable for topical administration, with liposomes exhibiting several advantages over other formulations. These advantages include reduced side effects due to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer the RNAi agent into the skin. In some embodiments, liposomes are used to deliver the RNAi agent to epidermal cells and also to enhance the penetration of the RNAi agent into dermal tissues, such as the skin. For example, liposomes can be applied topically. Local delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C.Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:78517855, 1987).

Non-ionic liposomal systems were also investigated to determine their suitability for drug delivery to the skin, specifically systems containing a non-ionic surfactant and cholesterol. Non-ionic liposomal formulations containing Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver the drug to the dermis layer of mouse skin. These RNAi-enhanced formulations are useful for treating dermatological disorders.

Liposomes that incorporate mRNA can be produced with high deformability. This deformability can allow the liposomes to penetrate pores smaller than the average liposome radius. For example, transfersomes are a type of deformable liposome. Transfersomes can be produced by adding surface interface activators, typically surfactants, to a standard liposomal formulation. Transfersomes that incorporate an RNAi agent can be delivered, for example, by subcutaneous injection to deliver the RNAi agent to keratinocytes in the skin. To pass through intact mammalian skin, lipid vesicles must penetrate a series of small pores, each less than 50 nm in diameter, under the influence of a suitable intradermal gradient. Furthermore, due to the properties of lipids, such transferosomes can be self-optimizing (adapting to the shape of pores, for example, in the skin), self-renewing, and can often reach their targets without fragmenting, and can often be self-loading.

Other formulations useful in the present invention are described in U.S. Provisional Application Serial Nos. 61/018,616, filed January 2, 2008; 61/018,611, filed January 2, 2008; 61/039,748, filed March 26, 2008; 61/047,087, filed April 22, 2008; and 61/051,528, filed May 8, 2008. PCT Application No. PCT/US2007/080331, filed October 3, 2007, also describes formulations useful in the present invention.

Transfersomes are another type of liposome and are highly deformable lipid aggregates that are promising candidates for drug delivery. Transfersomes can be described as lipid droplets that are so deformable that they can easily penetrate pores smaller than the droplets. Transfersomes are adaptable to the environment in which they are used; for example, they are self-optimizing (adapting to the shape of skin pores), self-healing, often reach targets without fragmentation, and can often be self-loading. Transfersomes can be produced by adding surface activators, typically surfactants, to a standard liposomal formulation. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrolipid balance (HLB). The nature of the hydrophilic group (also known as the head group) provides the most effective means for categorizing the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceutical and cosmetic products and can be used over a wide range of pH values. Typically, their HLB values range from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and esters, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block copolymers, are also included in this class. Polyoxyethylene surfactants are the most common

- 65 044245 non-ionic surfactants.

If a surfactant molecule carries a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates, such as saponifiers, acyl lactylates, amino acid acylamides, sulfuric acid esters, such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates, such as alkyl benzene sulfonates, acyl isethionates, acyl taurates, and sulfosuccinates, and phosphates. The most important representatives of the class of anionic surfactants are alkyl sulfates and saponifiers.

If a surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most widely used members of this class.

If a surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phosphatides.

The use of surfactants in finished dosage forms, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

mRNA for use in the methods of the present invention may also be provided in micellar formulations. Micelles are defined herein as a specific type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecule face inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The opposite arrangement occurs if the surrounding medium is hydrophobic.

A mixed micellar formulation suitable for delivery through intradermal membranes can be prepared by mixing an aqueous solution of the composition with siRNA, _C8 - _C22 -alkyl alkali metal sulfate and micelle-forming compounds. Illustrative micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening primrose oil, menthol, trihydroxyoxocholanyl glycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compounds can be added during or after the addition of the alkali metal alkyl sulfate. Mixed micelles will form with essentially any type of mixing of ingredients, except vigorous stirring to produce smaller micelles.

In one method, a first micellar composition is prepared that contains an siRNA composition and at least an alkali metal alkyl sulfate. The first micellar composition is then mixed with at least three micelle-forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by the addition of the remaining micelle-forming compounds with vigorous mixing.

Phenol and/or m-cresol can be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol can be added with the micelle-forming ingredients. An isotonic agent, such as glycerin, can also be added after the mixed micellar composition is formed.

To deliver a micellar formulation as a spray, the formulation can be placed in an aerosol dispenser and charged with propellant. The propellant, which is pressurized, is contained in liquid form in the dispenser. The ratio of ingredients is adjusted so that the aqueous phase and the propellant phase are one, i.e., a single phase is present. If two phases are present, the dispenser should be shaken before dispensing a portion of the contents, for example, using a metering valve. The sprayed dose of the pharmaceutical product is ejected from the metering valve as a fine mist.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

Specific concentrations of the main ingredients can be determined through relatively simple experiments. For oral absorption, it is often necessary to increase—for example, at least double or triple—the dose intended for injection or gastrointestinal administration.

B. Lipid particles.

- 66 044245 mRNA, such as dsRNA of the present invention, may be fully encapsulated in a lipid formulation, such as an LNP or other nucleic acid-lipid particle.

As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents particle aggregation (e.g., a PEG-lipid conjugate). LNPs are highly suitable for systemic applications because they are characterized by a long lifetime in the bloodstream after intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the injection site). LNPs include pSPLP, which contains an encapsulated condensing agent-nucleic acid complex, as described in PCT Publication No. WO 00/03683. The particles of the present invention typically have an average diameter of from about 50 nm to about 150 nm, more typically from about 60 nm to about 130 nm, more typically from about 70 nm to about 110 nm, most typically from about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, the nucleic acids, when present in the nucleic acid-lipid particles of the present invention, are resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and a method for producing them are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (weight/weight ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or from about 6:1 to about 9:1. Ranges intermediate to the ranges listed above are also contemplated as part of the present invention.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,Ntrimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-SDMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(III)-methylpiperazino)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis-(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (T ech G1) or a mixture thereof. The cationic lipid may comprise from about 20 mol% to about 50 mol% or about 40 mol% of the total lipid content present in the particle.

In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to produce lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61/107,998, filed October 23, 2008, which is incorporated herein by reference.

In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane; 10% DSPC; 40% cholesterol; 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and an siRNA/lipid ratio of 0.027.

The ionizable/non-cationic lipid may be an anionic lipid or a neutral lipid, including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl-PE, 16-O-dimethyl-PE, 18-1-trans-PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may comprise from about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% if cholesterol is included, of the total lipid content present in the particle.

The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid conjugate, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The conjugate

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PEG-DAA can be, for example, PEG-dilauryloxypropyl (Ci ₂ ), PEG-dimyristoyloxypropyl (Ci ₄ ), PEG-dipalmityloxypropyl (Ci ₆ ) or PEG-distearyloxypropyl (Ci ₈ ).

The conjugated lipid that prevents particle aggregation may comprise from 0 mol% to about 20 mol% or about 2 mol% of the total lipid content present in the particle.

In some embodiments, the nucleic acid-lipid particles further comprise cholesterol in an amount of, for example, from about 10 mol% to about 60 mol% or about 48 mol% of the total lipid content present in the particle.

In one embodiment, the lipidoid ND98-4HCl (MW 1487) (see U.S. Patent Application No. 12/056,230, filed March 26, 2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/mL; cholesterol, 25 mg/mL; PEG-ceramide C16, 100 mg/mL. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be combined, for example, in a molar ratio of 42:48:10. The combined lipid solution can be mixed with an aqueous solution of dsRNA (e.g., in sodium acetate, pH 5) to produce a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., with a 100 nm size cutoff) using, for example, a thermal cylinder extruder such as the Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished, for example, by dialysis or tangential flow filtration. The buffer can be replaced, for example, with phosphate buffered saline (PBS) with a pH of about 7, such as a pH of about 6.9, a pH of about 7.0, a pH of about 7.1, a pH of about 7.2, a pH of about 7.3, or a pH of about 7.4.

ND98 isomer!

Formula I

LNP01 formulations are described, for example, in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional illustrative lipid-dsRNA formulations are described in Table 1.

____________________________________________Table 1

Ionizable/cationic lipid Cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Lipid:siRNA ratio 1,2-dilinolenyloxy-I,N-dimethylaminopropane (DLinDMA) DLinDMA/DPPC/cholesterol/ PEG-cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~ 7:1 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]dioxolane (XTC) XTC/DPPC/cholesterol/PEG- cDMA 57.1/7.1/34.4/1.4 lipid:siRNA ~ 7:1

- 68 044245

LNP05 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]dioxolane (XTC) XTC/DSPC/cholesterol/RES- DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~ 6:1 LNP0 6 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]dioxolane (XTC) XTC/DSPC/cholesterol/RES- DMG 57.5/7.5/31.5/3.5 lipid:siRNA ~ 11:1 LNP07 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTS) XTC/DSPC/cholesterol/RES- DMG 60/7.5/31/1.5, lipid:siRNA ~ 6:1 LNP0 8 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTS) HTS/OBRS/cholesterol/REb- DMG 60/7.5/31/1.5, lipid:siRNA ~ 11:1 LNP0 9 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTS) HTS/OBRS/cholesterol/REb- DMG 50/10/38.5/1.5 lipid:siRNA 10:1 LNP10 (3aR,5s,6aS )-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl) tetrahydro-3aH- cyclopenta[d][1,3]dioxol-5-amine (ALN100) ALNIOO/DSPC/cholesterol/R EG-DMG 50/10/38.5/1.5 lipid:siRNA 10:1 LNP11 (6Z,9Z,28Z,31Z)- heptatriakonta-6,9,28,31tetraen-19-yl-4- (dimethylamino)butanoate (MSZ) ms- 3/DSPC/XonecTepnn/PEG- DMG 50/10/38.5/1.5 lipid:siRNA 10:1

- 69 044245

1, 1’-(2-(4-(2-( (2-(bis(2- hydroxydodecyl)amino)ethyl)(2 Tech G1/DSPC/cholesterol/PEG- LNP12 hydroxydodecyl)amino)ethyl)pi perazine-1- yl)ethylazanediyl)didodecane-2- ol (Tech G1) DMG 50/10/38.5/1.5 lipid:siRNA 10:1 XTC/DSPC/xon/PEG-DMG LNP13 HTS 50/10/38.5/1.5 lipid:siRNA: 33:1 MC3/DSPC/xon/PEG-DMG LNP14 LNP15 MSZ MSZ 40/15/40/5 lipid:siRNA: 11:1 MC3/DSPC/xon/PEGDSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 lipid:siRNA: 11:1 MC3/DSPC/xon/PEG-DMG LNP16 MSZ 50/10/38.5/1.5 lipid:siRNA: 7:1 MSZ/OZRS/hol/RES-OZS LNP17 MSZ 50/10/38.5/1.5 lipid:siRNA: 10:1 MSZ/DSPC/cold/PEG-DMG LNP18 MSZ 50/10/38.5/1.5 lipid:siRNA: 12:1 MC3/DSPC/xon/PEG-DMG LNP19 MSZ 50/10/35/5 lipid:siRNA: 8:1 MC3/DSPC/COL/PEG-DPG LNP2 0 MSZ 50/10/38.5/1.5 lipid:siRNA: 10:1 C12-200/DSPC/xon/PEG-DSG LNP21 C12-200 50/10/38.5/1.5 lipid:siRNA: 7:1 XTC/DSPC/xon/PEG-DSG LNP22 HTS 50/10/38.5/1.5 lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine;

DPPC: dipalmitoylphosphatidylcholine;

PEG-DMG: PEG-didymyristoylglycerol (C14-PEG or PEG-C14) (PEG with an average molecular weight of 2000);

PEG-DSG: PEG-distyrylglycerol (C18-PEG or PEG-C18) (PEG with an average molecular weight of 2000);

PEG-cDMA: PEG-carbamoyl-1,2-dimyristoyloxypropylamine (PEG with an average molecular weight of 2000).

Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in International Publication No. WO 2009/127060, filed April 15, 2009, which is hereby incorporated by reference.

XTC-containing compositions are described, for example, in U.S. Provisional Application Serial No. 61/148,366, filed January 29, 2009; U.S. Provisional Application Serial No. 61/156,851, filed March 2, 2009; U.S. Provisional Application Serial No. 61/228,373, filed July 24, 2009; U.S. Provisional Application Serial No. 61/239,686, filed September 3, 2009; and International Application No. PCT/US2010/022614, filed January 29, 2010, which are hereby incorporated by reference.

MC3-containing formulations are described, for example, in U.S. Publication No. 2010/0324120, filed June 10, 2010, the entire contents of which are hereby incorporated by reference.

ALNY-100-containing compositions are described, for example, in International Patent Application No. PCT/US09/63933, filed November 10, 2009, which is hereby incorporated by reference.

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C12-200-containing compositions are described in U.S. Provisional Application Serial No. 61/175,770, filed May 5, 2009, and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of ionizable/cationic lipids.

Any of the compounds, such as cationic lipids and the like, used in the nucleic acid-lipid particles of the present invention can be prepared using known organic synthetic techniques, including those described in more detail in the examples. All substituents are as defined below unless otherwise noted.

Alkyl means a straight-chain or branched, non-cyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Typical saturated straight-chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Typical saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

Alkenyl means an alkyl group, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis- and trans-isomers. Typical straight-chain and branched alkenyls include ethyleneyl, propyleneyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

Alkynyl means any alkyl or alkenyl group, as defined above, that further contains at least one triple bond between adjacent carbon atoms. Typical straight-chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

Acyl means any alkyl, alkenyl, or alkynyl group in which the carbon atom at the point of attachment is replaced by an oxo group, as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and C(=O)alkynyl are acyl groups.

A heterocycle means a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic ring that is either saturated, unsaturated, or aromatic and that contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen, and sulfur, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized, including bicyclic rings in which any of the above heterocycles is fused to a benzene ring. A heterocycle may be attached through any heteroatom or carbon atom. Heterocycles include heteroaryls, which are defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, etc.

The expressions optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted acyl, and optionally substituted heterocycle mean that, upon substitution, at least one hydrogen atom is replaced by the substituent. In the case of an oxo substituent (=O), two hydrogen atoms are replaced. In this regard, the substituents include oxo, halogen, heterocycle, -CN, -OR ^x , -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO2R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SONR ^x and -SONNR ^x R ^y , where n is 0, 1 or 2, R ^x and R ^y are the same or different and independently represent hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, -OH, -CN, alkyl, -OR ^x , heterocycle, -NR ^x R ^y , -NR ^x C(=O)R ^y , -NR ^x SO2R ^y , -C(=O)R ^x , -C(=O)OR ^x , -C(=O)NR ^x R ^y , -SONR ^x and -SOnNR ^x R ^y

Halogen stands for fluorine, chlorine, bromine and iodine.

In some embodiments, the methods of the present invention may require the use of protecting groups. Techniques using protecting groups are well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T.W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups in the context of the present invention are any group that reduces or eliminates undesirable chemical reactivity of a functional group. A protecting group can be added to a functional group to block its chemical reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments, an alcohol protecting group is used. An alcohol protecting group is any group that reduces or eliminates undesirable chemical reactivity of the alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

- 71 044245

Synthesis of formula A.

In some embodiments, the nucleic acid-lipid particles of the present invention are formulated using a cationic lipid of formula A:

^R 3 \

wherein R ¹ and R ² are independently alkyl, alkenyl, or alkynyl, each optionally substituted, and R ³ and R ⁴ are independently lower alkyl, or R ³ and R ⁴ can be combined to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). Generally, the lipid of Formula A above can be prepared using the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless otherwise noted.

Scheme 1

Lipid A obtained according to scheme 1, wherein R ¹ and R ² independently represent alkyl, alkenyl or alkynyl, wherein each can be optionally substituted, and R ³ and R ⁴ independently represent lower alkyl or R ³ and R ⁴ can be combined to form an optionally substituted heterocyclic ring. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to one skilled in the art. The reaction between 1 and 2 gives ketal 3. Treatment of ketal 3 with amine 4 gives lipids of formula A. Lipids of formula A can be converted into the corresponding ammonium salt using an organic salt of formula 5, where X is an anion, a counterion selected from halogen, hydroxide, phosphate, sulfate or the like.

Scheme 2.

Alternatively, the starting material, ketone 1, can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. The reaction between 6 and 7 yields ketone 1. The conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Scheme 2

Mg-R ₁ 4 . o/' ²

Ri ^! рз $2

MC3 synthesis.

The preparation of DLin-M-C3-DMA (i.e., (62,92,282,312)-heptatriaconta-6,9,28,31-tetraen-19-yl-4(dimethylamino)butanoate) was as follows. A solution of (62,92,282,312)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 ml) was stirred at room temperature overnight. The solution was washed

- 72 044245 with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulfate, filtered, and the solvent was removed using a rotary vacuum evaporator. The residue was passed through a silica gel column (20 g) using an elution gradient of 1-5% methanol/dichloromethane. Fractions containing purified product were combined, and the solvent was removed to yield a colorless oil (0.54 g). Synthesis of ALNY-100

The synthesis of ketal 519 [ALNY-100] was carried out using the following scheme 3.

Scheme 3

Synthesis 515.

To a stirred suspension of LiAlH ₄ (3.74 g, 0.09852 mol) in 200 mL anhydrous THF in a two-necked RBF (1 L) was slowly added a solution of 514 (10 g, 0.04926 mol) in 70 mL THF at 0 °C under nitrogen atmosphere. After complete addition, the reaction mixture was warmed to room temperature and then heated until condensation occurred for 4 h. The reaction progress was monitored by TLC. After completion of the reaction (determined by TLC), the mixture was cooled to 0 °C and quenched by careful addition of saturated Na ₂ SO ₄ solution. The reaction mixture was stirred for 4 h at room temperature and filtered. The residue was washed well with THF. The filtrate and precipitate from the washings were combined and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 min at room temperature. The volatiles were distilled in vacuo to give the hydrochloride salt 515 as a white solid. Yield: 7.12 g. ¹ I-NMR (DMSO, 400 MHz): δ = 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis 516.

To a stirred solution of compound 515 in 100 mL dry DCM in 250 mL two-necked RBF was added NEt ₃ (37.2 mL, 0.2669 mol) and cooled to 0 °C under nitrogen atmosphere. After slow addition of N(benzyloxycarbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, the reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h, determined by TLC), the mixture was washed successively with 1 N HCl solution (1x100 mL) and saturated NaHCO ₃ solution (1x50 mL). The organic layer was then dried over anhydrous. _Na2SO4 and solvent were evaporated to give _a crude material which was purified by silica gel column chromatography to afford 516 as a sticky mass. Yield: 11 g (89%). ¹ H-NMR ( _CDCl3 , 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br, 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] - 232.3 (96.94%).

Synthesis of 517A and 517B.

Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL of acetone and water (10:1) in a single-neck 500 mL RBF bottle and to it was added N-methyl-morpholine-N-oxide (7.6 g, 0.06492 mol), followed by 4.2 mL of 7.6% _OsO4 solution (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (~3 h), the mixture was quenched by _adding solid _Na2SO3 and the resulting mixture was stirred for 1.5 h at room temperature. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 x 100 mL), followed by saturated NaHCO3 solution ( ₁ x 50 mL), water (1 x 30 mL), and finally brine (1 x 50 mL). The organic phase was dried over anhydrous Na2SO4 and the solvent was removed in vacuo. Purification of the crude material by silica gel column chromatography afforded a mixture of diastereoisomers, which were separated by prep. HPLC. Yield: 6 g of crude product.

517A - peak-1 (white solid), 5.13 g (96%). 1H NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS - [M+H] 266.3, [M+NH4+] - 283.5 present, HPLC 97.86%. Stereochemistry was confirmed by X-ray diffraction.

Synthesis 518.

Using a procedure similar to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. ¹ H-NMR (CDCl ₃ , 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br.m, 36H), 0.87 (m, 6H). HPLC - 98.65%.

General procedure for the synthesis of compound 519.

A solution of compound 518 (1 eq.) in hexane (15 ml) was added dropwise to ice-cooled

- 73 044245 LAH solution in THF (1 M, 2 equiv). After complete addition, the mixture was heated at 40°C for 0.5 h, then cooled again in an ice bath. The mixture was carefully hydrolyzed _with saturated aqueous _Na2SO4 , then filtered through Celite and converted into an oil. Column chromatography afforded pure 519 (1.3 g, 68%), which was obtained as a colorless oil. ^13C NMR δ=130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ calcd 654.6, found 654.6.

Formulations prepared using either the standard or non-extrusion methods are characterized in the same manner. For example, formulations are typically characterized by visual inspection. They should be whitish, clear solutions free of aggregates or sediment. Particle size and particle size distribution of lipid nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). Particle size should be approximately 20-300 nm, for example, 40-100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the captured fraction, are determined using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting surfactant, such as 0.5% Triton-X100. Total dsRNA in the formulation can be determined by the signal from the sample containing a surfactant relative to the calibration curve. The captured fraction is determined by subtracting the free dsRNA content (measured from the signal in the absence of surfactant) from the total dsRNA content. The percentage of captured dsRNA is typically >85%. For the SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. A suitable range is typically from about at least 50 nm to about at least 110 nm, from about at least 60 nm to about at least 100 nm, or from about at least 80 nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gelatin capsules, powder sachets for solution, tablets, or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersants, or binders may be necessary. In some embodiments, oral formulations are those in which the dsRNA described herein is administered in combination with one or more penetration enhancers, surfactants, and chelators. Suitable surfactants include fatty acids and/or their esters or salts, bile acids and/or their salts. Suitable bile acids/bile salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucolic acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or a monoglyceride, diglyceride, or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, such as fatty acids/fatty acid salts in combination with bile acids/bile acid salts. One exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The dsRNAs described in the present invention can be delivered orally, in the form of granules, including spray-dried particles, or complexed to form micro- or nanoparticles. Complexing agents for dsRNA include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxethanes, polyalkyl cyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethylene glycols (PEG) and starches; polyalkyl cyanoacrylates; DEAE-derived polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethyl chitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene (PTDAE), polyaminostyrene (e.g., p-amino), poly(methyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), poly(isohexyl cyanoacrylate), DEAE-methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAEdextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), DL-74 044245 copolymer of lactic and glycolic acid (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations for dsRNA and their preparation are described in detail in U.S. Patent No. 6,887,906, U.S. Publication No. 20030027780, and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions that may also contain buffers, diluents and other appropriate additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. Such compositions can be prepared from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. Particularly preferred are formulations that target the liver for the treatment of liver diseases such as hepatocellular carcinoma.

The pharmaceutical compositions of the present invention, which may conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include bringing the active ingredients into contact with the pharmaceutical carrier(s) or excipient(s). Typically, the compositions are prepared by uniformly and intimately bringing the active ingredients into contact with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated in any of a variety of possible dosage forms, such as, but not limited to, tablets, capsules, gelatin capsules, liquid syrups, plastic gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions can additionally contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension can also contain stabilizers.

C. Additional compositions.

i. Emulsions.

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another, in the form of droplets typically larger than 0.1 µm in diameter (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV, Popovich NG, and Ansel HC, 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often two-phase systems containing two immiscible liquid phases intimately mixed and dispersed in one another. Typically, emulsions can be either water-in-oil (w/o) or oil-in-water (o/w) emulsions. When the aqueous phase is finely atomized within the bulk oil phase and dispersed as tiny droplets in it, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when the oil phase is finely atomized within the bulk of the aqueous phase and dispersed as tiny droplets therein, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug, which may be present as a solution either in the aqueous phase, the oil phase, or as a separate phase. Pharmaceutical excipients, such as emulsifiers, stabilizers, colorants, and antioxidants, may be present in emulsions if desired. Pharmaceutical emulsions may also be multiple emulsions consisting of more than two phases, such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often offer advantages not afforded by simple two-component emulsions. Multiple emulsions, in which individual oil droplets of an o/w emulsion contain small water droplets, constitute a w/o/w emulsion. Similarly, a system of oil droplets encapsulated in water droplets stabilized in an oil dispersant phase produces an o/w/o emulsion.

Emulsions are characterized by low or absent thermodynamic stability. Often, the dispersed or discontinuous phase of an emulsion is well dispersed in the continuous or dispersing phase and is maintained in this form by emulsifiers or the viscosity of the composition. Any phase of an emulsion can be semi-solid or solid, as is the case with similar emulsions in ointment bases and creams. Other methods of emulsion stabilization include the use of emulsifiers, which can be included in any phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely divided solids (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich N.G., and Ansel H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surfactants, have found wide application in emulsion formulations and have been reviewed in the literature (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich, N.G., and Ansel, H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and contain a hydrophilic and a hydrophobic moiety. The ratio of the hydrophilic to hydrophobic nature of a surfactant is called the hydrolipid balance (HLB) and is a valuable tool in categorizing and selecting surfactants for formulation. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic, and amphoteric (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich, N.G., and Ansel, H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and gum arabic. Absorption bases, such as anhydrous lanolin and hydrophilic petrolatum, have hydrophilic properties, allowing them to absorb water to form w/o emulsions while maintaining their semi-solid consistency. Finely divided solids have also been used as suitable emulsifiers, particularly in combination with surfactants and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and non-polar solids such as carbon or glyceryl tristearate.

A wide variety of non-emulsifying materials are also included in emulsion formulations and improve the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., gum arabic, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers). They disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the droplets of the dispersed phase and by increasing the viscosity of the continuous phase.

Because emulsions often contain a number of ingredients, such as carbohydrates, proteins, sterols, and phosphatides, which can easily support microbial growth, such formulations often include preservatives. Commonly used preservatives included in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent degradation. Antioxidants used can be free radical scavengers, such as tocopherols, alkyl gallates, butylated hydroxyanisole, and butylated hydroxytoluene; reducing agents, such as ascorbic acid and sodium metabisulfite; and antioxidant synergists, such as citric acid, tartaric acid, and lecithin.

The use of emulsion formulations via dermatological, oral, and parenteral routes and methods for their preparation have been reviewed in the literature (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV, Popovich NG, and Ansel HC, 2004, Lippincott Williams & Wilkins ( ^8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of formulation as well as their effectiveness in

- 76 044245 absorption and bioavailability (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich N.G., and Ansel H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral oil-based laxatives, fat-soluble vitamins, and high-fat nutritional preparations are among the materials commonly administered orally as o/w emulsions.

ii. Microemulsions.

In one embodiment of the present invention, the mRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and an amphiphilic substance that is a single optically isotropic and thermodynamically stable liquid solution (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich, N.G., and Ansel, H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Microemulsions are typically systems prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, typically a medium-chain alcohol, to form a transparent system. Thus, microemulsions have also been described as thermodynamically stable, isotropically pure dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are typically prepared by combining three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is a water-in-oil (w/o) or an oil-in-water (o/w) emulsion depends on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach using a phase diagram has been extensively studied and has provided extensive data on how to formulate microemulsions by those in the field (see, e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L.V., Popovich, N.G., and Ansel, H.C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to traditional emulsions, microemulsions have the advantage of stabilizing water-insoluble drugs within thermodynamically stable droplets that form spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sesquioleate (SO750), decaglycerol decaoleate (DAO750) alone or in combination with secondary surfactants. A secondary surfactant, typically a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase interfacial fluidity by penetrating the surfactant film and, consequently, creating a disordered film due to the void spaces created among the surfactant molecules. However, microemulsions can be prepared without the use of secondary surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be water, an aqueous drug solution, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and ethylene glycol derivatives. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain ( _C8 - _C12 ) mono-, di- and triglycerides, polyoxyethylated fatty acid esters of glyceryl, fatty alcohols, polyglycolized glycerides, saturated polyglycolized _C8 - _C10 glycerides, vegetable oils and silicone oil.

Microemulsions are of particular interest from the standpoint of drug solubility and enhanced drug absorption. Lipid microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see, e.g., U.S. Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions provide the advantages of improved drug solubility,

- 77 044245 shields the drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to a solid dosage form, improved clinical efficacy and reduced toxicity (see, e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions can often form spontaneously when their components are combined at ambient temperature. This can be particularly useful when formulating heat-labile drugs, peptides, or mRNA. Microemulsions have also been effective in the transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of mRNA and nucleic acids from the gastrointestinal tract, as well as improve local cellular uptake of mRNA and nucleic acids.

The microemulsions of the present invention may also contain additional components and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve the properties of the formulation and to enhance the absorption of the mRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five main categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles.

The RNAi agent of the present invention can be incorporated into a particle, such as a microparticle. Microparticles can be produced by spray drying, but can also be produced by other methods, including lyophilization, evaporation, fluidized bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration enhancing agents.

In one embodiment, the present invention utilizes a variety of penetration enhancers to influence the efficient delivery of nucleic acids, particularly mRNA, to animal skin. Most drugs are present in solution in both ionized and non-ionized forms. However, typically, only lipid-soluble or lipophilic drugs readily penetrate cell membranes. It has been found that even non-lipophilic drugs can penetrate cell membranes if the membrane through which the drug penetrates is treated with a penetration enhancer. In addition to facilitating the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five main categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, e.g., Malmsten, M., Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above classes of penetration enhancers is described in more detail below.

Surface-active agents (or surfactants) are chemical entities that, when dissolved in an aqueous solution, reduce the surface tension of the solution, or the interfacial tension between the aqueous solution and another liquid, thereby increasing the absorption of mRNA through the mucosa. In addition to bile salts and fatty acids, penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9 lauryl ether, and polyoxyethylene-20 cetyl ether (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92) and perforated emulsions such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (undecanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, their C1_ ₂₀ alkyl esters (e.g., methyl, isopropyl, and t-butyl) and their mono- and diglycerides (i.e., oleate, laurate, caprate, myistate, palmitate, stearate, linoleate, etc.) (see, for example, Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 133; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

- 78 044245

The physiological role of bile includes facilitating the distribution and absorption of lipids and fat-soluble vitamins (see, e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934–935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Thus, the term bile salt includes any naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glutcholic acid (sodium glucholate), glycocholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (see, for example, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents used in connection with the present invention can be defined as compounds that remove metal ions from solution by forming complexes with them, resulting in increased absorption of mRNA through the mucosa. In relation to their use as penetration enhancers, chelating agents in the present invention have the additional advantage of also acting as DNase inhibitors, since most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovalinate), N-acyl collagen derivatives, laureth-9, and N-aminoacyl beta-diketone derivatives (enamines) (see, e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating, non-surfactant penetration enhancers may be defined as compounds that exhibit little activity as chelating agents or as surfactants but that nevertheless enhance the absorption of mRNA through the gastrointestinal mucosa (see, e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cycloureas, 1-alkyl 1-alkenylazacycloalkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92), and nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance mRNA uptake at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids such as lipofectin (Junichi et al., U.S. Patent No. 5,705,188), cationic glycerol derivatives, and polycationic molecules such as polylysine (Lollo et al., PCT Application WO 97/30731) are also known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, but are not limited to, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; Carlsbad, CA), Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), Freestyle™ MAX (Invitrogen; Carlsbad, CA), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), RNAiMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Optifect™ (Invitrogen; Carlsbad, CA), X-tremeGENE Q2 Transfection Reagent (Roche; Grensacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grensacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grensacherstrasse, Switzerland) or Fugene (Grensacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Promega; Madison, WI), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, MA, USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, CA, USA), PerFectin transfection reagent (Genlan

- 79 044245 tis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER Transfection Reagent (Genlantis; San Diego, CA, USA), GenePORTER 2 Transfection Reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™ Transfection Reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International; Mountain View, CA, USA), SureFECTOR (B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, California, USA).

Other agents that can be used to enhance the penetration of injected nucleic acids include glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azones, and terpenes such as limonene and menthone.

v. Carriers.

Certain compositions of the present invention also contain carrier compounds. As used herein, the terms "carrier compound" or "carrier" may refer to a nucleic acid or analog thereof that is inert (i.e., lacks biological activity per se) but is recognized as a nucleic acid in vivo by processes that reduce the bioavailability of the biologically active nucleic acid, such as by degrading the biologically active nucleic acid or by promoting its removal from the bloodstream. Coadministration of a nucleic acid and a carrier compound, typically in excess of the latter substance, can result in a significant reduction in the amount of nucleic acid processed by the liver, kidneys, or other extravascular depots, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the uptake of partially phosphorothioate dsRNA by liver tissue can be reduced when it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4' isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Fillers.

In contrast to a carrier compound, a pharmaceutical carrier or excipient is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert medium for delivering one or more nucleic acids to an animal. The excipient may be a liquid or a solid and is selected based on the intended route of administration to provide the required volume, consistency, etc., when combined with the nucleic acid and other components of the pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or dibasic calcium phosphate, etc.); lubricants (e.g. magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrating agents (e.g. starch, sodium starch glycolate, etc.); and wetting agents (e.g. sodium lauryl sulfate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not adversely react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of nucleic acids in liquid or solid oil bases. Solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for administration other than parenteral may be used, which do not adversely interact with nucleic acids.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

vii. Other components.

The compositions of the present invention may further comprise other auxiliary components conventionally found in pharmaceutical compositions at levels of use established in the art. Thus, for example, the compositions may comprise an additional

- 80 044245 compatible pharmaceutically active materials, such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials suitable for the physical formulation of the compositions of the present invention in various dosage forms, such as colorants, flavoring agents, preservatives, antioxidants, opacifiers, thickeners and stabilizers. However, such materials, when added, should not excessively interfere with the biological activities of the components of the compositions of the present invention. The compositions may be sterile and, if necessary, may be mixed with auxiliary substances, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, flavoring agents and/or fragrances, etc., which do not interact adversely with the nucleic acid(s) of the composition.

Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension may also contain stabilizers.

In some embodiments, the pharmaceutical compositions described herein comprise (a) one or more mRNA compounds and (b) one or more agents that act by a mechanism other than RNAi and that are useful in the treatment of a hemolytic disorder. Examples of such agents include, but are not limited to, an anti-inflammatory agent, an antisteatosis agent, an antiviral agent, and/or an anti-fibrosis agent. Additionally, other substances commonly used for liver protection, such as silymarin, can also be used in combination with the mRNAs described herein. Other agents useful for treating liver disease include telbivudine, entecavir, and protease inhibitors such as telaprevir and others disclosed, for example, in U.S. Patent Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217 to Tung et al., and in U.S. Patent Application Publication No. 2004/0127488 to Hale et al.

The toxicity and therapeutic efficacy of such compounds can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD ₅₀ (the dose lethal to 50% of the population) and ED ₅₀ (the dose therapeutically effective to 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and can be expressed as the LD ₅₀ /ED ₅₀ ratio. Compounds characterized by a high therapeutic index are preferred.

Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for use in humans. In the present invention, the dose of the compositions described herein is typically within a range of circulating concentrations that includes the ED ₅₀ with little or no toxicity. The dose may vary within this range depending on the dosage form employed and the route of administration employed. For any compound used in the methods described herein, the therapeutically effective dose can be initially established based on the results of cell culture assays. The dose can be formulated for animal models to obtain a range of circulating plasma concentrations of the compound or, if necessary, the polypeptide product of the target sequence (e.g., to achieve a reduced concentration of the polypeptide) that includes the IC ₅₀ (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms), as determined in cell culture. Such information can be used to more accurately determine a suitable dose in humans. Plasma levels can be measured, for example, using high-performance liquid chromatography.

In addition to the administration discussed above, the mRNAs described in the present invention can be administered in combination with other known agents effective in the treatment of pathological processes mediated by C5 expression. In any case, the supervising physician may adjust the amount and timing of mRNA administration based on the results observed using standard efficacy measurement tools known in the art or described herein.

VI. Methods for inhibiting C5 expression.

The present invention provides methods for inhibiting C5 expression in a cell. The methods comprise contacting the cell with an RNAi agent, such as a double-stranded RNAi agent, in an amount effective to inhibit C5 expression in the cell, thereby inhibiting C5 expression in the cell.

Contacting a cell with a double-stranded RNAi agent can be performed in vitro or in vivo. Contacting a cell with an RNAi agent in vivo involves contacting a cell or group of cells of a subject, such as a human subject, with the RNAi agent. Combinations of in vitro and in vivo contacting methods are also possible. Contacting can be direct or indirect, as discussed above. Furthermore, contacting a cell can

- 81 044245 carried out by means of a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, such as a ligand that is

GalNAc ₃ , or any other ligand that directs the RNAi agent to the site of interest, such as the subject's liver.

The term inhibition as used herein is used interchangeably with reduction, silencing, down-regulation and other similar terms and includes any level of inhibition.

The phrase "inhibition of C5 expression" is intended to mean inhibition of the expression of any C5 gene (such as, for example, a mouse C5 gene, a rat C5 gene, a monkey C5 gene, or a human C5 gene), as well as variants or mutants of the C5 gene. Thus, a C5 gene may be a wild-type C5 gene, a mutant C5 gene, or a transgenic C5 gene in the context of cells, a group of cells, or an organism subjected to genetic manipulation.

Inhibition of C5 gene expression includes any level of C5 gene inhibition, such as at least partial suppression of C5 gene expression. C5 gene expression can be assessed based on the level or change in the level of any variable associated with C5 gene expression, such as the C5 mRNA level, the C5 protein level, or, for example, _CH50 activity as a measure of total complement hemolytic activity, _AH50 to measure the alternative complement pathway hemolytic activity, and/or lactate dehydrogenase (LDH) level as a measure of intravascular hemolysis and/or hemoglobin level. C5a, C5b, and soluble C5b-9 complex levels can also be measured to assess C5 expression. This level can be assessed in a single cell or in a group of cells, including, for example, a sample obtained from a subject.

Inhibition can be assessed by measuring the absolute or relative reduction in one or more variables associated with C5 expression compared to a control level. The control level can be any type of control level used in the art, such as a baseline level before drug administration or a level determined in a similar subject, cell, or sample that is untreated or treated with a control (such as a buffer-only control or an inactive agent control).

In some embodiments of the methods of the present invention, C5 gene expression is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

Evidence of inhibition of C5 gene expression may be a reduction in the amount of mRNA expressed by a first cell or first group of cells (such cells may be present, for example, in a sample obtained from a subject) in which the C5 gene is transcribed and which has been treated (for example, by contacting the cell or cells with an RNAi agent of the present invention or by administering an RNAi agent of the present invention to a subject in whose body the cells are or have been) such that the expression of the C5 gene is inhibited compared to a second cell or second group of cells that is substantially identical to the first cell or group of cells but which has not been treated (control cell(s)). In preferred embodiments, inhibition is assessed by expressing the mRNA level in the treated cells as a percentage of the mRNA level in the control cells using the following formula:

(mRNA level at checkpoints) - (mRNA level in treated cells) (mRNA level at checkpoints)

Alternatively, inhibition of C5 gene expression can be assessed by a decrease in a parameter functionally related to C5 gene expression, such as C5 protein expression, hepcidin gene or protein expression, or tissue or serum iron levels. C5 gene silencing can be detected in any cell expressing C5, either constitutively or through genetic engineering, and using any assay known in the art. The liver is the primary site of C5 expression. Other important sites of expression include the kidneys and uterus.

Evidence of inhibition of C5 protein expression can be provided by a decrease in the level of C5 protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample obtained from a subject). As explained above with respect to the assessment of mRNA suppression, inhibition of protein expression levels in a treated cell or group of treated cells can similarly be expressed as a percentage of the protein level in a control cell or group of control cells.

A control cell or group of control cells that can be used to assess inhibition of C5 gene expression includes a cell or group of cells that have not yet been exposed to the RNAi agent of the present invention. For example, a control cell or group of control cells can be obtained from an individual subject (e.g., a human subject or an animal subject) prior to treatment of the subject with the RNAi agent.

The level of C5 mRNA expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of C5 expression in a sample is determined by detecting a transcribed polynucleotide or a portion thereof, such as C5 gene mRNA. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray assays.

In one embodiment, the level of C5 expression is determined using a nucleic acid probe. The term "probe," as used herein, refers to any molecule capable of selectively binding to a specific C5. Probes can be synthesized by a person skilled in the art or obtained from suitable biological preparations. Probes can be specifically designed to contain a label. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

The isolated mRNAs can be used in hybridization- or amplification-based assays, including, but not limited to, Southern or Northern blot assays, polymerase chain reaction (PCR) assays, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to C5 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA through an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, on an Affymetrix gene microarray. One skilled in the art can readily adapt known mRNA detection methods for use in determining C5 mRNA levels.

An alternative method for determining the level of C5 expression in a sample involves a method of nucleic acid amplification and/or reverse transcription (to produce cDNA), e.g., mRNA in the sample, such as by RT-PCR (an experimental embodiment is set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustaining sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription-mediated amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Patent No. 5,854,033), or any other method of amplifying nucleic acid, followed by detection of the amplified molecules using techniques well known to one of skill in the art. Such detection schemes are particularly suitable for detecting nucleic acid molecules when such molecules are present in very low quantities. In particular aspects of the present invention, C5 expression levels are determined using fluorogenic RT-PCR (i.e., the TaqMan™ System).

C5 mRNA expression levels can be monitored using a membrane blot (such as those used in hybridization assays such as Northern, Southern, dot, etc.) or microwells, assay tubes, gels, beads, or fibers (or any solid support containing bound nucleic acid). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining C5 expression levels can also involve the use of nucleic acid probes in solution.

In preferred embodiments, mRNA expression levels are assessed using branched DNA (bDNA) or real-time PCR (qPCR) assays. The use of such methods is described and illustrated in the Examples section of this document.

The expression level of the C5 protein can be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, liquid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays, electrochemiluminescence assays, etc.

The term "sample" as used herein refers to a collection of similar fluids, cells, or tissues isolated from a subject's body, as well as fluids, cells, or tissues present in the subject's body. Examples of biological fluids include blood, serum and serous fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, intraocular fluids, and the like. Tissue samples may include samples from tissues, organs, or localized areas. For example, samples may be obtained from specific organs, parts of organs, or fluids or cells within these organs. In certain embodiments, samples may be obtained from the liver (e.g., the entire liver, or certain segments of the liver, or certain cell types within the liver, such as, for example, hepatocytes). In preferred embodiments, a sample obtained from a subject means blood or plasma obtained from the subject. In further embodiments, a sample obtained from a subject means liver tissue obtained from the subject.

In some embodiments of the methods of the present invention, an RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific location in the subject's body. Inhibition of C5 expression can be assessed by measuring the level or change in the level of C5 mRNA or C5 protein in a sample obtained from fluid or tissue from a specific location in the subject's body. In preferred embodiments, the location is the liver. The location can also be a subsection or subset of cells from any of the aforementioned locations. The location can also include cells that express a specific type of receptor.

The phrase "contacting a cell with an RNAi agent, such as dsRNA," as used herein, includes contacting the cell by any conceivable means. Contacting a cell with an RNAi agent includes contacting the cell with mRNA in vitro or contacting the cell with mRNA in vivo. Contacting can be accomplished directly or indirectly. Thus, for example, the RNAi agent can be physically contacted with the cell by a separate method, or, alternatively, the RNAi agent can be placed in an environment that allows or causes the agent to contact the cell.

In vitro cell contact can be accomplished, for example, by incubating the cell with an RNAi agent. In vivo cell contact can be accomplished, for example, by injecting the RNAi agent into or near the tissue in which the cell resides, or by injecting the RNAi agent into another location, such as the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue in which the cell to be contacted is located. For example, the RNAi agent may contain and/or be linked to a ligand, such as GalNAc ₃ , which directs the RNAi agent to a site of interest, such as the liver. Combinations of in vitro and in vivo contacting methods are also possible. For example, a cell can also be contacted with an RNAi agent in vitro and subsequently transplanted into a subject.

In one embodiment, contacting a cell with mRNA includes introducing or delivering mRNA to the cell, facilitating or effecting uptake or absorption into the cell. Uptake or uptake of mRNA can occur through random diffuse or active cellular processes, or through auxiliary means or devices. Introduction of mRNA to a cell can be in vitro and/or in vivo. For example, for in vivo administration, mRNA can be administered parenterally to a tissue site or administered systemically. In vivo delivery can be accomplished using a beta-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and Publication No. US 2005/0281781, the entire contents of which are incorporated herein by reference. Introduction to a cell in vitro includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below and/or as known in the art.

VII. Methods for treating or preventing disease associated with complement component C5.

The present invention also provides therapeutic and prophylactic methods comprising administering to a subject suffering from a disease associated with complement component C5, such as PNH or aHUS, an mRNA agent, a pharmaceutical composition comprising an mRNA agent, or a vector comprising mRNA, according to the present invention. In some aspects of the present invention, the methods further comprise administering to the subject an additional therapeutic agent, such as an antibody to complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab).

In one aspect, the present invention relates to methods of treating a subject suffering from a disorder in which a reduction in C5 expression would have a beneficial effect, such as a disease associated with the complement component C5, such as PNH or aHUS. The methods of treatment (and uses) of the present invention comprise administering to a subject, such as a human, a therapeutically effective amount of an mRNA agent targeting a C5 gene, or a pharmaceutical composition comprising an mRNA agent targeting a C5 gene, thereby treating the subject suffering from a disease in which a reduction in C5 expression would have a beneficial effect.

In another aspect, the present invention relates to methods of treating a subject suffering from a disorder in which a reduction in C5 expression would have a beneficial effect, such as a disease associated with the complement component C5, such as PNH or aHUS, which comprise administering to a subject, such as a human, a therapeutically effective amount of an mRNA agent targeting a C5 gene, or a pharmaceutical composition comprising an mRNA agent targeting a C5 gene, and an additional therapeutic agent, such as an antibody to the complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab), thereby treating the subject suffering from a disease in which a reduction in C5 expression would have a beneficial effect.

In one aspect, the present invention relates to methods for preventing at least one symptom in a subject suffering from a disorder in which a reduction in C5 expression would have a beneficial effect, such as a disease associated with the complement component C5, such as PNH or aHUS. The methods comprise administering to the subject a prophylactically effective amount of an agent that is an mRNA, such as a dsRNA, or a vector of the present invention, thereby preventing at least one symptom in a subject suffering from a disease in which a reduction in C5 expression would have a beneficial effect. For example, the present invention provides methods for preventing hemolysis in a subject suffering from a disorder in which a reduction in C5 expression would have a beneficial effect, such as a disease associated with the complement component C5, such as PNH or aHUS.

In another aspect, the present invention relates to methods for preventing at least one symptom in a subject suffering from a disorder in which a reduction in C5 expression would have a beneficial effect, such as a disease associated with the complement component C5, such as PNH or aHUS. The methods comprise administering to the subject a prophylactically effective amount of an agent that is an mRNA, such as dsRNA, or a vector of the present invention, and an additional therapeutic agent, such as an antibody to the complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab), thereby preventing at least one symptom in a subject suffering from a disease in which a reduction in C5 expression would have a beneficial effect.

A therapeutically effective amount as used herein is meant to include an amount of an RNAi agent or an antibody to complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab) that, when administered to a subject suffering from a C5-associated disease, is sufficient to effectively treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease).

The therapeutically effective amount may vary depending on the RNAi agent, antibody or antigen-binding fragment thereof, the route of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, types of previous or concomitant treatment, if any, and other individual characteristics of the subject to be treated.

A prophylactically effective amount as used herein is understood to include an amount of an RNAi agent or an antibody to complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab) that, when administered to a subject suffering from a disease associated with complement component C5 but who has not yet (or currently) experienced or does not exhibit symptoms of the disease and/or who is at risk of developing a disease associated with complement component C5, such as a subject suffering from a transplant rejection reaction and/or after a transplant, such as sensitized or allogeneic rejection, a subject with sepsis, and/or a subject with myocardial infarction, is sufficient to prevent or alleviate the disease or one or more symptoms of the disease. Alleviating the disease includes slowing the progression of the disease or reducing the severity of the disease that will develop later. The prophylactically effective amount may vary depending on the agent, which is an mRNA, or an antibody to the complement component C5 or its antigen-binding fragment, the route of administration of the agent, the antibody to the complement component C5 or its antigen-binding fragment, the degree of risk of developing the disease and the history of the disease, age, weight, family history, genetic makeup, types of previous or concomitant treatment, if any, and other individual characteristics of the patient to be treated.

A therapeutically effective amount or a prophylactically effective amount also includes an amount of an RNAi agent or an antibody to complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab) that produces some desired local or systemic effect at an acceptable benefit/risk ratio relative to any treatment. The mRNA agents used in the methods of the present invention can be administered in an amount sufficient to obtain an acceptable benefit/risk ratio relative to such treatment.

In another aspect, the present invention provides the use of a therapeutically effective amount of an mRNA agent of the present invention in the treatment of a subject, such as a subject in whom a reduction and/or inhibition of C5 expression would have a beneficial effect.

In another aspect, the present invention provides the use of a therapeutically effective amount of an mRNA agent of the present invention and an additional therapeutic agent, such as an antibody to complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab), in the treatment of a subject, such as a subject in whom a reduction and/or inhibition of C5 expression would have a beneficial effect.

In another aspect, the present invention relates to the use of an mRNA agent, such as a dsRNA, of the present invention targeting a C5 gene, or a pharmaceutical composition comprising an mRNA agent targeting a C5 gene, in the manufacture of a medicament for the treatment of a subject, such as a subject in whom a reduction and/or inhibition of C5 expression would have a beneficial effect, such as a subject suffering from a disease in which a reduction in C5 expression would have a beneficial effect, such as a disease associated with a complement component C5, such as PNH or aHUS.

In another aspect, the present invention relates to the use of an mRNA agent, e.g., dsRNA, of the present invention targeting a C5 gene, or a pharmaceutical composition comprising an mRNA agent targeting a C5 gene, in the manufacture of a medicament for use in combination with an additional therapeutic agent, such as an antibody to complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab), for the treatment of a subject, e.g., a subject in whom a reduction and/or inhibition of C5 expression would have a beneficial effect, e.g., with a disease associated with complement component C5, e.g., PNH or aHUS.

In another aspect, the present invention relates to the use of an mRNA, such as a dsRNA, of the present invention for preventing at least one symptom in a subject suffering from a disorder in which a reduction and/or inhibition of C5 expression would have a beneficial effect, such as a disease associated with the complement component C5, such as PNH or aHUS.

In another aspect, the present invention relates to the use of an mRNA agent, such as a dsRNA of the present invention, and an additional therapeutic agent, such as an antibody to complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab), for preventing at least one symptom in a subject suffering from a disorder in which a reduction and/or inhibition of C5 expression would have a beneficial effect, such as a disease associated with complement component C5, such as PNH or aHUS.

In another aspect, the present invention relates to the use of an mRNA agent of the present invention in the manufacture of a medicament for the prevention of at least one symptom in a subject suffering from a disorder in which a reduction and/or inhibition of C5 expression would have a beneficial effect, such as a disease associated with the complement component C5, such as PNH or aHUS.

In another aspect, the present invention relates to the use of an mRNA agent of the present invention in the manufacture of a medicament for use in combination with an additional therapeutic agent, such as an antibody to complement component C5 or an antigen-binding fragment thereof (e.g., eculizumab), for preventing at least one symptom in a subject suffering from a disorder in which a reduction and/or inhibition of C5 expression would have a beneficial effect, such as a disease associated with complement component C5, such as PNH or aHUS.

In one embodiment, an mRNA agent targeting C5 is administered to a subject suffering from a disease associated with the complement component C5 such that the levels of C5, for example, in a cell, tissue, blood, urine, or other tissue or fluid of the subject are reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,

60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,

79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98% or at least about 99% or more, and then administering to the subject an additional therapeutic agent (as described below).

An additional therapeutic agent may be an antibody to complement component C5,

- 86 044245 or an antigen-binding fragment or derivative thereof. In one embodiment, the antibody to complement component C5 is eculizumab (SOLIRIS®), or an antigen-binding fragment or derivative thereof. Eculizumab is a humanized monoclonal IgG2/4, kappa light chain antibody that specifically binds to complement component C5 with high affinity and inhibits the cleavage of C5 to C5a and C5b, thereby inhibiting the formation of the final complement complex C5b-9. Eculizumab is described in U.S. Patent No. 6,355,245, the entire disclosure of which is incorporated herein by reference.

The methods of the present invention comprising administering to a subject an mRNA agent of the present invention and eculizumab may further comprise administering to the subject a meningococcal vaccine.

An additional therapeutic agent, such as eculizumab and/or a meningococcal vaccine, may be administered to a subject concurrently with the C5-targeted mRNA agent or at different times.

In addition, an additional therapeutic agent, such as eculizumab, may be administered to a subject in the same formulation as the C5-targeting mRNA agent or in a different formulation than the C5-targeting mRNA agent.

The dosing regimen for eculizumab is described, for example, in the eculizumab (SOLIRIS®) product labeling and in U.S. Patent Application No. 2012/0225056, the entire contents of each of which are incorporated herein by reference. In exemplary methods of the present invention for treating a disease associated with a complement component C5, e.g., PNH or aHUS, an mRNA agent targeting C5 is first administered (e.g., subcutaneously) to a subject such that C5 levels in the subject are reduced (e.g., by at least about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,

42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,

61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,

80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more), and then eculizumab is administered at doses less than those described in the SOLIRIS® product labeling. For example, eculizumab may be administered to a subject once a week at a dose of less than about 600 mg for 4 weeks, followed by a fifth dose about one week later at less than about 900 mg, and then at a dose of less than about 900 mg approximately every two weeks thereafter. Eculizumab may also be administered to the subject once a week at a dose of less than about 900 mg for 4 weeks, followed by a fifth dose about one week later at a dose of less than about 1200 mg, and then at a dose of less than about 1200 mg about every two weeks thereafter. If the subject is under 18 years of age, eculizumab may be administered to the subject once a week at a dose of less than about 900 mg for 4 weeks, followed by a fifth dose one week later at a dose of less than about 1200 mg, and then at a dose of less than about 1200 mg about every two weeks thereafter; or if the subject is under 18 years of age, eculizumab may be administered to the subject once a week at a dose of less than about 600 mg for 2 weeks, followed by a third dose about one week later at a dose of less than about 900 mg, and then at a dose of less than about 900 mg every two weeks thereafter; or if the subject is under 18 years of age, eculizumab may be administered to the subject once a week at a dose of less than about 600 mg for 2 weeks, followed by a third dose about one week later at a dose of less than about 600 mg, and then at a dose of less than about 600 mg about every two weeks thereafter; or if the subject is under 18 years of age, eculizumab may be administered to the subject once a week at a dose of less than about 600 mg for 1 week, followed by a second dose about one week later at a dose of less than about 300 mg, and then at a dose of less than about 300 mg about every two weeks thereafter; or if the subject is under 18 years of age, eculizumab may be administered to the subject once a week at a dose of less than about 300 mg for 1 week, followed by a second dose about one week later at a dose of less than about 300 mg, and then at a dose of less than about 300 mg about every two weeks thereafter. If a subject is undergoing plasmapheresis or plasma exchange, eculizumab may be administered to the subject at a dose of less than approximately 300 mg (e.g., if the last dose of eculizumab was approximately 300 mg) or less than approximately 600 mg (e.g., if the last dose of eculizumab was approximately 600 mg or more). If a subject is undergoing plasma infusion, eculizumab may be administered to the subject at a dose of less than approximately 300 mg (e.g., if the last dose of eculizumab was approximately 300 mg or more). Lower doses of eculizumab may be administered either subcutaneously or intravenously.

In the combination therapy of the present invention comprising eculizumab, eculizumab can be administered to a subject, for example, subcutaneously at a dose of from about 0.01 mg/kg to about 10 mg/kg, or from about 5 mg/kg to about 10 mg/kg, or from about 0.5 mg/kg to about 15 mg/kg. For example, eculizumab can be administered to a subject, for example, subcutaneously at a dose of

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0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, at a dose of 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 10.5 mg/kg, mg/kg, 11.5 mg/kg, 12 mg/kg, 12.5 mg/kg, 13 mg/kg, 13.5 mg/kg, 14 mg/kg, 14.5 mg/kg or 15 mg/kg.

The methods and uses of the present invention comprise administering a composition described herein such that the expression of the C5 target gene is reduced, for example, for a period of about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76 or 80 hours. In one embodiment, the expression of the C5 target gene is reduced over a long period of time, for example, at least about two, three, four, five, six, seven days or more, for example, about one week, two weeks, three weeks or about four weeks or longer.

Administration of dsRNA according to the methods and uses of the present invention can result in a reduction in the severity, signs, symptoms and/or markers of such diseases or disorders in a patient with a disease associated with the complement component C5. Reduction in this context means a statistically significant reduction in such level. The reduction can occur, for example, by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or approximately 100%.

The effectiveness of treating or preventing a disease can be assessed, for example, by determining disease progression, remission, symptom severity, pain relief, quality of life, the dose of medication required to maintain the treatment effect, the level of a disease marker, or any other measurable parameter appropriate for the disease being treated or aimed at preventing. It is well within the ability of one skilled in the art to monitor the effectiveness of treatment or prevention by measuring any of these parameters or any combination of parameters. For example, the effectiveness of treating a hemolytic disorder can be assessed, for example, by periodically monitoring LDH and _CH50 levels. Comparing late readings with initial readings provides the physician with an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor the effectiveness of treatment or prevention by measuring any of these parameters or any combination of parameters. In this regard, administration of mRNA targeting C5 or a pharmaceutical composition thereof effective against a disease associated with complement component C5 demonstrates that administration in a clinically appropriate manner results in a beneficial effect on at least a statistically significant proportion of patients, such as improvement of symptoms, cure, reduction of disease, prolongation of life, improvement of quality of life, or other effect generally recognized as positive by physicians familiar with the treatment of disease associated with complement component C5 and related causes.

A treatment effect or preventative action is evident when a statistically significant improvement in one or more disease markers is observed, or by the absence of worsening or progression of otherwise predictable symptoms. For example, a favorable change in a measurable disease marker of at least 10%, and preferably at least 20%, 30%, 40%, 50%, or more, may be indicative of effective treatment. The efficacy of a given iRNA medicinal product or formulation thereof can also be assessed using an animal model for the given disease, which is known in the art. When using an animal model, treatment efficacy is demonstrated when a statistically significant reduction in a marker or symptom alleviation is observed.

Furthermore, efficacy can be assessed by a reduction in disease severity, as determined by a specialist in the diagnostic field using a clinically accepted disease severity scale, such as the Rheumatoid Arthritis Pain Severity Scale (RASS). Any positive change, such as a reduction in disease severity assessed using an appropriate scale, constitutes adequate treatment with mRNA or an mRNA formulation, as described herein.

The subject may be administered a therapeutic amount of mRNA, such as about 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg, 0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA, 2.8 mg/kg dsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2 mg/kg dsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6 mg/kg dsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0 mg/kg dsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4 mg/kg dsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8 mg/kg dsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2 mg/kg dsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6 mg/kg dsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0 mg/kg dsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4 mg/kg dsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8 mg/kg

- 88 044245 dsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2 mg/kg dsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6 mg/kg dsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0 mg/kg dsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4 mg/kg dsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8 mg/kg dsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2 mg/kg dsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6 mg/kg dsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kg dsRNA, 30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA or approximately 50 mg/kg dsRNA. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

In some embodiments, for example, when a composition of the present invention comprises dsRNA as described herein and a lipid, subjects can be administered a therapeutic amount of mRNA, e.g., from about 0.01 mg/kg to about 5 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.05 mg/kg to about 5 mg/kg, from about 0.05 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 5 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 0.2 mg/kg to about 5 mg/kg, from about 0.2 mg/kg to about 10 mg/kg, from about 0.3 mg/kg to about 5 mg/kg, from about 0.3 mg/kg to about 10 mg/kg, from about 0.4 mg/kg to about 5 mg/kg, from about 0.4 mg/kg to about 10 mg/kg, from about 0.5 mg/kg to about 5 mg/kg, from about 0.5 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1.5 mg/kg to about 5 mg/kg, from about 1.5 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 2.5 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 3 mg/kg to about 5 mg/kg, from about 3 mg/kg to about 10 mg/kg, from about 3.5 mg/kg to about 5 mg/kg, from about 4 mg/kg to about 5 mg/kg, from about 4.5 mg/kg to about 5 mg/kg, from about 4 mg/kg to about 10 mg/kg, from about 4.5 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 5.5 mg/kg to about 10 mg/kg, from about 6 mg/kg to about 10 mg/kg, from about 6.5 mg/kg to about 10 mg/kg, from about 7 mg/kg to about 10 mg/kg, from about 7.5 mg/kg to about 10 mg/kg, from about 8 mg/kg to about 10 mg/kg, from about 8.5 mg/kg to about 10 mg/kg, from about 9 mg/kg to about 10 mg/kg, or from about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

For example, dsRNA may be administered at a dose of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,

1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,

3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3,

6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9,

9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or approximately 10 mg/kg. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

In other embodiments, for example, when a composition of the present invention comprises dsRNA as described herein and N-acetylgalactosamine, a therapeutic amount of mRNA can be administered to the subject, e.g., at a dose of from about 0.1 to about 50 mg/kg, from about 0.25 to about 50 mg/kg, from about 0.5 to about 50 mg/kg, from about 0.75 to about 50 mg/kg, from about 1 to about 50 mg/kg, from about 1.5 to about 50 mg/kg, from about 2 to about 50 mg/kg, from about 2.5 to about 50 mg/kg, from about 3 to about 50 mg/kg, from about 3.5 to about 50 mg/kg, from about 4 to about 50 mg/kg, from about 4.5 to about 50 mg/kg, from about 5 to about 50 mg/kg, from about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, from about 2.5 to about 45 mg/kg, from about 3 to about 45 mg/kg, from about 3.5 to about 45 mg/kg, from about 4 to about 45 mg/kg, from about 4.5 to about 45 mg/kg, from about 5 to about 45 mg/kg, from

- 89 044245 about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, from about 0.5 to about 30 mg/kg, from about 0.75 to about 30 mg/kg, from about 1 to about 30 mg/kg, from about 1.5 to about 30 mg/kg, from about 2 to about 30 mg/kg, from about 2.5 to about 30 mg/kg, from about 3 to about 30 mg/kg, from about 3.5 to about 30 mg/kg, from about 4 to about 30 mg/kg, from about 4.5 to about 30 mg/kg, from about 5 to about 30 mg/kg, from about 7.5 to about 30 mg/kg, from about 10 to about 30 mg/kg, from about 15 to about 30 mg/kg, from about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, from about 25 to about 30 mg/kg, from about 0.1 to about 20 mg/kg, from about 0.25 to about 20 mg/kg, from about 0.5 to about 20 mg/kg, from about 0.75 to about 20 mg/kg, from about 1 to about 20 mg/kg, from about 1.5 to about 20 mg/kg, from about 2 to about 20 mg/kg, from about 2.5 to about 20 mg/kg, from about 3 to about 20 mg/kg, from about 3.5 to about 20 mg/kg, from about 4 to about 20 mg/kg, from about 4.5 to about 20 mg/kg, from about 5 to about 20 mg/kg, from about 7.5 to about 20 mg/kg, from about 10 to about 20 mg/kg, or from about 15 to about 20 mg/kg. In one embodiment, wherein the composition of the present invention comprises dsRNA as described herein and N-acetylgalactosamine, a therapeutic amount of from about 10 to about 30 mg/kg of dsRNA can be administered to the subject. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

For example, subjects can be administered a therapeutic amount of mRNA, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or approximately 50 mg/kg. Values and ranges intermediate to the listed values are also intended to be part of the present invention.

The mRNA may be administered by intravenous infusion over a period of time, such as a 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, or approximately 25-minute period. Administration may be repeated, for example, regularly, such as once a week, once every two weeks (i.e., every two weeks) for one month, two months, three months, four months, or longer. After the initial treatment regimen, treatments may be administered less frequently. For example, after administration once a week or once every two weeks for three months, administration may be repeated once a month for six months, or a year, or longer.

Administration of mRNA can reduce the level of C5, for example, in a cell, tissue, blood, urine, or other compartments of a patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,

33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,

71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least approximately 99% or more.

Before the full dose of mRNA is administered, patients may be given a smaller dose, such as

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5% infusion and monitor them for adverse effects, such as allergic reactions. Alternatively, the patient can be monitored for undesirable immunostimulatory effects, such as increased cytokine levels (e.g., TNF-alpha or INF-alpha).

Due to the inhibitory effect on the expression of C5, the composition of the present invention or a pharmaceutical composition prepared therefrom can improve the quality of life.

The mRNA of the present invention can be administered in naked form or as free mRNA. Naked mRNA is administered in the absence of a pharmaceutical composition. The naked mRNA can be in a suitable buffer solution. The buffer solution can contain acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmolarity of the mRNA buffer solution can be adjusted to suit administration to a subject.

Alternatively, the mRNA of the present invention may be administered as a pharmaceutical composition, such as in liposomal dsRNA.

Subjects who would benefit from reducing and/or inhibiting C5 expression include those suffering from a disease or disorder associated with complement component C5, as described herein. In one embodiment, a subject suffering from a disease associated with complement component C5 suffers from paroxysmal nocturnal hemoglobinuria (PNH). In another embodiment, a subject suffering from a disease associated with complement component C5 suffers from asthma. In another embodiment, a subject suffering from a disease associated with complement component C5 suffers from rheumatoid arthritis. In yet another embodiment, a subject suffering from a disease associated with complement component C5 suffers from systemic lupus erythematosus. In one embodiment, a subject suffering from a disease associated with complement component C5 suffers from glomerulonephritis. In another embodiment, a subject suffering from a disease associated with complement component C5 suffers from psoriasis. In another embodiment, the subject suffering from a disease associated with the complement component C5 suffers from dermatomyositis bullosa pemphigoid. In one embodiment, the subject suffering from a disease associated with the complement component C5 suffers from atypical hemolytic uremic syndrome. In another embodiment, the subject suffering from a disease associated with the complement component C5 suffers from hemolytic uremic syndrome caused by a Shiga-like toxin of E. coli. In another embodiment, the subject suffering from a disease associated with the complement component C5 suffers from myasthenia gravis. In yet another embodiment, the subject suffering from a disease associated with the complement component C5 suffers from neuromyelitis optica. In one embodiment, the subject suffering from a disease associated with the complement component C5 suffers from dense deposit disease. In one embodiment, the subject suffering from a disease associated with complement component C5 suffers from C3 nephropathy.

In another embodiment, a subject suffering from a disease associated with complement component C5 suffers from age-related macular degeneration. In another embodiment, a subject suffering from a disease associated with complement component C5 suffers from cold agglutinin disease. In one embodiment, a subject suffering from a disease associated with complement component C5 suffers from vasculitis associated with antibodies to neutrophil cytoplasm. In another embodiment, a subject suffering from a disease associated with complement component C5 suffers from a transplant rejection reaction caused by humoral and vascular mechanisms. In one embodiment, a subject suffering from a disease associated with complement component C5 suffers from transplant dysfunction. In one embodiment, a subject suffering from a disease associated with complement component C5 suffers from myocardial infarction. In another embodiment, the disease suffered by the subject suffering from a disease associated with complement component C5 is sensitized transplant rejection. In yet another embodiment, the subject suffering from a disease associated with complement component C5 suffers from sepsis.

Treatment of a subject who would benefit from reduction and/or inhibition of C5 gene expression includes therapeutic and prophylactic (e.g., the subject has undergone sensitized (or allogeneic) transplantation) treatment.

The invention further relates to methods and use of an mRNA agent or a pharmaceutical composition thereof (including methods and use of an mRNA agent or a pharmaceutical composition comprising an mRNA agent and an antibody to complement component C5 or an antigen-binding fragment thereof) for treating a subject in whom a reduction and/or inhibition of C5 expression would have a beneficial effect, such as a subject suffering from a disease associated with complement component C5, in combination with other pharmaceuticals and/or other therapeutic methods, such as known pharmaceutical and/or therapeutic methods, such as, for example, those currently used in the treatment of these disorders. For example, in some embodiments, mRNA targeting C5 is administered in combination with, for example, an agent useful in the treatment of a disease associated with complement component C5, as described elsewhere herein.

For example, additional therapeutic agents and therapeutic methods useful for treating a subject who would benefit from a reduction in C5 expression, such as a subject suffering from a disease associated with complement component C5, include plasmapheresis, thrombolytic therapy (e.g., with streptokinase), antiplatelet agents, folic acid, corticosteroids; immunosuppressants; Estrogens, methotrexate, 6-MP, azathioprine, sulfasalazine, mesalazine, olsalazine, chloroquine/hydroxychloroquine, penicillamine, aurothiomalate (intramuscular and oral), azathioprine, colchicine, corticosteroids (oral, inhalation, and local injection), beta-2 adrenergic agonists (salbutamol, terbutaline, salmeterol), xanthines (theophylline, aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium and oxitropium, cyclosporine, FK-506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs such as ibuprofen, corticosteroids such as prednisolone, phosphodiesterase inhibitors, agonists, adenosine, antithrombotic agents, complement inhibitors, adrenergic agents, agents that interfere with the signaling function of proinflammatory cytokines such as TNF-α or IL-1 (e.g., IRAK, NIK, IKK, p38, or MAP kinase inhibitors), GE-1β-converting enzyme inhibitors, TNFa-converting enzyme (TACE) inhibitors, T cell signaling inhibitors such as kinase inhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine, 6-mercantopurines, angiotensin-converting enzyme inhibitors, soluble cytokine receptors and derivatives thereof (e.g., soluble p55 or p75 TNF receptors and p75TNFRIgG derivatives (Enbrel™ and p55TNFRIgG (Lenercept)), sIL-1RI, sIL-1RII, and sIL-6R), anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13, and TGFe), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib, etanercept, inflixi monoclonal antibody, naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam, methylprednisolone acetate, gold, sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene napsylate/APAP, folate, nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium, oxaprozin, oxycodone hydrochloride, hydrocodone bitartrate/APAP, diclofenac sodium/misoprostol, fentanyl, anakinra, Recombinant human immunoglobulin, tramadol hydrochloride, salsalate, sulindac, cyanocobalamin/fa/pyridoxine, acetaminophen, sodium alendronate, prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin, glucosamine sulfate/chondroitin, amitriptyline hydrochloride, sulfadiazine, oxycodone hydrochloride/acetaminophen, olopatadine hydrochloride, misoprostol, naproxen sodium, omeprazole, cyclophosphamide, monoclonal antibody rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18 antibody, anti-IL15 antibody, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740, roflumilast, IC-485, CDC-801, mesopram, cyclosporine, cytokine-suppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNFa antibody; Celltech/Bayer); cA2/inflixi monoclonal antibody (chimeric anti-TNFa antibody; Centocor); 75 kdTNFRIgG/etanercept (75 kDa TNF receptor-IgG fusion protein; Immunex, see, e.g., (1994) Arthr. Rheum. 37: S295; (1996) J. Invest. Med. 44: 235A); 55 kDa TNF-IgG (55 kDa TNF receptor-IgG fusion protein Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody; IDEC/SmithKline; see, e.g., (1995) Arthr. Rheum. 38: S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; see, e.g., (1993) Arthrit. Rheum. 36: 1223); anti-Tac antibody (humanized anti-IL-2Ra antibody; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen); anakinra (Kineret®/Amgen); TNF-bp/s-TNF (soluble TNF-binding protein; see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement)): S284; (1995) Amer. J. Physiol. - Heart and Circ. Physiol. 268: 37–42); R973401 (phosphodiesterase type IV inhibitor; see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S282); MK-966 (COX-2 inhibitor; see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S81); iloprost (see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S82); methotrexate; thalidomide (see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S282) and thalidomide-related drugs (e.g., Celgen); leflunomide (anti-inflammatory and cytokine inhibitor, see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S131; (1996) Inflamm. Res. 45: 103–107); tranexamic acid (plasminogen activation inhibitor, see e.g. (1996) Arthr. Rheum. 39 (9 (supplement): S284); T-614 (cytokine inhibitor; see e.g. (1996) Arthr. Rheum. 39 (9 (supplement): S282); prostaglandin E1 (see e.g. (1996) Arthr. Rheum. 39 (9 (supplement): S282); tenidap (non-steroidal anti-inflammatory drug; see e.g. (1996) Arthr. Rheum. 39 (9 (supplement): S280); naproxen (non-steroidal anti-inflammatory drug; see e.g. (1996) Neuro. Report 7: 1209–1213); meloxicam (non-steroidal anti-inflammatory drug); ibuprofen (non-steroidal anti-inflammatory drug); piroxicam (non-steroidal anti-inflammatory drug); diclofenac (non-steroidal anti-inflammatory drug); indomethacin (non-steroidal anti-inflammatory drug); sulfasalazine (see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S281); azathioprine (see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S281); ICE inhibitor (interleukin-1a converting enzyme inhibitor); zap-70 and/or lck inhibitor (zap-70 or lck tyrosine kinase inhibitor); VEGF inhibitor and/or VEGF-R inhibitor (cell growth factor inhibitors vascular endothelial cell growth factor receptor or vascular endothelial cell growth factor receptor; angiogenesis inhibitors); corticosteroid anti-inflammatory drugs (e.g. SB203580); TNF convertase inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies; interleukin-11 (see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S296); interleukin-13 (see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S308); interleukin-17 inhibitors (see, e.g., (1996) Arthr. Rheum. 39 (9 (supplement): S120); gold; penicillamine; chloroquine; chlorambucil; hydroxychloroquine; cyclosporine; cyclophosphamide; whole body irradiation lymphoid tissue; anti-T-cell antibody globulin; anti-CD4 antibodies; CD5 toxins; orally administered peptides and collagen; disodium lobenzarit; cytokine regulatory agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.); prednisolone; orgotein; glycosaminoglycan polysulfate; minocycline; anti-IL2R antibodies, lipids of marine and botanical origin (fish and seed fatty acids, see, e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21: 759–777); auranofin; phenylbutazone; meclofenamic acid; flufenamic acid; intravenous immunoglobulin; zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amipriloz (terafectin); cladribine (2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see Bruncko, M. et al. (2007) J. Med. Chem. 50(4): 641-662); antivirals and immunomodulators, small molecule KDR inhibitor, small molecule Tie-2 inhibitor; methotrexate; prednisolone; celecoxib; folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept; monoclonal antibody inflix; leflunomide; naproxen; valdecoxib; sulfasalazine; methylprednisolone; ibuprofen; meloxicam; methylprednisolone acetate; gold sodium thiomalate; aspirin; azathioprine; triamcinolone acetonide; propxifene napsylate/apap; folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium; oxaprozin; oxycodone hydrochloride; Hydrocodone bitartrate/APAP; Diclofenac sodium/misoprostol; Fentanyl; Anakinra, recombinant human globulin; Tramadol hydrochloride; Salsalate; Sulindac; Cyanocobalamin/FA/pyridoxine; Acetaminophen; Alendronate sodium; Prednisolone; Morphine sulfate; Lidocaine hydrochloride; Indomethacin; Glucosamine sulfate/chondroitin; Cyclosporine; Amitriptyline hydrochloride; Sulfadiazine; Oxycodone hydrochloride/acetaminophen; Olopatadine hydrochloride; Misoprostol; Naproxen sodium; Omeprazole; Mycophenolate mofetil; Cyclophosphamide; Rituxin monoclonal antibody; IL-1 TRAP; MRA; CTLA4-IG; IL-18 BP; IL-12/23; Anti-IL 18 antibody; Anti-IL 15 antibody; BIRB-796; SCIO469; VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801; Mesopram, albuterol, salmeterol/fluticasone, montelukast sodium, fluticasone propionate, budesonide, prednisone, salmeterol xinafoate, levalbuterol hydrochloride, albuterol sulfate/ipratropium, prednisolone sodium phosphate, triamcinolone acetonide, beclomethasone dipropionate, ipratropium bromide, azithromycin, pirbuterol acetate, prednisolone, theophylline anhydrous, sodium succinate methylprednisolone, clerythromycin, zafirlukast, formoterol fumarate, influenza virus vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide, allergy injection, cromolyn sodium, fexofenadine hydrochloride, flunisolide/menthol, Amoxicillin/Clavulanate, Levofloxacin, Inhalation Aid Device, Guaifenesin, Dexamethasone Sodium Phosphate, Moxifloxacin Hydrochloride, Doxycycline Hyclate, Guaifenesin/D-Methorphan, R-Ephedrine/Codeine/Chlorphenir, Gatifloxacin, Cetirizine Hydrochloride, Mometasone Furoate, Salmeterol Xinafoate, Benzonatate, Cephalexin, Phenylephrine/Hydrocodone/Chlorpheniramine, Cetirizine Hydrochloride/Pseudoephedrine, Phenylephrine/Codeine/Promethazine, Codeine/Promethazine, Cefprozil, Dexamethasone, Guaifenesin/Pseudoephedrine, Chlorpheniramine/Hydrocodone, Nedocromil Sodium, Terbutaline Sulfate, Epinephrine, methylprednisolone, metaproterenol sulfate, aspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine sulfate, metoprolol succinate, warfarin sodium, lisinopril, isosorbide mononitrate, digoxin, quinapril hydrochloride/mag carb, bumetanide, alteplase, enalaprilat, amiodarone hydrochloride, tirofiban hydrochloride m-hydrate, diltiazem hydrochloride, captopril, irbesartan, valsartan, propranolol hydrochloride furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate, torsemide, retavaz, losartan potassium, fosinopril sodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium, atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalol hydrochloride, potassium chloride, docusate sodium, dobutamine hydrochloride, alprazolam, pravastatin sodium, atorvastatin calcium, midazolam hydrochloride, hydromorphone hydrochloride, isosorbide dinitrate, epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe, and cariporide.

The mRNA agent (and/or antibody to complement component C5) and the additional therapeutic agent and/or treatment may be administered simultaneously and/or in a single combination, such as parenterally, or the additional therapeutic agent may be administered as part of a separate formulation, or at different times, and/or in another manner known in the art or described herein.

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The present invention also relates to methods of using an mRNA agent of the present invention and/or a composition comprising an mRNA agent of the present invention to reduce and/or inhibit the expression of complement component C5 in a cell. In other aspects, the present invention relates to an mRNA of the present invention and/or a composition comprising an mRNA of the present invention for use in reducing and/or inhibiting the expression of complement component C5 in a cell. In other aspects, the use of an mRNA of the present invention and/or a composition comprising an mRNA of the present invention in the manufacture of a medicament for reducing and/or inhibiting the expression of complement component C5 in a cell is provided.

The methods and uses include contacting a cell with an mRNA, such as a dsRNA, of the present invention and maintaining the cell for a time sufficient to ensure cleavage of the mRNA transcript of the C5 gene, thereby inhibiting expression of the C5 gene in the cell.

A decrease in gene expression can be assessed by any of the methods known in the art. For example, a decrease in C5 expression can be determined by determining the level of C5 mRNA expression using conventional methods known to those of ordinary skill in the art, such as Northern blotting, qRT-PCR, by determining the level of C5 protein using conventional methods known to those of ordinary skill in the art, such as Western blotting, immunological methods, flow cytometry, ELISA, and/or by determining the biological activity of C5, such as in the _CH50 or _AH50 hemolysis reaction, and/or by determining the biological activity of one or more molecules associated with the complement system, such as C5 products such as C5a and C5b (or in vivo, such as under hemolytic conditions).

In the methods and uses of the present invention, the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject. In embodiments of the present invention where the cell is within a subject, the methods may further comprise contacting the cell with an antibody to complement component C5, such as eculizumab.

A cell suitable for treatment using the methods of the present invention can be any cell expressing the C5 gene. A cell suitable for treatment using the methods of the present invention can be a mammalian cell, such as a primate cell (e.g., a human cell or a non-human primate cell, such as a monkey cell or a chimpanzee cell), a non-primate cell (e.g., a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster cell, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), an avian cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, such as a human liver cell.

C5 expression can be inhibited in a cell by at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,

31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,

50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,

69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or approximately 100%.

The in vivo methods and uses of the present invention may comprise administering to a subject a composition comprising mRNA, wherein the mRNA comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of the C5 gene of a mammal to be treated. When the organism to be treated is a mammal, such as a human, the composition may be administered by any route known in the art, including, but not limited to, subcutaneous, intravenous, oral, intraperitoneal, or parenteral administration, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, respiratory (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In some embodiments, the compositions are administered by subcutaneous or intravenous infusions or injections.

In some embodiments, administration is by injection of a depot agent. Depot injection can release mRNA sustainably over an extended period of time. Thus, depot injection can reduce the frequency of dosing required to achieve the desired effect, such as desired C5 inhibition, or a therapeutic or prophylactic effect. Depot injection can also provide more stable serum concentrations. Depot injections can include subcutaneous or intramuscular injections. In preferred embodiments, the depot injection is subcutaneous.

In some embodiments, administration is accomplished via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments

- 94 044245 In some embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. The infusion pump can be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implantable pump that delivers mRNA to the liver.

The route of administration can be selected based on whether local or systemic treatment is required and the area to be treated. The route and location of administration can be tailored to maximize targeted action.

In one aspect, the present invention also provides methods for inhibiting the expression of the C5 gene in a human. The present invention also relates to a composition comprising mRNA, for example, dsRNA, targeting the C5 gene in a mammalian cell for use in inhibiting the expression of the C5 gene in a mammal. In another aspect, the present invention relates to the use of mRNA, for example, dsRNA, targeting the C5 gene in a mammalian cell, in the manufacture of a medicament for inhibiting the expression of the C5 gene in a mammal.

Methods and uses include administering to a mammal, such as a human, a composition comprising mRNA, such as dsRNA, targeting the C5 gene in a mammalian cell and maintaining the mammal for a time sufficient to ensure cleavage of the mRNA transcript of the C5 gene, thereby inhibiting expression of the C5 gene in the mammal. In some embodiments, the methods further comprise administering to the subject an antibody to the complement component C5, such as eculizumab.

A decrease in gene expression can be assessed by any method known in the art, such as qRT-PCR described herein. A decrease in protein synthesis can be assessed by any method known in the art, such as ELISA or Western blotting described herein. In one embodiment, a liver biopsy serves as a tissue sample for monitoring a decrease in C5 gene and/or protein expression. In another embodiment, a blood sample serves as a tissue sample for monitoring a decrease in C5 gene and/or protein expression. In other embodiments, inhibition of C5 gene expression is monitored indirectly, such as by determining the expression and/or activity of a gene in the C5 biosynthetic pathway, including, for example, C5a, C5b, and soluble C5b-9 (see, for example, Fig. 1). For example, CD59 activity can be monitored to determine inhibition of C5 gene expression. It is also possible to measure _CH50 , _AH50 , thrombus formation, and/or lactate dehydrogenase (LDH) in a serum sample, such as blood or liver. Suitable assays are further described in the examples section below.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the mRNA and methods described in this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other literature references cited herein are incorporated by reference in their entirety. In case of conflict, the present description, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Examples

Example 1. Synthesis of mRNA.

Origin of reagents.

Unless the origin of a reagent is specifically stated in this document, such reagent may be obtained from any supplier of molecular biology reagents of standard quality/purity for molecular biology use.

Transcripts.

SiRNA construction was performed to identify siRNA targeting C5 transcripts from human, rhesus macaque (Macaca mulatta), mouse, and rat annotated in the NCBI gene database (http://www.ncbi.nlm.nih.gov/gene/). The following transcripts from the NCBI RefSeq collection were used in the construction: human - NM_001735.2; rhesus macaque - XM_001095750.2; mouse - NM_010406.2; rat - XM_345342.4. SiRNA duplexes were constructed in several separate lots, including, but not limited to, lots containing duplexes corresponding to human and rhesus macaque transcripts only; human, rhesus macaque, and mouse transcripts only; human, rhesus macaque, mouse, and rat transcripts only; and mouse and rat transcripts only. All siRNA duplexes were designed to achieve 100% identity to the specified human transcript and transcripts from other species considered in each construction lot (above).

siRNA design, specificity and efficacy prediction.

The predicted specificities of all possible 19mers were predicted from each sequence. Then, candidate 19mers were selected that did not contain repeats longer than 7 nucleotides. These 2971 can

- 95 044245 human/rhesus macaque, 142 human/rhesus macaque/mouse, 54 human/rhesus macaque/mouse/rat, and 807 mouse/rat siRNAs were applied in large-scale analyses to the corresponding transcriptomes (defined as the set of NM_ and XM_ entries within the NCBI RefSeq sets of human, rhesus macaque, dog, mouse, or rat) using an exhaustive brute-force algorithm implemented in the BruteForce.ru python script. The script was then parsed into transcript-oligo alignments to create a score based on the position and number of mismatches between the siRNA and any potential 'off-target' transcript. The off-target score was weighted to emphasize differences in the original region of siRNAs, at positions 2-9 from the 5' end of the molecule.

Each oligo-transcript pair from the exhaustive search was assigned a mismatch score by summing the individual mismatch scores; mismatches at positions 2-9 were scored as 2.8, mismatches at cleavage site positions 10-11 were scored as 1.2, and mismatches in the 12-19 region were scored as 1.0. Additional off-target prediction was performed by comparing the frequency of heptamers and octomers generated from three different derivatives of the parent hexamers of each oligonucleotide. Hexamers from positions 2-7 relative to the 5' start were used to generate two heptamers and one octamer. Heptamer 1 was generated by adding a 3'-A to the hexamer; heptamer 2 was generated by adding a 5'-A to the hexamer; and an octomer was generated by adding an A to both the 5' and 3' ends of the hexamer. The frequencies of octamers and heptamers in the 3'-UTRome of humans, rhesus macaques, mice, or rats (defined as a transcriptome subsequence from the NCBI RefSeq database where the end of the coding region, CDS, is clearly defined) were preliminarily calculated. Octamer frequencies were matched to heptamer frequencies using the average value from the octamer frequency range. The mirSeedScore was then calculated by calculating the sum of (3 x matched octamer count) (2 x heptamer count 2) (1 x heptamer count 1)).

Both siRNA strands were assigned to specificity categories based on calculated scores: a score greater than 3 is considered highly specific, a score of 3 is considered specific, and a score between 2.2 and 2.8 is considered moderately specific. Duplexes were sorted by antisense strand specificity, and those whose antisense oligonucleotides did not contain a GC at position 1, did not contain a G at both positions 13 and 14, and contained three or more U or A residues in the starting region were selected.

For GalNaC-conjugated duplexes, sense 21mer and antisense 23mer oligonucleotides were constructed by extending the antisense 19mers (described above) to 23 nucleotides in the target complementary sequence. All transcript species included in the batch construction were checked for complementarity. Only 23mers that retained 100% sequence complementarity in at least two species were used. For each duplex, the sense 21mer was determined as the reverse complementarity of the first 21 nucleotides of the antisense strand.

Selection of siRNA sequence.

A total of 23 human/rhesus macaque-derived sense and 23 antisense, 6 human/rhesus macaque-derived sense and 6 antisense, and 13 mouse/rat-derived sense and 13 mouse/rat-derived antisense 19mer siRNA oligomers were synthesized and formed into duplexes.

The above 19mer sets were expanded to 21/23mer duplexes for the GalNac-conjugated construct and reclassified according to their new species assignments. Twenty-seven human/rhesus macaque-derived sense and 27 antisense, 1 human/rhesus macaque/mouse-derived sense and 1 antisense, 3 human/rhesus macaque/rat-derived sense and 3 antisense, 4 human/rhesus macaque/mouse/rat-derived sense and 4 antisense, and 13 mouse/rat-derived sense and 13 antisense 21mer (sense) and 23mer (antisense) oligonucleotides were synthesized and formed into duplexes.

A detailed list of the sequences of the sense and antisense strands of C5 are given in Tables 3–6.

Synthesis of siRNA.

General methods for the synthesis of small and medium-sized RNA.

RNA oligonucleotides were synthesized at scales between 0.2-500 μmol using commercially available 5'-O-(4,4'-dimethoxytrityl)-2'-O-t-butyldimethylsilyl-3'-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite monomers of uridine, 4-N-acetylcytidine, 6-N-benzoyladenosine, and 2-N-isobutyrguanosine and the corresponding 2'-O-methyl and 2'-fluorophosphoramidites, according to standard solid-phase oligonucleotide synthesis protocols. Amidite solutions were prepared at concentrations of 0.1-0.15 M, and 5-ethylthio-1H-tetrazole (0.25-0.6 M in acetonitrile) was used as an activator. During the synthesis, phosphorothioate modifications of the backbone were introduced using 0.2 M phenylacetyl disulfide (PADS) in lutidine:acetonitrile (1:1) (v:v) or 0.1 M 3-(dimethylaminomethylene)amino-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine for the oxidation step. After completion of the synthesis, the sequences were cleaved from the solid support and deprotected using methylamine followed by trimethylamine 3HF to remove the present 2'-O-t-butyldimethylsilyl protecting groups.

For intersequence synthesis scales (5-500 µmol and fully 2'-modified with 2'-fluoro and/or 2'-O-methyl or combinations thereof), oligonucleotides were deprotected using 3:1 (v/v) ethanol and concentrated (28-32%) aqueous ammonia or

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35°C for 16 h, or at 55°C for 5.5 h. Before removing the ammonia protecting group, the oligonucleotides were treated with 0.5 M piperidine in acetonitrile for 20 min on a solid support. Crude oligonucleotides were analyzed by LC-MS and anion exchange HPLC (IEX-HPLC). Oligonucleotide purification was performed by IEX HPLC using: 20 mM phosphate, 10%-15% ACN, pH 8.5 (buffer A) and 20 mM phosphate, 10%-15% ACN, 1 M NaBr, pH 8.5 (buffer B). Fractions were analyzed for purity by analytical HPLC. Product-containing fractions with acceptable purity were pooled and concentrated on a rotary evaporator prior to desalting. Samples were desalted using size-exclusion chromatography and lyophilized to dryness. Equal molar quantities of sense and antisense strands were annealed in 1x PBS buffer to generate the corresponding siRNA duplexes.

For small scale (0.2-1 μmol), synthesis was performed on a MerMade 192 synthesizer in a 96-well format. For fully 2'-modified sequences (2'-fluoro and/or 2'-O-methyl or combinations thereof), oligonucleotides were deprotected using methylamine at room temperature for 30-60 min followed by incubation at 60°C for 30 min or using 3:1 (v/v) ethanol and concentrated (28-32%) aqueous ammonia at room temperature for 30-60 min followed by incubation at 40°C for 1.5 h. Crude oligonucleotides were then precipitated in acetonitrile:acetone (9:1) and isolated by centrifugation and decantation of the supernatant. The crude oligonucleotide pellet was resuspended in 20 mM NaOAc buffer and analyzed by LC-MS and anion-exchange HPLC. Crude oligonucleotide sequences were desalted in deep 96-well plates using a 5 ml HiTrap Sephadex G25 column (GE Healthcare). A 1.5 ml sample corresponding to an individual sequence was collected from each well. These purified, desalted oligonucleotides were analyzed by LC-MS and anion-exchange chromatography. Duplexes were generated by annealing equimolar amounts of sense and antisense sequences on a Tecan robot. The duplex concentration was adjusted to 10 μM in 1x PBS buffer.

Synthesis of GalNAc-conjugated oligonucleotides for in vivo analysis.

Oligonucleotides conjugated with a GalNAc ligand at their 3' end were synthesized at scales between 0.2-500 μmol using a solid support preloaded with a Y-shaped linker bearing a 4,4-dimethoxytrityl (DMT)-protecting primary hydroxy group for oligonucleotide synthesis and a GalNAc ligand attached via a moiety.

For the synthesis of GalNAc conjugates at scales between 5-500 μmol, the above RNA synthesis protocol was supplemented with the following modifications: For polystyrene-based synthesis supports, 5% dichloroacetic acid in toluene was used for DMT cleavage during synthesis. Support cleavage and deprotection were performed as described above. Phosphothioate-rich sequences (typically >5 phosphorothioates) were synthesized without removal of the terminal 5'-DMT group (with DMT) and, after cleavage and deprotection as described above, were purified by reverse-phase HPLC using 50 mM ammonium acetate in water (buffer A) and 50 mM ammonium acetate in 80% acetonitrile (buffer B). Fractions were analyzed for purity using analytical HPLC and/or LC-MS. Product-containing fractions of acceptable purity were pooled and concentrated on a rotary evaporator. The DMT group was removed using 20-25% acetic acid in water until completion. Samples were desalted using size-exclusion chromatography and lyophilized to dryness. Equal molar amounts of sense and antisense strands were annealed in 1x PBS buffer to yield the corresponding siRNA duplexes.

For small-scale synthesis of GalNAc conjugates (0.2-1 μmol), including sequences with multiple phosphorothioate linkages, the protocols described above for the synthesis of RNA or fully 2'-P/2'-OMe-containing sequences on the MerMade platform were used. Synthesis was performed on prepacked columns containing a GalNAc-functionalized, steerable glass-pore support.

Example 2. In vitro screening.

Cell culture and transfections.

Hep3B cells (ATCC, Manassas, VA) were grown to near confluence at 37°C under 5% _CO2 in Eagle's Minimum Essential Medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before detachment from the dish by trypsinization. Cells were washed and resuspended to 0.25 x 106 cells/mL. For transfections, cells were seeded in 96-well plates at approximately 20,000 cells per well.

Primary mouse hepatocytes (PMH) were isolated immediately prior to transfection from female C57BL/6 mice (Charles River Laboratories International, Inc., Willmington, MA), less than 1 h before transfections, and grown in primary hepatocyte medium. Cells were resuspended at 0.11 x 106 cells/mL in InVitroGRO CP Rat (coated) medium (Celsis In Vitro Technologies, catalog # S01494). During transfections, cells were seeded on BD Biocoat 96-well collagen plates (BD, 356407), 10,000 cells per well, and incubated at 37°C under 5% _CO2 .

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Cryopreserved primary cynomolgus macaque hepatocytes (Celsis In Vitro Technologies,

M003055-P) were thawed at 37°C in a water bath immediately before use and resuspended at 0.26 x 106 cells/mL in InVitroGRO CP (coated) medium (Celsis In Vitro Technologies, catalog # Z99029). For transfections, cells were seeded on a BD Biocoat 96-well collagen plate (BD, 356407), 25,000 cells per well, and incubated at 37°C under 5% _CO2 .

For Hep3B, RMN, and primary cynomolgus hepatocytes, transfection was performed by adding 14.8 μl of Opti-MEM with 0.2 μl of RNAiMax Lipofectamine per well (Invitrogen, Carlsbad, California, catalog #13778-150) to 5 μl of each siRNA duplex in a separate well in a 96-well plate. The mixture was then incubated at room temperature for 20 min. Eighty μl of complete culture media without antibiotics containing the appropriate number of cells was then added to the siRNA mixture. Cells were incubated for 24 h before RNA purification.

Single dose experiments were performed at a final duplex concentration of 10 nM and 0.1 nM for GalNAc-modified sequences or at a final duplex concentration of 1 nM and 0.01 nM for all other sequences. Dose-response experiments were performed at 3, 1, 0.3, 0.1, 0.037, 0.0123, 0.00412, and 0.00137 nM final duplex concentrations for primary mouse hepatocytes and at 3, 1, 0.3, 0.1, 0.037, 0.0123, 0.00412, 0.00137, 0.00046, 0.00015, 0.00005, and 0.000017 nM final duplex concentrations for Hep3B cells.

Transfection by free uptake.

Free uptake experiments were performed by adding 10 μl of siRNA duplexes in PBS per well in a 96-well plate. 90 μl of complete growth medium containing the appropriate number of cells for the cell type was then added to the siRNA. Cells were incubated for 24 h before RNA purification. Single dose experiments were performed at final duplex concentrations of 500 nM and 5 nM, and dose-response experiments were performed at final duplex concentrations of 1000, 333, 111, 37, 12.3, 4.12, 1.37, and 0.46 nM.

Total RNA was isolated using the DYNABEADS mRNA Isolation Kit (Invitrogen, catalog number 610-12).

Cells were collected and lysed in 150 μl of lysis/binding buffer, then mixed for 5 min at 850 rpm using an Eppendorf Thermomixer (mixing speed was constant throughout). 10 μl of magnetic beads and 80 μl of lysis/binding buffer mixture were added to a round-bottomed plate and mixed for 1 min. The magnetic beads were fixed using a magnetic stand, and the supernatant was removed without dislodging the beads. After removing the supernatant, lysed cells were added to the remaining beads and mixed for 5 min. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 min. The beads were fixed, and the supernatant was removed. The beads were then washed with 150 μl of wash buffer B, fixed, and the supernatant was removed. The beads were then washed with 150 µl of wash buffer B, fixed, and the supernatant removed. Finally, the beads were allowed to dry for 2 min. After drying, 50 µl of elution buffer was added and mixed for 5 min at 70°C. The beads were fixed on a magnet for 5 min. 45 µl of supernatant was removed and added to another 96-well plate.

cDNA synthesis using the ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Forster City, CA, Cat. #4368813)

A master mix was prepared with 2 μl of 10X buffer, 0.8 μl of 25X dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNase inhibitor, and 3.2 μl _{of H2O} per reaction. Equal volumes of master mix and RNA were mixed to a final volume of 12 μl for in vitro screening or 20 μl for in vivo screening of samples. cDNA was prepared using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, California) using the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 s, and storage at 4°C.

Real-time PCR.

µl cDNA was added to a master mix containing 2 µl _H2O , 0.5 µl TaqMan probe for GAPDH (Life Technologies, catalog #4326317E for Hep3B cells, catalog #352339E for primary mouse hepatocytes, or regular probe for primary cynomolgus macaque hepatocytes), 0.5 µl TaqMan probe for C5 (Life Technologies, catalog #Hs00156197_m1 for Hep3B cells or mm00439275_m1 for primary mouse hepatocytes, or regular probe for primary cynomolgus macaque hepatocytes), and 5 µl LightCycler 480 master mix probe (Roche, catalog #04887301001) per well in 384-well plates. plates (Roche, catalog number 04887301001). Real-time PCR was performed on the Roche LC480 Real Time PCR system (Roche) using the AACt(RQ) assay. For in vitro screening, each duplex was tested in two biological replicates, unless otherwise stated, and real-time PCR was performed in identical technical replicates for each run. For in vitro screening, each duplex was tested in one or more experiments (3 mice per group), and real-time PCR was performed in identical technical replicates for each run.

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To calculate the relative fold change in C5 mRNA levels, real-time data were analyzed using the ΔΔCt method and normalized to data from assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells. IC ₅₀ was calculated using a 4-parameter fit model using XLFit and normalized to that of cells transfected with AD-1955 over the same dose range or to its lowest dose.

The sense and antisense sequences of AD-1955 are: SENSE: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 13);

ANTISENSE: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 14).

Table 7 shows the results of a single-dose assay in Hep3B cells transfected with the indicated GalNAC-conjugated modified mRNAs. Data are expressed as a percentage of the remaining transcript relative to untreated cells.

Table 8 shows the results of a single-dose transfection assay in primary mouse hepatocytes transfected with the indicated GalNAC-conjugated modified mRNAs. Data are expressed as the percentage of remaining transcript relative to untreated cells.

Table 9 shows the results of a single-dose free uptake assay in primary cynomolgus macaque hepatocytes for the indicated GalNAC-conjugated modified mRNAs. Data are expressed as the percentage of remaining transcript relative to untreated cells.

Table 10 shows the results of a single-dose free uptake assay in primary mouse hepatocytes with the indicated GalNAC-conjugated modified mRNAs. Data are expressed as the percentage of remaining transcript relative to untreated cells.

Table 11 shows the dose-response relationship in a free uptake assay in primary cynomolgus macaque hepatocytes with the indicated GalNAC-conjugated modified mRNAs. The IC ₅₀ values shown represent the IC ₅₀ values relative to untreated cells.

Table 12 shows the dose-response relationship in the free uptake assay in primary mouse hepatocytes with the indicated GalNAC-conjugated modified mRNAs. The IC ₅₀ values shown represent the IC ₅₀ values relative to untreated cells.

Table 13 shows the results of a single-dose assay in Hep3B cells transfected with the indicated modified and unmodified mRNAs. Data are expressed as the percentage of the remaining transcript relative to untreated cells. A single transfection was performed at 0.01 nM, and a repeat transfection at 1 nM was performed.

Table 14 shows the results of a single-dose assay in primary mouse hepatocytes transfected with the indicated modified and unmodified mRNAs. Data are expressed as the percentage of remaining transcript relative to untreated cells.

Table 15 shows the dose-response relationship in Hep3B cells transfected with the indicated modified and unmodified mRNAs. The IC ₅₀ values shown represent the IC ₅₀ values relative to untreated cells.

Table 16 shows the dose-response relationship in primary mouse hepatocytes transfected with the indicated modified and unmodified mRNAs. The IC ₅₀ values shown represent the IC ₅₀ values relative to untreated cells.

Table 2

Abbreviations for nucleotide monomers used in representing nucleic acid sequences. It should be understood that these monomers, if present in an oligonucleotide, are linked to each other by 5'-3' phosphodiester bonds.

Reduction Nucleotide(s) A Adenosine 3'-phosphate Af 2'-fluoroadenosine-3'-phosphate Af s 2'-fluoroadenosine-3'-phosphothioate As adenosine 3'-phosphothioate WITH cytidine 3'-phosphate Cf 2'-fluorocytidine-3'-phosphate

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CFS 2'-fluorocytidine-3 ^t -phosphothioate Cs cytidine-3'-phosphothioate G guanosine-3- ^t -phosphate Gf 2'-fluoroguanosine-3'-phosphate Gf s 2'-fluoroguanosine-3'-phosphothioate Gs guanosine-3 ^t -phosphothioate T 5'-methyluridine-3 ^t -phosphate Tf 2'-fluoro-5-methyluridine-3'-phosphate Tfs 2'-fluoro-5-methyluridine-3'-phosphorothioate Ts 5-methyluridine-3'-phosphothioate U uridine 3'-phosphate Uf 2'-fluorouridine-3'-phosphate Uf s 2'-fluorouridine-3'-phosphothioate Us uridine-3'-phosphothioate N any nucleotide (G, A, C, T, or U) a 2'-O-methyladenosine-3'-phosphate as 2'-O-methyladenosine-3'-phosphothioate c 2'-O-methylcytidine-3'-phosphate cs 2'-O-methylcytidine-3'-phosphothioate g 2'-0-methylguanosine-3'-phosphate gs 2'-0-methylguanosine-3'-phosphothioate t 2'-O-methyl-5-methyluridine-3'-phosphate ts 2'-O-methyl-5-methyluridine-3'-phosphothioate u 2'-O-methyluridine-3'-phosphate US 2'-O-methyluridine-3'-phosphorothioate S phosphorothioate bond L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol-Nur-(GalNAc-alkyl)3 (dt) deoxythymine

Table 3

Sequences of the unmodified sense and antisense strands of dsRNA C5_____

Duplex ID The semantic thread Unmodified semantic sequence SEQ ID NO: Antisense Antisense unmodified sequence SEQ ID NO: Types_name of oligonucleotide ¹ AD- 58093.1 ² UM ³ A-118310.1 AAUAACUCACUA UAAUUACUU 15 A-118311.1 AAGUAAUUAUAGUG AGUUAUUUU 66 NM_001735.2_151 7-1539_as AD-58099.1 im A-118312.1 UGACAAAAUAAC UCACUAUAA 16 A-118313.1 UUAUAGUGAGUUAU UUUGUCAAU 67 NM_001735.2_151 l-1533_as AD-58105.1 im A-118314.1 CUUCCUCUGGAA AUUGGCCUU 17 A-118315.1 AAGGCCAAUUUCCA GAGGAAGCA 68 NM_001735.2_273 3-2755_as AD-58111.1 im A-118316.1 GACAAAAUAACU CACUAUAAU 18 A-118317.1 AUUAUAGUGAGUUA UUUUGUCAA 69 NM_001735.2_151 2-1534_as

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AD-58117.1 UM A-118318.1 UCCUCUGGAAAU UGGCCUUCA 19 A-118319.1 UGAAGGCCAAUUUC CAGAGGAAG 70 NM_001735.2_273 5-2757_as AD-58123.1 UM A-118320.1 AAGCAAGAUAUU UUUAUAAUA 20 A-118321.1 UAUUAUAAAAAUAU CUUGCUUUU 71 NM_001735.2_784 -806_as AD-58129.1 UM A-118322.1 AAAAUGUUUUUG UCAAGUACA 21 A-118323.1 UGUACUUGACAAAA ACAUUUUCU 72 NM_001735.2_474 4-4766_as AD-58088.1 UM A-118324.1 AUUUAAACAACA AGUACCUUU 22 A-118325.1 AAAGGUACUUGUUG UUUAAAUCU 73 NM_001735.2_982 -1004_as AD-58094.1 UM A-118326.1 AUUCAGAAAGUC UGUGAAGGA 23 A-118327.1 UCCUUCACAGACUU UCUGAAUUU 74 NM_001735.2_457 8-4 600_as AD-58100.1 UM A-118328.1 ACACUGAAGCAU UUGAUGCAA 24 A-118329.1 UUGCAUCAAAUGCU UCAGUGUAU 75 NM_001735.2_169 -191_as AD-58106.1 UM A-118330.1 GCAGUUCUGUGU UAAAAUGUC 25 A-118331.1 GACAUUUUAACACA GAACUGCAU 76 NM_001735.2_259 l-2613_as AD-58112.1 UM A-118332.1 AGGAUUUUGAGU GUAAAAGGA 26 A-118333.1 UCCUUUUACACUCA AAAUCCUUU 77 NM_001735.2_295 5-2977_as AD-58118.1 UM A-118334.1 AAUGAUGAACCU UGUAAAGAA 27 A-118335.1 UUCUUUACAAGGUU CAUCAUUUU 78 NM_001735.2_202 5-2047_as AD-58124.1 UM A-118336.1 AUCAUUGGAACA UUUUUCAUU 28 A-118337.1 AAUGAAAAAUGUUC CAAUGAUUU 79 NM_001735.2_311 8-3140_as AD-58130.1 UM A-118338.1 AGCCAGAAAUUC GGAGUUAUU 29 A-118339.1 AAUAACUC C GAAUU UCUGGCUUG 80 NM_001735.2_231 7-2339_as AD-58089.1 UM A-118340.1 UCCCUGGGAGAU AAAACUCAC 30 A-118341.1 GUGAGUUUUAUCUC CCAGGGAAA 81 NM_001735.2_361 8-3 64 0_as AD-58095.1 UM A-118342.1 GAAAAUGAUGAA CCUUGUAAA 31 A-118343.1 UUUACAAGGUUCAU CAUUUUCUU 82 NM_001735.2_202 2-2044_as AD-58101.1 UM A-118344.1 AUUGCUCAAGUC ACAUUUGAU 32 A-118345.1 AUCAAAUGUGACUU GAGCAAUUC 83 NM_001735.2_918 -940 as AD-58107.1 UM A-118346.1 GAGAUUGCAUAU GCUUAUAAA 33 A-118347.1 UUUAUAAGCAUAUG CAAUCUCUG 84 NM_001735.2_469 8-4720_as AD-58113.1 UM A-118348.1 GUUAUCCUGAUA AAAAAUUUA 34 A-118349.1 UAAAUUUUUUAUCA GGAUAACUU 85 NM_001735.2_205 -227_as AD-58119.1 UM A-118350.1 AGGAAGUUUGCA GCUUUUAUU 35 A-118351.1 AAUAAAAGCUGCAA ACUUCCUCA 86 NM_001735.2_414 7-4169_as AD-58125.1 UM A-118352.1 GAAGAAAUUGAU CAUAUUGGA 36 A-118353.1 UC CAAUAUGAUCAA UUUCUUCUA 87 NM_001735.2_555 -577_as AD-58131.1 UM A-118354.1 AUCCUGAUAAAA AAUUUAGUU 37 A-118355.1 AACUAAAUUUUUUA UCAGGAUAA 88 NM_001735.2_208 -230_as AD-58090.1 UM A-118356.1 UGGAAAAGAAAU CUUAGUAAA 38 A-118357.1 UUUACUAAGAUUUC UUUUCCAAA 89 NM_001735.2_278 6-2808_as AD-58096.1 UM A-118358.1 UCUUAUCAAAGU AUAAACAUU 39 A-118359.1 AAUGUUUAUACUUU GAUAAGAUG 90 NM_001735.2_159 6-1618_as AD-58102.1 UM A-118360.1 UCCCUACAAACU GAAUUUGGU 40 A-118361.1 AC CAAAUUCAGUUU GUAGGGAGA 91 NM_001735.2_108 2-1104_as

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AD-58108.1 im A-118362.1 CAGGAGCAAACA UAUGUCAUU 41 A-118363.1 AAUGACAUAUGUUU GCUCCUGUC 92 NM_001735.2 109_as _87- AD-58114.1 im A-118364.1 ACAUGUAACAAC UGUAGUUCA 42 A-118365.1 UGAACUACAGUUGU UACAUGUAC 93 NM_001735.2_ 9-4131_as _410 AD-58120.1 im A-118366.1 CAGGAAAUCAUU GGAACAUUU 43 A-118367.1 AAAUGUUCCAAUGA UUUCCUGUU 94 NM_001735.2_ 2-3134_as _311 AD-58126.1 im A-118368.1 UUUAAGAAUUUU GAAAUUACU 44 A-118369.1 AGUAAUUUCAAAAU UCUUAAAGU 95 NM_001735.2 -781_as _759 AD-58132.1 im A-118370.1 UAUUCUGCAACU GAAUUCGAU 45 A-118371.1 AUCGAAUUCAGUUG CAGAAUAAC 96 NM_001735.2_ 2-4434_as _441 AD-58091.1 im A-118372.1 GCCCUUGGAAAG AGUAUUUCA 46 A-118373.1 UGAAAUACUCUUUC CAAGGGCUU 97 NM_001735.2 6-1908_as _18 8 AD-58097.1 im A-118374.1 CCUGAUAAAAAA UUUAGUUAC 47 A-118375.1 GUAACUAAAUUUUU UAUCAGGAU 98 NM_001735.2 -232_as _210 AD-58103.1 im A-118376.1 CCCUUGGAAAGA GUAUUUCAA 48 A-118377.1 UUGAAAUACUCUUU CCAAGGGCU 99 NM_001735.2_ 7-1909_as _18 8 AD-58121.1 im A-118382.1 UGCAGAUCAAAC ACAAUUUCA 49 A-118383.1 UGAAAUUGUGUUUG AUCUGCAGA 100 NM_010406.2 3-4965_as _494 AD-58133.1 im A-118386.1 CAGAUCAAACAC AAUUUCAGU 50 A-118387.1 ACUGAAAUUGUGUU UGAUCUGCA 101 NM_010406.2 5-4967_as _494 AD-58116.1 im A-118396.1 GUUCCGGAUAUU UGAACUUUU 51 A-118397.1 AAAAGUUCAAAUAU CCGGAACCG 102 NM_010406.2 0-4522_as _450 AD-58644.1 im A-119328.1 AUUUAAACAACA AGUACCUUU 52 A-119329.1 AAAGGUACUUGUUG UUUAAAUCU 103 NM_001735.2 -1004_as _982 AD-58651.1 im A-119328.2 AUUUAAACAACA AGUACCUUU 53 A-119339.1 AAAGGUACUUGUUG UUUAAAUCU 104 NM_001735.2_ -1004_as _982 AD-58641.1 im A-119322.1 UGACAAAAUAAC UCACUAUAA 54 A-119323.1 UUAUAGUGAGUUAU UUUGUCAAU 105 NM_001735.2 l-1533_as _151 AD-58648.1 im A-119322.2 UGACAAAAUAAC UCACUAUAA 55 A-119336.1 UUAUAGUGAGUUAU UUUGUCAAU 106 NM_001735.2 l-1533_as _151 AD-58642.1 im A-119324.1 GACAAAAUAACU CACUAUAAU 56 A-119325.1 AUUAUAGUGAGUUA UUUUGUCAA 107 NM_001735.2_ 2-1534_as _151 AD-58649.1 im A-119324.2 GACAAAAUAACU CACUAUAAU 57 A-119337.1 AUUAUAGUGAGUUA UUUUGUCAA 108 NM_001735.2 2-1534_as _151 AD-58647.1 im A-119334.1 GUUCCGGAUAUU UGAACUUUU 58 A-119335.1 AAAAGUUCAAAUAU CCGGAACCG 109 NM_010406.2_ 0-4522_as _450 AD-58654.1 im A-119334.2 GUUCCGGAUAUU UGAACUUUU 59 A-119342.1 AAAAGUUCAAAUAU CCGGAACCG 110 NM_010406.2 0-4522_as _450 AD-58645.1 im A-119330.1 UGCAGAUCAAAC ACAAUUUCA 60 A-119331.1 UGAAAUUGUGUUUG AUCUGCAGA 111 NM_010406.2 3-4965_as _494 AD-58652.1 im A-119330.2 UGCAGAUCAAAC ACAAUUUCA 61 A-119340.1 UGAAAUUGUGUUUG AUCUGCAGA 112 NM_010406.2 3-4965_as _494 AD-58643.1 im A-119326.1 AAGCAAGAUAUU UUUAUAAUA 62 A-119327.1 UAUUAUAAAAAUAU CUUGCUUUU 113 NM_001735.2 -806_as _784 AD-58650.1 im A-119326.2 AAGCAAGAUAUU UUUAUAAUA 63 A-119338.1 UAUUAUAAAAAUAU CUUGCUUUU 114 NM_001735.2 -806_as _784 AD-58646.1 im A-119332.1 CAGAUCAAACAC AAUUUCAGU 64 A-119333.1 ACUGAAAUUGUGUU UGAUCUGCA 115 NM_010406.2_ 5-4967_as _494 AD-58653.1 im A-119332.2 CAGAUCAAACAC AAUUUCAGU 65 A-119341.1 ACUGAAAUUGUGUU UGAUCUGCA 116 NM_010406.2 5-4967_as _494

¹ The name of the oligonucleotide species reflects the GenBank entry (e.g., NM_001735.2) and the position in the nucleotide sequence of the GenBank entry (e.g., 1517-1539) to which the antisense strand is targeted.

² The number following the decimal point refers to the batch number.

³ NM = Unmodified.

- 102 044245

Table 4

Sequences of the modified GalNAC-conjugated and antisense strand of dsRNA C5

Duplex ID Semantic thread Semantic sequence SEQ ID NO: Antisense Antisense sequence SEQ ID NO: Types_name of oligonucleotide ⁴ AD- 58093.1 A- 118310.1 AfaUfaAfcUfcAfCfUfa UfaAfuUfaCfuUfL96 117 A- 118311.1 aAfgUfaAfuUfaUfaguGfa GfuUfaUfusUfsu 168 AD- 58099.1 A- 118312.1 UfgAfcAfaAfaUfAfAfc UfcAfcUfaUfaAfL96 118 A- 118313.1 uUfaUfaGfuGfaGfuuaUfu UfuGfuCfasAfsu 169 AD- 58105.1 A- 118314.1 CfuUfcCfuCfuGfGfAfa AfuUfgGfcCfuUfL96 119 A- 118315.1 aAfgGfcCfaAfuUfuccAfg AfgGfaAfgsCfsa 170 AD- 58111.1 A- 118316.1 GfaCfaAfaAfuAfAfCfu CfaCfuAfuAfaUfL96 120 A- 118317.1 aUfuAfuAfgUfgAfguuAfu UfuUfgUfcsAfsa 171 AD- 58117.1 A- 118318.1 UfcCfuCfuGfgAfAfAfu UfgGfcCfuUfcAfL96 121 A- 118319.1 uGfaAfgGfcCfaAfuuuCfc AfgAfgGfasAfsg 172 AD- 58123.1 A- 118320.1 AfaGfcAfaGfaUfAfUfu UfuUfaUfaAfuAfL96 122 A- 118321.1 uAfuU f aUf aAf aAf auaUf c UfuGfcUfusUfsu 173 AD- 58129.1 A- 118322.1 AfaAfaUfgUfuUfUfUfg U f cAf aG fuAf cAfL 9 6 123 A- 118323.1 uGfuAfcUfuGfaCfaaaAfa CfaUfuUfusCfsu 174 AD- A- AfuUfuAfaAfcAfAfCfa 124 A- aAfaGfgUfaCfuUfguuGfu 17 5 58088.1 118324.1 AfgUfaCfcUfuUfL96 118325.1 UfuAfaAfusCfsu AD- 58094.1 A- 118326.1 AfuUfcAfgAfaAfGfUfc UfgUfgAfaGfgAfL96 125 A- 118327.1 uCfcUfuCfaCfaGfacuUfu CfuGfaAfusUfsu 176 AD- 58100.1 A- 118328.1 AfcAfcUfgAfaGfCfAfu UfuGfaUfgCfaAfL96 126 A- 118329.1 uUfgCfaUfcAfaAfugcUfu CfaGfuGfusAfsu 177 AD- 58106.1 A- 118330.1 GfcAfgUfuCfuGfUfGfu UfaAfaAfuGfuCfL96 127 A- 118331.1 gAfcAfuUfuUfaAfcacAfg Af aCfuGfcsAfsu 178 AD- 58112.1 A- 118332.1 AfgGfaUfuUfuGfAfGfu GfuAfaAfaGfgAfL96 128 A- 118333.1 uCfcUfuUfuAfcAfcucAfa AfaUfcCfusUfsu 179 AD- 58118.1 A- 118334.1 AfaUfgAfuGfaAfCfCfu UfgUfaAfaGfaAfL96 129 A- 118335.1 uUf cUfuUf aC f aAf gguU f c AfuCfaUfusUfsu 180 AD- 58124.1 A- 118336.1 AfuCfaUfuGfgAfAfCfa UfuUfuUfcAfuUfL96 130 A- 118337.1 aAfuG f aAf aAf aU f guuC f c AfaUfgAfusUfsu 181 AD- 58130.1 A- 118338.1 Af gC f cAf gAf aAfU fUf c GfgAfgUfuAfuUfL96 131 A- 118339.1 aAfuAfaCfuCfcGfauUfu CfuGfgCfusUfsg 182 AD- 58089.1 A- 118340.1 UfcCfcUfgGfgAfGfAfu AfaAfaCfuCfaCfL96 132 A- 118341.1 gUfgAfgUfuUfuAfucuCfc CfaGfgGfasAfsa 183

- 103 044245

AD- 58095.1 A- 118342.1 GfaAfaAfuGfaUfGfAfa CfcUfuGfuAfaAfL96 133 A- 118343.1 uUfuAfcAfaGfgUfucaUfc AfuUfuUfcsUfsu 184 AD- 58101.1 A- 118344.1 AfuUfgCfuCfaAfGfUfc AfcAfuUfuGfaUfL96 134 A- 118345.1 aUfcAfaAfuGfuGfacuUfg AfgCfaAfusUfsc 185 AD- 58107.1 A- 118346.1 GfaGfaUfuGfcAfUfAfu GfcUfuAfuAfaAfL96 135 A- 118347.1 uUfuAfuAfaGfcAfuauGfc AfaUfcUfcsUfsg 186 AD- 58113.1 A- 118348.1 GfuUfaUfcCfuGfAfUfa AfaAfaAfuUfuAfL96 136 A- 118349.1 uAfaAfuUfuUfuUfaucAfg GfaUfaAfcsUfsu 187 AD- 58119.1 A- 118350.1 AfgGfaAfgUfuUfGfCfa GfcUfuUfuAfuUfL96 137 A- 118351.1 aAfuAfaAfaGfcUfgcaAfa CfuUfcCfusCfsa 188 AD- 58125.1 A- 118352.1 GfaAfgAfaAfuUfGfAfu CfaUfaUfuGfgAfL96 138 A- 118353.1 uCfcAfaUfaUfgAfucaAfu UfuCfuUfcsUfsa 189 AD- 58131.1 A- 118354.1 AfuCfcUfgAfuAfAfAfa AfaUfuUfaGfuUfL96 139 A- 118355.1 aAfcUfaAfaUfuUfuuuAfu CfaGfgAfusAfsa 190 AD- 58090.1 A- 118356.1 Uf gG f aAf aAf gAfAfAfu CfuUfaGfuAfaAfL96 140 A- 118357.1 uUfuAfcUfaAfgAfuuuCfu UfuUfcCfasAfsa 191 AD- 58096.1 A- 118358.1 UfcUfuAfuCfaAfAfGfu AfuAfaAfcAfuUfL96 141 A- 118359.1 aAfuGfuUfuAfuAfcuuUfg AfuAfaGfasUfsg 192 AD- 58102.1 A- 118360.1 UfcCfcUfaCfaAfAfGfu GfaAfuUfuGfgUfL96 142 A- 118361.1 aC f cAf aAfuU f cAf guuU f g UfaGfgGfasGfsa 193 AD- 58108.1 A- 118362.1 GfaGfgAfgGfaAfAfGfa UfaUfgUfcAfuUfL96 143 A- 118363.1 aAfuGfaCfaUfaUfguuUfg CfuCfcUfgsUfsc 194 AD- 58114.1 A- 118364.1 AfcAfuGfuAfaGfAfAfc UfgUfaGfuUfcAfL96 144 A- 118365.1 uGfaAfcUfaCfaGfuugUfu AfcAfuGfusAfsc 195 AD- 58120.1 A- 118366.1 CfaGfgAfaAfuCfAfUfu GfgAfaGfaUfuUfL96 145 A- 118367.1 aAfaUfgUfuCfcAfaugAfu UfuCfcUfgsUfsu 196 AD- 58126.1 A- 118368.1 UfuUfaAfgAfaUfUfUfu GfaAfaUfuAfcUfL96 146 A- 118369.1 aGfuAfaUfuUfcAfaaaUfu CfuUfaAfasGfsu 197 AD- 58132.1 A- 118370.1 UfaUfuCfuGfcAfAfGfu GfaAfuUfcGfaUfL96 147 A- 118371.1 aUfcGfaAfuUfcAfguuGfc AfgAfaUfasAfsc 198 AD- 58091.1 A- 118372.1 GfcCfcUfuGfgAfAfAfg AfgUfaUfuUfcAfL96 148 A- 118373.1 uGfaAfaUfaCfuCfuuuCfc Af aGfgGfcsUfsu 199 AD- 58097.1 A- 118374.1 C f cUf gAfuAf aAfAfAf a UfuUfaGfuUfaCfL96 149 A- 118375.1 gU f aAf cU f aAf aUfuuuUfuAf uCfaGfgsAfsu 200 AD- 58103.1 A- 118376.1 GfcCfuUfgGfaAfAfGfa GfuAfuUfuCfaAfL96 150 A- 118377.1 uU f gAf aAfuAf cU f cuuU f cC f a AfgGfgsCfsu 201 AD- 58121.1 A- 118382.1 UfgGfaGfaUfcAfAfAfc Af cAf aUfuUf cAfL 9 6 151 A- 118383.1 uGfaAfaUfuGfuGfuuuGfaUfc UfgCfasGfsa 202 AD- 58133.1 A- 118386.1 CfaGfaUfcAfaAfCfAfc AfaUfuUfcAfgUfL96 152 A- 118387.1 aCfuGfaAfaUfuGfuguUfuGfa UfcUfgsCfsa 203 AD- 58116.1 A- 118396.1 GfuUfcCfgGfaUfAfUfu UfgAfaCfuUfuUfL96 153 A- 118397.1 aAf aAf gUfuC f aAf auaUf cC f g GfaAfcsCfsg 204 AD- 58644.1 A- 119328.1 AfsusUfuAfaAfcAfAfUfa Af gUfaCfcUfuUfL96 154 A- 119329.1 asAfsaGfgUfaCfuUfguuGfu UfuAfaAfuscsu 205

- 104 044245

AD- 58651.1 A- 119328.2 Af susUfuAfaAfcAfAfCfa AfgUfaCfcUfuUfL96 155 A- 119339.1 asAfsaGfsgUfsaCfsuUfs guu GfsuUfsuAfsaAfsuscsu 206 AD- 58641.1 A- 119322.1 U f s gsAf cAf aAf aU fAfAf c UfcAfcUfaUfaAfL9 6 156 A- 119323.1 usUfsaUfaGfuGfaGfuuaUfu UfuGfuCfasasu 207 AD- 58648.1 A- 119322.2 U f s gsAf cAf aAf aU fAfAf c UfcAfcUfaUfaAfL9 6 157 A- 119336.1 usUfsaUfsaGfsuGfsaGfsuua UfsuUfsuGfsuCfsasasu 208 AD- 58642.1 A- 119324.1 GfsasCfaAfaAfuAfAfCfu CfaCfuAfuAfaUfL9 6 158 A- 119325.1 asUfsuAfuAfgUfgAfguuAfu UfuUfgUfcsasa 209 AD- 58649.1 A- 119324.2 GfsasCfaAfaAfuAfAfCfu CfaCfuAfuAfaUfL96 159 A- 119337.1 asUfsuAfsuAfsgUfsgAfsguu AfsuUfsuUfsgUfscsasa 210 AD- 58647.1 A- 119334.1 GfsusUfcCfgGfaUfAfUfu UfgAfaCfuUfuUfL96 160 A- 119335.1 asAfsaAfgUfuCfaAfauaUfc CfgGfaAfcscsg 211 AD- 58654.1 A- 119334.2 GfsusUfcCfgGfaUfAfUfu UfgAfaCfuUfuUfL96 161 A- 119342.1 asAfsaAfsgUfsuCfsaAfsaua UfscCfsgGfsaAfscscsg 212 AD- 58645.1 A- 119330.1 UfsgsCfaGfaUfcAfAfAfc AfcAfaUfuUfcAfL9 6 162 A- 119331.1 usGfsaAfaUfuGfuGfuuuGfa UfcUfgCfasgsa 213 AD- 58652.1 A- 119330.2 UfsgsCfaGfaUfcAfAfAfc AfcAfaUfuUfcAfL9 6 163 A- 119340.1 usGfsaAfsaUfsuGfsuGfsuuu GfsaUfscUfsgCfsasgsa 214 AD- 58643.1 A- 119326.1 AfsasGfcAfaGfaUfAfUfu UfuUfaUfaAfuAfL96 164 A- 119327.1 usAfsuUfaUfaAfaAfauaUfc UfuGfcUfususu 215 AD- 58650.1 A- 119326.2 AfsasGfcAfaGfaUfAfUfu UfuUfaUfaAfuAfL96 165 A- 119338.1 usAfsuUfsaUfsaAfsaAfsaua UfscUfsuGfscUfsususu 216 AD- 58646.1 A- 119332.1 CfsasGfaUfcAfaAfCfAfc AfaUfuUfcAfgUfL9 6 166 A- 119333.1 asCfsuGfaAfaUfuGfuguUfuGfa UfcUfgscsa 217 AD- 58653.1 A- 119332.2 CfsasGfaUfcAfaAfCfAfc AfaUfuUfcAfgUfL9 6 167 A- 119341.1 asCfsuGfsaAfsaUfsuGfsugu UfsuGfsaUfscUfsgscsa 218

⁴ The name of the oligonucleotide species and the position in the nucleotide sequence of the GenBank entry to which the antisense strand corresponding to it is targeted are shown in Table 3.

Table 5

Sequences of the unmodified sense and antisense strands of dsRNA C5

Duplex ID Semantic chain Unmodified semantic sequence SEQ ID NO: Antisense Antisense unmodified sequence SEQ ID NO: Types_ oligonucleotide name AD- 58143.1 UM A- 118423.1 CACUAUAAUUAC UUGAUUU 219 A- 118424.1 AAAUCAAGUAAUUAUAGUG 302 NM_001735.2_15221540_as AD- 58149.1 UM A- 118425.1 UAACUCACUAUA AUUACUU 220 A- 118426.1 AAGUAAUUAUAGUGAGUUA 303 NM_001735.2_15171535_as AD- 58155.1 UM A- 118427.1 ACAAAAUAACUC ACUAUAA 221 A- 118428.1 UUAUAGUGAGUUAUUUUGU 304 NM—001735.2_15111529—as

- 105 044245

AD- 58161.1 UM A- 118429.1 UCCUCUGGAAAU UGGCCUU 222 A- 118430.1 AAGGCCAAUUUCCAGAGGA 305 NM_001735.2_2733- 2751_as AD- 58167.1 UM A- 118431.1 CAAAAUAACUCA CUAUAAU 223 A- 118432.1 AUUAUAGUGAGUUAUUUUG 306 NM_001735.2_1512- 1530_as AD- 58173.1 UM A- 118433.1 CUCUGGAAAUUG GCCUUCA 224 A- 118434.1 UGAAGGCCAAUUUCCAGAG 307 NM_001735.2_2735- 2753_as AD- 58179.1 UM A- 118435.1 GCAAGAUAUUUU UAUAAUA 225 A- 118436.1 UAUUAUAAAAAUAUCUUGC 308 NM_001735.2_784802_as AD- 58185.1 UM A- 118437.1 AAUGUUUUUGUC AAGUACA 226 A- 118438.1 UGUACUUGACAAAAACAUU 309 NM_001735.2_4744- 4762_as AD- 58144.1 UM A- 118439.1 UUAAACAACAAG UACCUUU 227 A- 118440.1 AAAGGUACUUGUUGUUUAA 310 NM_001735.2_9821000_as AD- 58150.1 UM A- 118441.1 UCAGAAAGUCUG UGAAGGA 228 A- 118442.1 UCCUUCACAGACUUUCUGA 311 NM_001735.2_45784596_as AD- 58156.1 UM A- 118443.1 ACUGAAGCAUUU GAUGCAA 229 A- 118444.1 UUGCAUCAAAUGCUUCAGU 312 NM—001735.2_169187—as AD- 58162.1 UM A- 118445.1 AGUUCUGUGUU AAAAUGUC 230 A- 118446.1 GACAUUUUAACACAGAACU 313 NM—001735.2_25912609—as AD- 58168.1 UM A- 118447.1 GAUUUUGAGUGU AAAAGGA 231 A- 118448.1 UCCUUUUACACUCAAAAUC 314 NM—001735.2_29552973—as AD- 58174.1 UM A- 118449.1 UGAUGAACCUUG UAAAGAA 232 A- 118450.1 UUCUUUACAAGGUUCAUCA 315 NM—001735.2_2025- 2043—as AD- 58180.1 UM A- 118451.1 CAUUGGAACAUU UUUCAUU 233 A- 118452.1 AAUGAAAAAUGUUC CAAUG 316 NM—001735.2_31183136—as AD- 58186.1 UM A- 118453.1 CCAGAAAUUCGG AGUUAUU 234 A- 118454.1 AAUAACUCCGAAUUUCUGG 317 NM—001735.2_23172335—as AD- 58145.1 UM A- 118455.1 CCUGGGAGAUAAA ACUCAC 235 A- 118456.1 GUGAGUUUUAUCUCCCAGG 318 NM_001735.2_3618- 3 63 6_as

- 106 044245

AD- 58151.1 UM A- 118457.1 AAAUGAUGAACC UUGUAAA 236 A- 118458.1 UUUACAAGGUUCAUCAUUU 319 NM_001735.2_2022- 2040_as AD- 58157.1 UM A- 118459.1 UGCUCAAGUCACA UUUGAU 237 A- 118460.1 AUCAAAUGUGACUUGAGCA 320 NM_001735.2_918- 936_as AD- 58163.1 UM A- 118461.1 GAUUGCAUAUGCU UAUAAA 238 A- 118462.1 UUUAUAAGCAUAUGCAAUC 321 NM_001735.2_46984716_as AD- 58169.1 UM A- 118463.1 UAUCCUGAUAAAA AAUUUA 239 A- 118464.1 UAAAUUUUUUAUCAGGAUA 322 NM_001735.2_205- 223_as AD- 58175.1 UM A- 118465.1 GAAGUUUGCAGCU UUUAUU 240 A- 118466.1 AAUAAAAGCUGCAAACUUC 323 NM_001735.2_4147- 4165_as AD- 58181.1 UM A- 118467.1 AGAAAUUGAUCAU AUUGGA 241 A- 118468.1 UC CAAUAUGAUCAAUUUCU 324 NM_001735.2_555573_as AD- 58187.1 UM A- 118469.1 CCUGAUAAAAAAU UUAGUU 242 A- 118470.1 AACUAAAUUUUUUAUCAGG 325 NM_001735.2_208226_as AD- 58146.1 UM A- 118471.1 GAAAAGAAAUCUU AGUAAA 243 A- 118472.1 UUUACUAAGAUUUCUUUUC 326 NM—001735.2_27862804—as AD- 58152.1 UM A- 118473.1 UUAUCAAAGUAUA AACAUU 244 A- 118474.1 AAUGUUUAUACUUUGAUAA 327 NM—001735.2_15961614—as AD- 58158.1 UM A- 118475.1 CCUACAAACUGAA UUUGGU 245 A- 118476.1 ACCAAAUUCAGUUUGUAGG 328 NM—001735.2_10821100—as AD- 58164.1 UM A- 118477.1 GGAGCAAACAUAU GUCAUU 246 A- 118478.1 AAUGACAUAUGUUUGCUCC 329 NM_001735.2_87105—as AD- 58170.1 UM A- 118479.1 AUGUAACAACUGU AGUUCA 247 A- 118480.1 UGAACUACAGUUGUUACAU 330 NM—001735.2_41094127—as AD- 58176.1 UM A- 118481.1 GGAAAUCAUUGGA ACAUUU 248 A- 118482.1 AAAUGUUCCAAUGAUUUCC 331 NM—001735.2_31123130—as AD- 58182.1 UM A- 118483.1 UAAGAAUUUUGAA AUUACU 249 A- 118484.1 AGUAAUUUCAAAAUUCUUA 332 NM—001735.2_759- 777_as

- 107 044245

AD- 58188.1 UM A- 118485.1 UUCUGCAACUGAA UUCGAU 250 A- 118486.1 AUCGAAUUCAGUUGCAGAA 333 NM_001735.2_4412- 4430_as AD- 58147.1 UM A- 118487.1 CCUUGGAAAGAGU AUUUCA 251 A- 118488.1 UGAAAUACUCUUUCCAAGG 334 NM_001735.2_18861904_as AD- 58153.1 UM A- 118489.1 UGAUAAAAAAUUU AGUUAC 252 A- 118490.1 GUAACUAAAUUUUUUAUCA 335 NM_001735.2_210- 228_as AD- 58159.1 UM A- 118491.1 CUUGGAAAGAGU UUUCAA 253 A- 118492.1 UUGAAAUACUCUUUCCAAG 336 NM_001735.2_1887- 1905_as AD- 58190.1 UM A- 118519.1 CACUAUAAUUACU UGAUUU 254 A- 118520.1 AAAUCAAGUAAUUAUAGUG 337 NM_001735.2_1522- 1540_as AD- 58196.1 UM A- 118521.1 UAACUCACUAUAA UUACUU 255 A- 118522.1 AAGUAAUUAUAGUGAGUUA 338 NM_001735.2_15171535_as AD- 58202.1 UM A- 118523.1 ACAAAAUAACUCA CUAUAA 256 A- 118524.1 UUAUAGUGAGUUAUUUUGU 339 NM_001735.2_15111529—as AD- 58208.1 UM A- 118525.1 UCCUCUGGAAAUU GGCCUU 257 A- 118526.1 AAGGCCAAUUUCCAGAGGA 340 NM—001735.2_27332751—as AD- 58214.1 UM A- 118527.1 CAAAAUAACUCAC UAUAAU 258 A- 118528.1 AUUAUAGUGAGUUAUUUUG 341 NM—001735.2_1512- 1530—as AD- 58220.1 UM A- 118529.1 CUCUGGAAAUUGG CCUUCA 259 A- 118530.1 UGAAGGCCAAUUUCCAGAG 342 NM—001735.2_27352753—as AD- 58226.1 UM A- 118531.1 GCAAGAUAUUUUU AUAAUA 260 A- 118532.1 UAUUAUAAAAAUAUCUUGC 343 NM—001735.2_784- 8 02—as AD- 58231.1 UM A- 118533.1 AAUGUUUUUGUCA AGUACA 261 A- 118534.1 UGUACUUGACAAAAACAUU 344 NM—001735.2_4744- 4762_as AD- 58191.1 UM A- 118535.1 UUAAACAACAAGU ACCUUU 262 A- 118536.1 AAAGGUACUUGUUGUUUAA 345 NM—001735.2_9821000—as AD- 58197.1 UM A- 118537.1 UCAGAAAGUCUGU GAAGGA 263 A- 118538.1 UCCUUCACAGACUUUCUGA 346 NM—001735.2_45784596_as

- 108 044245

AD- 58203.1 UM A- 118539.1 ACUGAAGCAUUUG AUGCAA 264 A- 118540.1 UUGCAUCAAAUGCUUCAGU 347 NM_001735.2_169187_as AD- 58209.1 UM A- 118541.1 AGUUCUGUGUUAA AAUGUC 265 A- 118542.1 GACAUUUUAACACAGAACU 348 NM_001735.2_2591- 2 609_as AD- 58233.1 UM A- 118565.1 CACUAUAAUUACU UGAUUU 266 A- 118566.1 AAAUCAAGUAAUUAUAGUG 349 NM_001735.2_1522- 1540_as AD- 58193.1 UM A- 118567.1 UAACUCACUAUAA UUACUU 267 A- 118568.1 AAGUAAUUAUAGUGAGUUA 350 NM_001735.2_1517- 1535_as AD- 58199.1 UM A- 118569.1 ACAAAAUAACUCA CUAUAA 268 A- 118570.1 UUAUAGUGAGUUAUUUUGU 351 NM_001735.2_1511- 1529_as AD- 58205.1 UM A- 118571.1 UCCUCUGGAAAUU GGCCUU 269 A- 118572.1 AAGGCCAAUUUCCAGAGGA 352 NM_001735.2_27332751_as AD- 58211.1 UM A- 118573.1 CAAAAUAACUCAC UAUAAU 270 A- 118574.1 AUUAUAGUGAGUUAUUUUG 353 NM_001735.2_15121530—as AD- 58217.1 UM A- 118575.1 CUCUGGAAAUUGG CCUUCA 271 A- 118576.1 UGAAGGCCAAUUUCCAGAG 354 NM—001735.2_27352753—as AD- 58223.1 UM A- 118577.1 GCAAGAUAUUUUU AUAAUA 272 A- 118578.1 UAUUAUAAAAAUAUCUUGC 355 NM—001735.2_784- 8 02—as AD- 58229.1 UM A- 118579.1 AAUGUUUUUGUC AGUACA 273 A- 118580.1 UGUACUUGACAAAAACAUU 356 NM—001735.2_4744- 4762_as AD- 58234.1 UM A- 118581.1 UUAAACAACAAGU ACCUUU 274 A- 118582.1 AAAGGUACUUGUUGUUUAA 357 NM—001735.2_9821000—as AD- 58194.1 UM A- 118583.1 UCAGAAAGUCUGU GAAGGA 275 A- 118584.1 UCCUUCACAGACUUUCUGA 358 NM—001735.2_4578- 4596_as AD- 58200.1 UM A- 118585.1 ACUGAAGCAUUU GAUGCAA 276 A- 118586.1 UUGCAUCAAAUGCUUCAGU 359 NM—001735.2_169187—as AD- 58206.1 UM A- 118587.1 AGUUCUGUGUUAA AAUGUC 277 A- 118588.1 GACAUUUUAACACAGAACU 360 NM—001735.2_25912609—as

- 109 044245

AD- 58236.1 UM A- 118423.2 CACUAUAAUUACU UGAUUU 278 A- 118644.1 AAAUCAAGUAAUUAUAGUG 361 NM_001735.2_1522- 1540_as AD- 58242.1 UM A- 118425.2 UAACUCACUAUA AUUACUU 279 A- 118645.1 AAGUAAUUAUAGUGAGUUA 362 NM_001735.2_1517- 1535_as AD- 58248.1 UM A- 118427.2 ACAAAAUAACUC ACUAUAA 280 A- 118646.1 UUAUAGUGAGUUAUUUUGU 363 NM_001735.2_1511- 1529_as AD- 58254.1 UM A- 118429.2 UCCUCUGGAAAU UGGCCUU 281 A- 118647.1 AAGGCCAAUUUCCAGAGGA 364 NM_001735.2_2733- 2751_as AD- 58260.1 UM A- 118431.2 CAAAAUAACUCA CUAUAAU 282 A- 118648.1 AUUAUAGUGAGUUAUUUUG 365 NM_001735.2_1512- 1530_as AD- 58266.1 UM A- 118433.2 CUCUGGAAAUUG GCCUUCA 283 A- 118649.1 UGAAGGCCAAUUUCCAGAG 366 NM_001735.2_27352753_as AD- 58272.1 UM A- 118435.2 GCAAGAUAUUUU UAUAAUA 284 A- 118650.1 UAUUAUAAAAAUAUCUUGC 367 NM_001735.2_784802—as AD- 58277.1 UM A- 118437.2 AAUGUUUUUGUC AAGUACA 285 A- 118651.1 UGUACUUGACAAAAACAUU 368 NM—001735.2_4744- 4762_as AD- 58237.1 UM A- 118439.2 UUAAACAACAAG UACCUUU 286 A- 118652.1 AAAGGUACUUGUUGUUUAA 369 NM—001735.2_9821000—as AD- 58243.1 UM A- 118441.2 UCAGAAAGUCUG UGAAGGA 287 A- 118653.1 UCCUUCACAGACUUUCUGA 370 NM—001735.2_45784596_as AD- 58249.1 UM A- 118443.2 ACUGAAGCAUUU GAUGCAA 288 A- 118654.1 UUGCAUCAAAUGCUUCAGU 371 NM—001735.2_169187—as AD- 58255.1 UM A- 118445.2 AGUUCUGUGUU AAAAUGUC 289 A- 118655.1 GACAUUUUAACACAGAACU 372 NM—001735.2_25912609—as AD- 58279.1 UM A- 118423.3 CACUAUAAUUA CUUGAUUU 290 A- 118667.1 AAAUCAAGUAAUUAUAGUG 373 NM—001735.2_15221540—as AD- 58239.1 UM A- 118425.3 UAACUCACUAUA AUUACUU 291 A- 118668.1 AAGUAAUUAUAGUGAGUUA 374 NM—001735.2_1517- 1535—as

- 110 044245

AD- 58245.1 im A- 118427.3 ACAAAAUAACUC ACUAUAA 292 A- 118669.1 UUAUAGUGAGUUAUUUUGU 375 NM_001735.2_1511- 1529_as AD- 58251.1 im A- 118429.3 UCCUCUGGAAAUU GGCCUU 293 A- 118670.1 AAGGCCAAUUUCCAGAGGA 376 NM_001735.2_2733- 2751_as AD- 58257.1 im A- 118431.3 CAAAAUAACUCAC UAUAAU 294 A- 118671.1 AUUAUAGUGAGUUAUUUUG 377 NM_001735.2_1512- 1530_as AD- 58263.1 im A- 118433.3 CUCUGGAAAUUG GCCUUCA 295 A- 118672.1 UGAAGGCCAAUUUCCAGAG 378 NM_001735.2_2735- 2753_as AD- 58269.1 im A- 118435.3 GCAAGAUAUUUU UAUAAUA 296 A- 118673.1 UAUUAUAAAAAUAUCUUGC 379 NM_001735.2_784802_as AD- 58275.1 im A- 118437.3 AAUGUUUUUGUC AAGUACA 297 A- 118674.1 UGUACUUGACAAAAACAUU 380 NM_001735.2_4744- 4762_as AD- 58280.1 im A- 118439.3 UUAAACAACAAGU ACCUUU 298 A- 118675.1 AAAGGUACUUGUUGUUUAA 381 NM_001735.2_9821000—as AD- 58240.1 im A- 118441.3 UCAGAAAGUCUGU GAAGGA 299 A- 118676.1 UCCUUCACAGACUUUCUGA 382 NM—001735.2_45784596_as AD- 58246.1 im A- 118443.3 ACUGAAGCAUUU GAUGCAA 300 A- 118677.1 UUGCAUCAAAUGCUUCAGU 383 NM—001735.2_169187—as AD- 58252.1 im A- 118445.3 AGUUCUGUGUUA AAAUGUC 301 A- 118678.1 GACAUUUUAACACAGAACU 384 NM—001735.2_2591- 2 609—as

Table 6

Sequences of the modified sense and antisense strands of dsRNA C5

Duplex ID Semantic chain Semantic sequence SEQ ID NO: Antisense Antisense sequence SEQ ID NO: Types_name of oligo Nucleotide ⁵ AD- 58143.1 A- 118423.1 cAcuAuAAuuAcuu GAuuudTsdT 385 A-118424.1 AAAUcAAGuAAUuAuA GUGdTsdT 468 AD- 58149.1 A- 118425.1 uAAcucAcuAuAAuu AcuudTsdT 386 A-118426.1 AAGuAAUuAuAGUGAG UuAdTsdT 469 AD- 58155.1 A- 118427.1 AcAAAAuAAc u cAc u AuAAdTsdT 387 A-118428.1 UuAuAGUGAGUuAUUU UGUdTsdT 470

- 111 044245

AD- 58161.1 A- 118429.1 uccucuGGAAAuuGG ccuudTsdT 388 A-118430.1 AAGGCcAAUUUCcAGAG GAdTsdT 471 AD- 58167.1 A- 118431.1 cAAAAuAAc u cAc uAu AAudTsdT 389 A-118432.1 AUuAuAGUGAGUuAUU UUGdTsdT 472 AD- 58173.1 A- 118433.1 cucuGGAAAuuGGccu ucAdTsdT 390 A-118434.1 UGAAGGCcAAUUUCcAGA GdTsdT 473 AD- 58179.1 A- 118435.1 GcAAGAuAuuuuuAu AAuAdTsdT 391 A-118436.1 uAUuAuAAAAAuAUCU UGCdTsdT 474 AD- 58185.1 A- 118437.1 AAuGuuuuuGucAAGu AcAdTsdT 392 A-118438.1 UGuACUUGAcAAAAAcA UUdTsdT 475 AD- 58144.1 A- 118439.1 uuAAAcAAcAAGuAccu uudTsdT 393 A-118440.1 AAAGGuACUUGUUGUUu AAdTsdT 476 AD- 58150.1 A- 118441.1 ucAGAAAGucuGuGAAG GAdTsdT 394 A-118442.1 UCCUUcAcAGACUUUC UGAdTsdT 477 AD- 58156.1 A- 118443.1 AcuGAAGcAuuuGAuGc AAdTsdT 395 A-118444.1 UUGcAUcAAAUGCUUcA GUdTsdT 478 AD- 58162.1 A- 118445.1 AGuucuGuGuuAAAAuG ucdTsdT 396 A-118446.1 GAcAUUUuAAcAcAGAA GUdTsdT 479 AD- 58168.1 A- 118447.1 GAuuuuGAGuGuAAAAG GAdTsdT 397 A-118448.1 UCCUUUuAcACUcAAAA UGdTsdT 480 AD- 58174.1 A- 118449.1 uGAuGAAccuuGuAAAG AAdTsdT 398 A-118450.1 UUCUUuAcAAGGUUcAU cAdTsdT 481 AD- 58180.1 A- 118451.1 cAuuGGAAcAuuuuucA uudTsdT 399 A-118452.1 AAUGAAAAAUGUUC cAA UGdTsdT 482 AD- 58186.1 A- 118453.1 ccAGAAAuucGGAGuuA uudTsdT 400 A-118454.1 AAuAACUCCGAAUUUCU GGdTsdT 483 AD- 58145.1 A- 118455.1 c c u G G GAGAuAAAAc u c AcdTsdT 401 A-118456.1 GUGAGUUUuAUCUCCcA GGdTsdT 484 AD- 58151.1 A- 118457.1 AAAuGAuGAAccuuGuA AAdTsdT 402 A-118458.1 UUuAcAAGGUUcAUcAU UUdTsdT 485 AD- 58157.1 A- 118459.1 uGcucAAGucAcAuuuGA udTsdT 403 A-118460.1 AUcAAAUGUGACUUGAGc AdTsdT 486 AD- 58163.1 A- 118461.1 GAuuGcAuAuGcuuAuAA AdTsdT 404 A-118462.1 UUuAuAAG cAuAUG cAAU GdTsdT 487 AD- 58169.1 A- 118463.1 uAuccuGAuAAAAAAuuu AdTsdT 405 A-118464.1 uAAAUUUUUuAUcAGGAu AdTsdT 488 AD- 58175.1 A- 118465.1 GAAGuuuGcAGcuuuu AuudTsdT 406 A-118466.1 AAuAAAAGCUGcAAACU UGdTsdT 489 AD- 58181.1 A- 118467.1 AGAAAuuGAucAuAuuG GAdTsdT 407 A-118468.1 UC cAAuAUGAUcAAUUU GUdTsdT 490 AD- 58187.1 A- 118469.1 c cuGAuAAAAAAuuuAG uudTsdT 408 A-118470.1 AACuAAAUUUUUuAUcA GGdTsdT 491 AD- 58146.1 A- 118471.1 GAAAAAGAAAucuuAGuAA AdTsdT 409 A-118472.1 UUuACuAAGAUUUCUU UUGdTsdT 492

- 112 044245

AD- 58152.1 A- 118473.1 uuAu cAAAGuAuAAAcA uudTsdT 410 A-118474.1 AAUGUUuAuACUUUGAu AAdTsdT 493 AD- 58158.1 A- 118475.1 ccuAcAAAcuGAAuuuGG udTsdT 411 A-118476.1 ACcAAAUUcAGUUUGuA GGdTsdT 494 AD- 58164.1 A- 118477.1 GGAGcAAAcAuAuGucAu udTsdT 412 A-118478.1 AAUGAcAuAUGUUUGCU GGdTsdT 495 AD- 58170.1 A- 118479.1 AuGuAAcAAcuGuAGuuc AdTsdT 413 A-118480.1 UGAACuAcAGUUGUuAc AUdTsdT 496 AD- 58176.1 A- 118481.1 GGAAAucAuuGGAAcAuu udTsdT 414 A-118482.1 AAAUGUUC cAAUGAUUU GGdTsdT 497 AD- 58182.1 A- 118483.1 uAAGAAuuuuGAAAuuAc udTsdT 415 A-118484.1 AGuAAUUUcAAAAUUC UuAdTsdT 498 AD- 58188.1 A- 118485.1 uucuGcAAcuGAAuucGA udTsdT 416 A-118486.1 AUGGAAUUcAGUUGcA GAAdTsdT 499 AD- 58147.1 A- 118487.1 ccuuGGAAAGAGuAuuuc AdTsdT 417 A-118488.1 UGAAAuACUCUUUC cAA GGdTsdT 500 AD- 58153.1 A- 118489.1 uGAuAAAAAAuuuAGuuA cdTsdT 418 A-118490.1 GuAACuAAAUUUUUuAU cAdTsdT 501 AD- 58159.1 A- 118491.1 cuuGGAAAGAGuAuuucA AdTsdT 419 A-1184 92.1 UUGAAAuACUCUUUC cA AGdTsdT 502 AD- 58190.1 A- 118519.1 CACUAUAAUUACUUGAUU UdTdT 420 A-118520.1 AAAUCAAGUAAUUAUA GUGdTdT 503 AD- 58196.1 A- 118521.1 UAACUCACUAUAAUUAC UUdTdT 421 A-118522.1 AAGUAAUUAUAGUGAGU UAdTdT 504 AD- 58202.1 A- 118523.1 ACAAAAUAACUCACUAU AAdTdT 422 A-118524.1 UUAUAGUGAGUUAUUUU UdTdT 505 AD- 58208.1 A- 118525.1 UCCUCUGGAAAUUGGCC UUdTdT 423 A-118526.1 AAGGCCAAUUUCCAGAG GAdTdT 506 AD- 58214.1 A- 118527.1 CAAAAUAACUCACUAUA AUdTdT 424 A-118528.1 AUUAUAGUGAGUUAUU UUGdTdT 507 AD- 58220.1 A- 118529.1 CUCUGGAAAUUGGCCUU CAdTdT 425 A-118530.1 UGAAGGCCAAUUUCCAG AGdTdT 508 AD- 58226.1 A- 118531.1 GCAAGAUAUUUUUAUA AUAdTdT 426 A-118532.1 UAUUAUAAAAAUAUCUU GCdTdT 509 AD- 58231.1 A- 118533.1 AAUGUUUUUGUCAAGUA CAdTdT 427 A-118534.1 UGUACUUGACAAAAACA UUdTdT 510 AD- 58191.1 A- 118535.1 UUAAACAACAAGUAC CU UUdTdT 428 A-118536.1 AAAGGUACUUGUUGUUU AAdTdT 511 AD- 58197.1 A- 118537.1 UCAGAAAGUCUGUGAAG GAdTdT 429 A-118538.1 UCCUUCACAGACUUUCU GAdTdT 512 AD- 58203.1 A- 118539.1 ACUGAAGCAUUUGAUGC AAdTdT 430 A-118540.1 UUGCAUCAAAUGCUUCAG UdTdT 513 AD- 58209.1 A- 118541.1 AGUUCUGUGUUAAAAUG UCdTdT 431 A-118542.1 GACAUUUUAACACAGAA CUdTdT 514

- 113 044245

AD- 58233.1 A- 118565.1 C fAC fU fAU fAAU fU fAC fUfUfGAUfUfUfdTsdT 432 A-118566.1 AAAUC fAAGU fAAUU fA UfAGUGdTsdT 515 AD- 58193.1 A- 118567.1 U fAAC fU f C fAC fU fAU fAAUfUfACfUfUfdTsdT 433 A-118568.1 AAGU fAAUU fAU fAGUG AGUUfAdTsdT 516 AD- 58199.1 A- 118569.1 AC fAAAAU fAAC fU f C fA CfUfAUfAAdTsdT 434 A-118570.1 UU fAU fAGUGAGUU fAU UUUGUdTsdT 517 AD- 58205.1 A- 118571.1 UfCfCfUfCfUfGGAAAU fUfGGCfCfUfUfdTsdT 435 A-118572.1 AAGGCC fAAUUUCC fAG AGGAdTsdT 518 AD- 58211.1 A- 118573.1 C fAAAAU fAAC fU f C fAC fUfAUfAAUfdTsdT 436 A-118574.1 AUU fAU fAGUGAGUU fA UUUUGdTsdT 519 AD- 58217.1 A- 118575.1 CfUfCfUfGGAAAUfUfG GCfCfUfUfCfAdTsdT 437 A-118576.1 UGAAGGCC fAAUUUCC fA GAGdTsdT 520 AD- 58223.1 A- 118577.1 GC fAAGAU fAU fU fU fU fUfAUfAAUfAdTsdT 438 A-118578.1 U fAUU fAU fAAAAAU fA UCUUGCdTsdT 521 AD- 58229.1 A- 118579.1 AAUfGUfUfUfUfUfGU fCfAAGUfACfAdTsdT 439 A-118580.1 UGU fACUUGAC fAAAAA CfAUUdTsdT 522 AD- 58234.1 A- 118581.1 U fU fAAAC fAAC fAAGU f ACfCfUfUfUfdTsdT 440 A-118582.1 AAAGGU fACUUGUUGUUU fAAdTsdT 523 AD- 58194.1 A- 118583.1 UfCfAGAAAGUfCfUfGU fGAAGGAdTsdT 441 A-118584.1 UCCUUC fAC fAGACUUU CUGAdTsdT 524 AD- 58200.1 A- 118585.1 AC fU f GAAGC fAU fUfUf GAUfGCfAAdTsdT 442 A-118586.1 UUGC fAUC fAAAUGCUU CfAGUdTsdT 525 AD- 58206.1 A- 118587.1 AGUfUfCfUfGUfGUfUf AAAAUfGUfCfdTsdT 443 A-118588.1 GAC fAUUUU fAAC fAC fA GAACUdTsdT 526 AD- 58236.1 A- 118423.2 cAcuAuAAuuAcuuGAuu udTsdT 444 A-118644.1 AAAUcAAGuAAUuAuAG uGdTsdT 527 AD- 58242.1 A- 118425.2 uAAc u cAc uAuAAuuAc u udTsdT 445 A-118645.1 AAGuAAUuAuAGuGAGU uAdTsdT 528 AD- 58248.1 A- 118427.2 AcAAAAuAAc u cAc uAuA AdTsdT 446 A-118646.1 UuAuAGuGAGUuAuUuu GUdTsdT 529 AD- 58254.1 A- 118429.2 uccucuGGAAAuuGGccu udTsdT 447 A-118647.1 AAGGCcAAuUUCcAGAGG AdTsdT 530 AD- 58260.1 A- 118431.2 cAAAAuAAc u cAc uAuAA udTsdT 448 A-118648.1 AUuAuAGuGAGUuAuUuu GdTsdT 531 AD- 58266.1 A- 118433.2 cucuGGAAAuuGGccuu cAdTsdT 449 A-118649.1 uGAAGGCcAAuUUCcAG AGdTsdT 532 AD- 58272.1 A- 118435.2 GcAAGAuAuuuuuAuAA uAdTsdT 450 A-118650.1 uAUuAuAAAAAuAUCuuG CdTsdT 533 AD- 58277.1 A- 118437.2 AAuGuuuuuGucAAGuA cAdTsdT 451 A-118651.1 uGuACuuGAcAAAAAcAu UdTsdT 534 AD- 58237.1 A- 118439.2 uuAAAcAAcAAGuAc cu uudTsdT 452 A-118652.1 AAAGGuACuuGuuGuUuA AdTsdT 535 AD- 58243.1 A- 118441.2 ucAGAAAGucuGuGAAG GAdTsdT 453 A-118653.1 UCCuUcAcAGACuUUCuGA dTsdT 536

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AD- 58249.1 A- 118443.2 AcuGAAGcAuuuGAuGc AAdTsdT 454 A-118654.1 uuGcAUcAAAuGCuUcAGU dTsdT 537 AD- 58255.1 A- 118445.2 AGuucuGuGuuAAAAuG ucdTsdT 455 A-118655.1 GAcAuUUuAAcAcAGAACU dTsdT 538 AD- 58279.1 A- 118423.3 cAcuAuAAuuAcuuGAu uudTsdT 456 A-118667.1 AAAU CAAGuAAuuAuAgu g dTsdT 539 AD- 58239.1 A- 118425.3 uAAcucAcuAuAAuuAc uudTsdT 457 A-118668.1 AAGuAAuUAuAGuGAGuua dTsdT 540 AD- 58245.1 A- 118427.3 AcAAAAuAAc u cAc uAu AAdTsdT 458 A-118669.1 UuAuAGuGAGuuAuuuugu dTsdT 541 AD- 58251.1 A- 118429.3 uccucuGGAAAuuGGcc uudTsdT 459 A-118670.1 AAGGCCAAuUuCCAGAgg dTsdT 542 AD- 58257.1 A- 118431.3 cAAAAuAAc u cAc uAuA AudTsdT 460 A-118671.1 AuUAuAGuGAGuuAuuuug dTsdT 543 AD- 58263.1 A- 118433.3 cucuGGAAAuuGGccuu cAdTsdT 461 A-118672.1 UGAAGGCCAAuuuCCAgag dTsdT 544 AD- 58269.1 A- 118435.3 GcAAGAuAuuuuuAuAA uAdTsdT 462 A-118673.1 UAuUAuAAAAAuAu C u u g c d TsdT 545 AD- 58275.1 A- 118437.3 AAuGuuuuuGucAAGuA cAdTsdT 463 A-118674.1 UGuACuUGACAAAAACauu dTsdT 546 AD- 58280.1 A- 118439.3 uuAAAcAAcAAGuAc cu uudTsdT 464 A-118675.1 AAAGGuACuUGuuGuuuaa dTsdT 547 AD- 58240.1 A- 118441.3 ucAGAAAGucuGuGAAG GAdTsdT 465 A-118676.1 UCCuUCACAGACuuuCuga dTsdT 548 AD- 58246.1 A- 118443.3 AcuGAAGcAuuuGAuGc AAdTsdT 466 A-118677.1 UuGCAUCAAAuGCuuCagu dTsdT 549 AD- 58252.1 A- 118445.3 AGuucuGuGuuAAAAuG ucdTsdT 467 A-118678.1 GACAuUuUAACACAGAa cud TsdT 550

⁵ The name of the oligonucleotide species and the position in the GenBank nucleotide sequence of the entry to which the antisense strand corresponding to it is targeted are shown in Table 5.

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Table 7

Single-dose C5 assay in Hep3B cells with GalNAC-conjugated mRNA

Duplex ID 10 nM AVG 0.1 nm AVG 10 nM STDEV 0.1 nM STDEV AD-58093.1 15, 62 21, 60 7.48 6, 52 AD-58099.1 9, 07 14.70 1.18 4, 65 AD-58105.1 36.71 60.23 5, 07 19, 83 AD-58111.1 11.83 22.78 3.51 12.75 AD-58117.1 12.43 33.46 2.00 23.56 AD-58123.1 8.05 15, 18 2.89 7.94 AD-58129.1 10.77 40, 06 1.30 19, 66 AD-58088.1 6, 55 16.40 1.24 4.58 AD-58094.1 19, 59 40, 68 7, 64 12:30 AD-58100.1 10, 92 20, 12 0.74 8.38 AD-58106.1 10, 97 37.23 2.49 19, 95 AD-58112.1 13.24 29, 32 2.90 14.08 AD-58118.1 6, 63 15, 23 0.54 5, 72 AD-58124.1 7.17 13:00 1.44 6.48 AD-58130.1 10.38 17.92 2.36 6, 92 AD-58089.1 8.81 30, 67 2.91 10.53 AD-58095.1 8.72 14, 66 1.04 3.37 AD-58101.1 8.17 19.36 1.30 5, 69 AD-58107.1 4.84 18.10 1, 66 7.21 AD-58113.1 8.78 14, 62 1.77 7.89 AD-58119.1 8.90 15, 01 0.91 7.35 AD-58125.1 11, 13 17.04 2, 61 9, 03 AD-58131.1 13.50 40, 14 1.08 12.07 AD-58090.1 7.90 21.57 2.95 6, 61 AD-58096.1 8.02 16, 56 1.54 6, 68 AD-58102.1 12.40 27.93 1.83 11.78 AD-58108.1 12.02 July 15 2.88 5, 74 AD-58114.1 11.86 May 25 1.48 9.46 AD-58120.1 7, 65 10.57 0.58 3.56 AD-58126.1 8.45 15, 39 2.08 7.42 AD-58132.1 8.50 19.26 2.52 9, 38 AD-58091.1 8, 68 18.05 2.95 6, 62 AD-58097.1 9, 31 23, 02 0.67 10, 10 AD-58103.1 8.53 17.23 2.90 7.27 AD-1955 57.41 81.16 10.76 5.29 Imitation 78, 61 75, 97 5.70 2.76 Unprocessed 100 100 6, 13 5, 98

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Table 8

Single-dose transfection assay of C5 in primary mouse hepatocytes with GalNAC-conjugated mRNA

Duplex ID 10 nM AVG 0.1 nm AVG 10 nM STDEV 0.1 nM STDEV AD-58093.1 1.53 1, 65 0.17 0.25 AD-58099.1 1, 65 1.50 0.61 0.22 AD-58105.1 11.20 46, 95 0.08 3, 89 AD-58111.1 2.49 2.13 0.26 0.20 AD-58117.1 3, 57 31, 91 0.93 0.62 AD-58123.1 4.29 2.97 0.11 2.22 AD-58129.1 1, 19 8.53 0.23 0.72 AD-58088.1 0.84 1.34 0.68 0.07 AD-58094.1 And, 34 66, 82 0.17 3, 01 AD-58100.1 2.78 1.51 0.43 0.33 AD-58106.1 6, 79 52, 91 4.42 6, 78 AD-58121.1 1, 94 2.15 0.04 0.91 AD-58133.1 1.74 3.25 0.19 1, 64 AD-58116.1 1.76 2.21 1.27 0.78 AD-1955 87.39 91.71 5.77 4, 68 Imitation 79, 67 89, 02 1.51 3, 91 Unprocessed 100 100 6, 39 13, 11

Table 9

Single-dose C5 assay in primary cynomolgus macaque hepatocytes with GalNAC-conjugated mRNA

Duplex ID 500 nM AVG 5 nM AVG 500 nM STDEV 5 nM STDEV AD-58093.1 63, 94 83, 09 2, 14 12, 65 AD-58099.1 61.34 85, 85 12.32 21.95 AD-58105.1 91.98 97.57 6, 09 11.48 AD-58111.1 71.27 92.28 1, 93 12.72 AD-58117.1 73.42 88.82 3.24 And, 08 AD-58123.1 75, 14 73, 06 7, 72 9.71 AD-58129.1 81, 66 90, 62 2, 13 4.77 AD-58088.1 53, 63 87.03 5, 93 19, 86 AD-58094.1 89, 62 93, 65 0.87 14.76 AD-58100.1 79, 56 96.70 4.31 1.10 AD-58106.1 116.24 125.99 14.28 40, 65 AD-58112.1 97.19 107.81 N/A 3, 13 AD-58118.1 67.40 97.38 5.28 22, 64 AD-58124.1 58.04 96, 14 8.72 10, 64 AD-58130.1 84.19 88, 65 10.50 4.34 AD-58089.1 83, 83 83.44 1, 91 12.26 AD-58095.1 58.53 78.02 July 15 12:45 AD-58101.1 76, 68 76, 73 3.95 6, 35 AD-58107.1 57.37 86.78 14.71 2.99 AD-58113.1 37.79 71.10 8.27 7.76 AD-58119.1 36, 77 83.16 3, 42 9, 66 AD-58125.1 72.40 96, 53 4.46 4.96 AD-58131.1 95, 58 101.69 10, 17 2.21 AD-58090.1 56, 37 75.00 3.21 4.97 AD-58096.1 44.33 57.99 11.46 25, 17 AD-58102.1 95.46 89, 35 0.83 1.76

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AD-58108.1 41.54 56.41 8.41 0.14 AD-58114.1 88.32 101.88 20, 02 30.29 AD-58120.1 37.34 56.41 0.73 2.14 AD-58126.1 84.97 105.90 2.39 7.96 AD-58132.1 81.55 85, 12 12.93 8.94 AD-58091.1 78.88 84, 60 44, 66 17:40 AD-58097.1 106.06 98.16 13.74 3, 14 AD-58103.1 57.21 89.46 6, 40 5, 93 Unprocessed 100 100 8.77 10.33

Table 10

Single-dose free C5 uptake assay in primary mouse hepatocytes with GalNAC-conjugated mRNA

Duplex ID 500 nM AVG 5 nM AVG 500 nM STDEV 5 nM STDEV AD-58093.1 31, 62 64.91 7.13 8.39 AD-58099.1 9.46 29, 63 1.29 5, 66 AD-58105.1 84.77 96.41 5, 22 1, 89 AD-58111.1 17.35 50.95 1.21 3, 16 AD-58117.1 94, 95 139.52 15, 43 43.39 AD-58123.1 July 13 44.58 2.11 3, 49 AD-58129.1 68, 87 85, 04 2, 62 4.42 AD-58088.1 17, 61 48.22 2.22 3, 40 AD-58094.1 95, 92 104.23 4.16 6, 53 AD-58100.1 34, 92 61.71 1.30 2, 15 AD-58106.1 85.26 107.53 2.30 3, 38 AD-58121.1 12, 88 43.76 1.41 1.28 AD-58133.1 20, 97 42.76 0.24 0.11 AD-58116.1 8.35 38.04 1.35 1.40 Unprocessed 100.00 100.00 3, 85 4.38

Table 11

IC ₅₀ values in primary cynomolgus monkey hepatocytes with GalNAC-conjugated mRNA

Duplex ID 1C ₅₀ (nM) STDEV AD-58099.1 3, 131 1, 141 AD-58111.1 12,750 5,280 AD-58123.1 0.679 7,587 AD-58088.1 0.218 3,487 AD-58113.1 7,296 3,540 AD-58119.1 33,240 14,740 AD-58096.1 10,380 4, 199 AD-58108.1 0.953 10,080 AD-58120.1 36,170 88,070

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Table 12

IC ₅ values in primary mouse hepatocytes with GalNAC-conjugated mRNA

Duplex ID 1C ₅₀ (nM) STDEV AD-58099 3,777 0.122 AD-58111 0.622 2,421 AD-58123 0.549 1,626 AD-58088 9,513 2,588 AD-58121 2,169 1,176 AD-58133 3,802 1,006 AD-58116 2,227 0, 604 AD-58644.1 4,596 0.3506 AD-58651.1 59.76 51.99 AD-58641.1 0.82 0.2618 AD-58648.1 7,031 1,256 AD-58642.1 0.5414 0.7334 AD-58649.1 3.32 4,922 AD-58647.1 1,356 0.5215 AD-58654.1 2.09 0.8338 AD-58645.1 2,944 0.3315 AD-58652.1 5, 316 2,477 AD-58643.1 2,179 1,112 AD-58650.1 8,223 3.76 AD-58646.1 2,581 0.8186 AD-58653.1 2,451 1,249

Single-dose C5 assay in Hep3B cells with modified and unmodified mRNA

Table 13

Duplex 1 nM AVG 0.01 nM AVG 1 nM STDEV 0.01 nM STDEV AD-58143.1 12.13 100.58 3.47 3, 94 AD-58149.1 10.46 64.97 0.98 0.00 AD-58155.1 44.88 76, 24 1.56 3.74 AD-58161.1 8.51 102.30 1.06 0.50 AD-58167.1 6, 54 76, 24 1.15 3.74 AD-58173.1 6, 85 107.44 0.85 4.74 AD-58179.1 10, 19 78, 07 0.59 1.15 AD-58185.1 29, 46 79, 99 3, 64 0.78 AD-58144.1 16, 82 81, 95 1.09 0.40 AD-58150.1 11, 05 76, 20 2.55 0.00 AD-58156.1 25, 92 76, 73 2.72 1.50 AD-58162.1 13, 25 71, 89 0.43 3, 87 AD-58168.1 9, 74 45, 16 0.52 1.11 AD-58174.1 4.84 70, 14 0.25 2.75 AD-58180.1 9.41 56, 77 1.91 1.95 AD-58186.1 9, 97 68, 91 1.03 0.34 AD-58145.1 14.29 103.38 1.94 2.03 AD-58151.1 10, 16 81, 17 1.71 4.77 AD-58157.1 4.72 63, 19 1.05 0.00

- 119044245

AD-58163.1 4.95 40, 13 1, 65 0.59 AD-58169.1 17.02 83, 10 1.88 2.04 AD-58175.1 8:30 62.54 0.28 0.31 AD-58181.1 21.89 55.26 4.22 3.52 AD-58187.1 61.96 71.12 2, 61 2.79 AD-58146.1 14.25 95, 23 2, 64 6, 53 AD-58152.1 11.22 70, 09 0.80 7.88 AD-58158.1 7, 96 98.86 0.76 4.36 AD-58164.1 11, 60 43, 83 2.06 3.43 AD-58170.1 12.28 39, 59 0.96 1.36 AD-58176.1 6, 89 38.77 1.04 1.33 AD-58182.1 18, 65 55.78 0.96 0.55 AD-58188.1 5, 40 69, 39 1.07 0.34 AD-58147.1 8.22 106.66 0.77 2, 61 AD-58153.1 68.10 104.17 4.44 18.29 AD-58159.1 8.76 81.41 1.54 2.79 AD-58190.1 21.94 77.26 2.23 0.76 AD-58196.1 15, 97 72.43 1.07 5, 32 AD-58202.1 11.99 93, 83 5, 34 2.76 AD-58208.1 18, 63 52.07 12.88 2.55 AD-58214.1 6, 85 94.15 0.51 2.31 AD-58220.1 11.50 78.34 3, 85 0.77 AD-58226.1 5, 77 57.75 1.71 1.13 AD-58231.1 7.23 75, 67 1.07 0.74 AD-58191.1 35.40 66, 17 5.50 4.21 AD-58197.1 12.05 67.49 1.70 0.33 AD-58203.1 15.16 66, 80 1.46 1.31 AD-58209.1 7.58 71.23 3.58 6.28 AD-58233.1 27.01 86, 02 0.86 0.42 AD-58193.1 15.37 99, 85 1.44 0.00 AD-58199.1 21.52 78.39 6, 02 16.40 AD-58205.1 24.13 78.88 5.46 0.77

- 120 044245

AD-58211.1 16.38 32.37 2, 61 0.48 AD-58217.1 12.23 70.16 0.29 3.44 AD-58223.1 8.51 72.85 3, 01 1.79 AD-58229.1 5.50 75, 93 1.96 0.37 AD-58234.1 46.86 101.94 15, 59 0.00 AD-58194.1 14.49 107.05 2.47 4.20 AD-58200.1 16.21 61.04 0.96 1.20 AD-58206.1 13.25 37.73 2.82 2.03 AD-58236.1 8.29 119.17 1.16 2.92 AD-58242.1 12.05 102.69 0.44 4.03 AD-58248.1 62.78 83.41 15, 22 3.27 AD-58254.1 11.18 100.54 1.59 0.00 AD-58260.1 8.42 71.84 1, 10 0.35 AD-58266.1 14.05 92.21 1.91 2.26 AD-58272.1 22, 63 81, 11 1, 62 1.59 AD-58277.1 70.51 75, 67 4.80 0.74 AD-58237.1 28.10 98.56 1.96 5.79 AD-58243.1 14.16 86, 05 1.11 2.95 AD-58249.1 77, 08 96, 45 15, 14 0.95 AD-58255.1 12.27 47.89 2.58 0.00 AD-58279.1 25.78 94.13 5, 52 0.46 AD-58239.1 22.98 83.45 0.28 4.91 AD-58245.1 89, 60 90, 93 15, 24 0.45 AD-58251.1 28.39 86, 32 7.29 0.00 AD-58257.1 48.97 64.53 9, 10 1.90 AD-58263.1 9, 14 83.39 1.27 1, 63 AD-58269.1 83.84 75, 94 15, 90 1.12 AD-58275.1 10.29 86, 32 0.73 0.85 AD-58280.1 72.77 110.04 7.44 3.24 AD-58240.1 65.42 75, 69 3, 82 2.23 AD-58246.1 59, 19 65, 88 28.95 0.65 AD-58252.1 15.35 97.26 1.14 7, 62 Imitation 76.53 66, 57 14.26 4.72 AD-1955 72.30 82.72 19, 54 49, 99 Unprocessed 100.00 100.00 21, 68 26.78

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Table 14

Single-dose C5 assay in primary mouse hepatocytes with modified and ^-modified mRNAs

1 nM 0.1 nM Duplex ID 1 nM AVG 0.1 nm AVG STDEV STDEV AD-58143.1 4.51 81.77 3, 13 8.75 AD-58149.1 4, 65 73.16 3, 14 20, 17 AD-58155.1 65, 56 79, 74 4, 66 9.36 AD-58161.1 16, 82 81.11 6, 22 7.43 AD-58167.1 4.72 77.12 1.17 14.25 AD-58173.1 5, 57 76.00 3, 14 13.52 AD-58179.1 14.55 77.88 1.44 18:40 AD-58185.1 15, 69 72.59 8, 67 7.81 AD-58144.1 8.70 91.49 0.90 7.08 AD-58150.1 12.51 84.01 1, 64 8.20 AD-58156.1 18.23 97.32 1.47 19, 50 AD-58162.1 7.72 78.89 5, 19 13, 80 AD-58190.1 11.86 92.80 2.82 4.41 AD-58196.1 7.27 82.71 1.39 31.81 AD-58202.1 10, 67 87.11 1, 04 35.79 AD-58208.1 32.21 74.39 8, 60 27.45 AD-58214.1 4.24 67, 63 0.45 17.85 AD-58220.1 13, 64 96, 14 4.56 14.36 AD-58226.1 3, 83 63.44 1.30 11.94 AD-58231.1 5.95 82.24 2.80 17.36 AD-58191.1 14.50 99, 50 5.48 5, 53 AD-58197.1 16, 12 93, 09 0.81 3.21 AD-58203.1 12.52 104.63 5, 98 6, 02

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AD-58209.1 8.79 59, 35 3, 05 July 13 AD-58233.1 9, 50 64.26 5, 69 8.70 AD-58193.1 8.88 89, 60 3.36 3, 08 AD-58199.1 13.56 87.14 2.18 6, 44 AD-58205.1 46, 84 89, 13 4.48 17.16 AD-58211.1 13, 10 111.62 1, 10 21.54 AD-58217.1 29.79 117.49 11.85 20.41 AD-58223.1 20.53 105.44 1.94 2.98 AD-58229.1 13.76 98.15 1, 05 9, 03 AD-58234.1 12.33 71.34 0.72 4.17 AD-58194.1 14.02 90, 60 1.39 15, 64 AD-58200.1 5, 25 90, 95 1.37 31.70 AD-58206.1 8, 19 109.47 3.99 21.75 AD-58236.1 2.07 70, 19 0.80 20.59 AD-58242.1 4.76 53.26 1.59 11.56 AD-58248.1 62.42 78.23 5, 47 25, 85 AD-58254.1 16, 47 70.22 2.92 21.74 AD-58260.1 2.84 75, 65 0.38 11.59 AD-58266.1 40.70 89, 88 16, 05 11.57 AD-58272.1 21.42 59, 44 13.29 10, 98 AD-58277.1 71.72 121.44 16.35 21.16 AD-58237.1 11.85 112.68 9, 22 12.88 AD-58243.1 10.46 90, 64 3.42 4.33 AD-58249.1 71.47 113.30 4.30 3, 84 AD-58255.1 6.86 78.55 2.22 28.37 AD-58279.1 7.15 74.96 2.84 4.72 AD-58239.1 13, 64 106, 45 1.87 8.25 AD-58245.1 68, 67 112.08 21.89 7.73 AD-58251.1 47.01 133.20 4, 69 7.14 AD-58257.1 30, 68 87.51 2.87 32.84 AD-58263.1 7.22 83.23 2.55 37.50 AD-58269.1 78.90 106, 06 5, 07 3, 04 AD-58275.1 8.92 95, 77 1.91 7.14 AD-58280.1 16, 67 78.47 4.15 6, 06 AD-58240.1 71.03 138.54 5, 32 10, 87 AD-58246.1 71.87 89, 02 4.95 8, 63 AD-58252.1 4.04 56, 10 1.23 12.02 Imitation 66, 84 82.81 2.75 17.19 AD-1955 87.44 102.07 3, 64 4.08 Unprocessed 100.00 100.00 15.25 18.37

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Table 15

IC ₅₀ values in Hep3B cells with modified and unmodified mRNA

Duplex ID 1C ₅₀ (PM) STDEV AD-58143.1 36, 35 12.26 AD-58149.1 5,735 6, 196 AD-58161.1 78.12 26, 64 AD-58167.1 31.03 18, 14 AD-58173.1 29, 12 16, 53 AD-58236.1 52.73 32.02 AD-58242.1 8,859 4,321 AD-58260.1 7,706 5,094 AD-58263.1 96, 64 47, 61

Table 16

IC50 values in primary mouse hepatocytes with modified and unmodified mRNA

Duplex ID 1C ₅₀ (PM) STDEV AD-58260.1 1,015 0.9676 AD-58149.1 1,309 1,749 AD-58167.1 1,991 2,477 AD-58242.1 0.5866 1.8 AD-58236.1 0.4517 0.06392 AD-58143.1 0.8876 0.1613 AD-58279.1 3, 116 0.7368 AD-58252.1 7, 153 1,021 AD-58173.1 7, 144 19, 88 AD-58263.1 3,224 5,478

Example 3. In vivo screening.

A subset of seven GalNAC-conjugated mRNAs was selected for further in vivo study.

C57BL/6 mice (n=3 per group) were injected subcutaneously with 10 mg/kg GalNAc-conjugated duplexes or an equal volume of 1× Dulbecco's phosphate-buffered saline (DPBS) (Life Technologies, catalog #14040133). Forty-eight hours later, mice were sacrificed, and the livers were excised and snap-frozen in liquid nitrogen. Livers were ground in a 2000 Geno/Grinder (SPEX, SamplePrep, Metuchen, NJ). Approximately 10 mg of liver powder per sample was used for RNA extraction. Samples were first homogenized in a TissueLyserII (Qiagen Inc., Valencia, CA), and then RNA was extracted with an RNeasy 96 Universal Tissue Kit (Qiagen Inc., catalog #74881) according to the manufacturer's protocol using vacuum/spin technology. RNA concentration was measured with a NanoDrop 8000 (Thermo Scientific, Wilmington, DE) and adjusted to 100 ng/µL. cDNA and RT-PCR were performed as described above.

The results of the single-dose assay are shown in Fig. 2. Table 17 shows the results of the in vivo single-dose assay with the indicated GalNAC-conjugated modified mRNAs. Data are expressed as the percentage of remaining mRNA relative to DPBS-treated mice. The Experiments column lists the number of experiments from which the mean value was calculated. The standard deviation was calculated for all mice in a group across all analyzed experiments.

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Table 17

In vivo single dose test of C5

Duplex ID Experiments AVG STDEV AD-58088.2 2 82, 66 13.54 AD-58644.1 1 37.79 9, 63 AD-58651.1 1 75, 33 5.21 AD-58099.2 2 71.94 15:45 AD-58641.1 1 20.09 4.09 AD-58648.1 1 48.43 9, 07 AD-58111.2 3 67.17 13, 60 AD-58642.1 2 21.78 5, 32 AD-58649.1 1 45, 30 14, 02 AD-58116.2 2 70.16 10, 32 AD-58647.1 1 26, 77 4.14 AD-58654.1 1 50, 06 27, 85 AD-58121.2 2 52.56 13:00 AD-58645.1 1 24, 60 1.29 AD-58652.1 1 52, 67 3, 87 AD-58123.2 2 65, 70 9, 60 AD-58643.1 1 23.21 2.41 AD-58 650.1 1 46, 75 14.10 AD-58133.2 3 51.98 13:45 AD-58646.1 2 28, 67 5, 34 AD-58 653.1 1 43, 02 10, 61 PBS 3 100.00 9, 03

The two most potent GalNa-conjugated mRNAs were further modified to incorporate additional phosphorothioate linkages (Table 18), and the potency of these duplexes was determined in vivo as described above. The results of the single-dose assay are shown in Fig. 3 and demonstrate that mRNA agents with additional phosphorothioate linkages are more potent than mRNA agents with no or fewer phosphorothioate linkages.

Table 18

GalNAC-conjugated C5 mRNA modified with phosphorothioate linkages

Duplex ID Semantic thread Semantic sequence SEQ ID NO: Antisense Antisense sequence SEQ ID NO: Cross-reactivity AD- 58642.1 A- 119324.1 GfsasCfaAfaAfuAfAf CfuCfaCfuAfuAfaUfL 96 551 A-119325.1 asUfsuAfuAfgUfg AfguuAfuUfuUfgU fcsasa 555 HumRheMusRat AD- 58111.2 A- 118316.1 GfaCfaAfaAfuAfAfCf uCfaCfuAfuAfaUfL96 552 A-118317.1 aUfuAfuAfgUfgAf guuAfuUfuUfgUfc sAf sa 556 HumRheMusRat AD- 58646.1 A- 119332.1 CfsasGfaUfcAfaAfCf Af cAfaUfuUfcAfgUfL 96 553 A-119333.1 asCfsuGfaAfaUfu GfuguUfuGfaUfcU fgscsa 557 MusRat AD- 58133.2 A- 118386.1 CfaGfaUfcAfaAfCfAf cAfaUfuUfcAfgUfL96 554 A-118387.1 aCfuGfaAfaUfuGf uguUfuGfaUfcUfg sCf sa 558 MusRat

Given the influence of additional phosphorothioate linkages on the silencing abilities of the mRNA agents described above, the efficacy of additional GalNAC-conjugated RNA duplexes containing phosphorothioate linkages (Table 19) was determined in vivo as described above. The results of this single-dose assay are shown in Fig. 4.

The duration of silencing by AD-58642 in vivo was determined by administering a single dose of 2.5 mg/kg, 10 mg/kg, or 25 mg/kg to rats and determining the amount of C5 protein (Fig. 5B) present.

- 125 044245 on day 7, and C5 protein activity (Fig. 5A) present on days 4 and 7. As shown in Fig. 5, there is a 50% decrease in C5 protein activity on day 4 at the 25 mg/kg dose and a greater than 70% decrease in C5 protein activity on day 7.

C5 protein was quantified by Western blotting of whole serum. C5 protein activity was determined in a hemolysis assay. Briefly, a fixed dilution of human C5 depleted in human serum was mixed with mouse serum and incubated with antibody-coated sheep red blood cells for 1 hour. Hemoglobin uptake was measured, and the % hemolysis was calculated compared to a reference curve (obtained using a series of mouse serum dilutions).

The in vivo efficacy of AD-58642 was also analyzed in mice following single subcutaneous administration of 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, and 25 mg/kg AD-58642. On day 5, C5 mRNA was analyzed in liver samples using qPCR, C5 activity was analyzed by hemolysis, and C5 protein was quantified by Western blot analysis of whole serum.

As shown in Figs. 6A and 6B, although there is only a modest improvement (i.e., approximately 5%) in the potency of AD-58642 to inhibit C5 mRNA at 25 mg/kg compared to 10 mg/kg, an average of 85% silencing is observed at 25 mg/kg. Furthermore, a dose-response effect is observed with IC ₅₀ values of approximately 2.5 mg/kg.

Figures 7A and 7B and 8 demonstrate that AD-58642 is effective in reducing the amount of C5 protein (Figure 8) and the activity of C5 protein (7A and 7B).

The duration of AD-58641 silencing in vivo was determined by administering a single dose of 0.625 mg/kg, 1.25 mg/kg, 2.5 mg/kg, 5.0 mg/kg, or 10 mg/kg AD-58641 to C57B1/6 mice (n=3) and determining the amount of C5 protein present in these animals on days 5 and 9 by ELISA. Briefly, day 0 pre-screened serum was collected, and C5 protein levels were quantified by ELISA on day 5 and day 9. C5 protein levels were normalized to the day 0 level in pre-screened serum. As shown in Fig. 9, the results demonstrate that there is a dose-dependent effect and durable knockdown of C5 protein in serum (single dose ED ₅₀ was 0.6 mg/kg).

AD-58641 was also tested for efficacy using a multiple-dose protocol in C57BL/6 mice. Mice were administered AD-58641 subcutaneously at 0.625 mg/kg, 1.25 mg/kg, or 2.5 mg/kg on days 0, 1, 2, and 3. Serum was collected on days 0 and 8, as shown in Fig. 10, and analyzed for C5 protein levels by ELISA. C5 levels were normalized to day 0 levels in pre-collected sera. Fig. 10 shows that multiple doses of AD-58641 resulted in C5 protein silencing at all doses tested, with greater than 90% C5 protein silencing at the 2.5 mg/kg dose.

AD-58641 was further tested for efficacy and the cumulative effect of the compound was assessed in rats using a repeated administration protocol. Wild-type Sprague Dawley rats were injected subcutaneously with AD-58641 at a dose of 2.5 mg/kg/dose or 5.0 mg/kg/dose twice weekly for 3 weeks (q2w x3). Serum was collected on days 0, 4, 7, 11, 14, 18, 25, and 32. Serum hemolytic activity was measured using a hemolysis assay in which a 1:150 dilution of rat serum was incubated with sensitized sheep and rat blood cells in GVB++ buffer for 1 h, and hemoglobin release was quantified by measuring the optical density at 415 nm (see Fig. 11A). The amount of C5 protein present in the samples was also determined using ELISA (Fig. 11B). The results demonstrate a dose-dependent effect and a long-lasting reduction in hemolytic activity, achieving approximately 90% inhibition of hemolytic activity.

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Table 19

Additional GalNAC-conjugated C5 mRNAs modified with phosphorothioate linkages

Duplex ID Semantic thread Semantic sequence SEQ ID NO: Antisense Antisense sequence SEQ ID NO: Starting position Cross-reactivity PS# AD- 58088.2 A- 118324.1 AfuUfuAfaAfc AfAfCfaAfgUf aCfcUfuUfL96 559 A- 118325.1 aAfaGfgUfaCfuU fguuGfuUfuAfaA fusCfsu 580 984 HumRheMus 2 AD- 58644.1 A- 119328.1 AfsusUfuAfaA fcAfAfCfaAfg UfaCfcUfuUfL 96 560 A- 119329.1 asAfsaGfgUfaCf uUfguuGfuUfuAf aAfuscsu 581 984 HumRheMus 6 AfsusUfuAfaA asAfsaGfsgUfsa AD- 58651.1 A- 119328.2 fcAfAfCfaAfg UfaCfcUfuUfL 561 A- 119339.1 CfsuUfsguuGfsu UfsuAfsaAfsusc 582 984 HumRheMus 14 96 su AD- 58099.2 A- 118312.1 U f gAf cAf aAf a UfAfAfcUfcAf cUfaUfaAfL96 562 A- 118313.1 uUfaUfaGfuGfaG fuuaUfuUfuGfuC fasAfsu 583 1513 HumRheMusRat 2 AD- 58641.1 A- 119322.1 UfsgsAfcAfaA faUfAfAfcUfc AfcUfaUfaAfL 96 563 A- 119323.1 usUfsaUfaGfuGf aGfuuaUfuUfuGf uCfasasu 584 1513 HumRheMusRat 6 UfsgsAfcAfaA usUfsaUfsaGfsu AD- 58648.1 A- 119322.2 faUfAfAfcUfc AfcUfaUfaAfL 564 A- 119336.1 GfsaGfsuuaUfsu UfsuGfsuCfsasa 585 1513 HumRheMusRat 14 96 su AD- 58111.2 A- 118316.1 GfaCfaAfaAfu AfAfCfuCfaCf uAfuAfaUfL96 565 A- 118317.1 aUfuAfuAfgUfgA fguuAfuUfuUfgU fcsAfsa 586 1514 HumRheMusRat 2 AD- 58642.1 A- 119324.1 GfsasCfaAfaA fuAfAfCfuCfa CfuAfuAfaUfL 96 566 A- 119325.1 asUfsuAfuAfgUf gAfguuAfuUfuUf gUfcsasa 587 1514 HumRheMusRat 6 GfsasCfaAfaA asUfsuAfsuAfsg AD- 58649.1 A- 119324.2 fuAfAfCfuCfa CfuAfuAfaUfL 567 A- 119337.1 UfsgAfsguuAf su UfsuUfsgUfscsa 588 1514 HumRheMusRat 14 96 sa AD- 58116.2 A- 118396.1 GfuUfcCfgGfa UfAfUfuUfgAf aCfuUfuUfL96 568 A- 118397.1 aAfaAfgUfuCfaA fauaUfcCfgGfaA fcsCfsg 589 4502 MusRat 2

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AD- 58647.1 A- 119334.1 GfsusUfcCfgG faUfAfUfuUfg AfaCfuUfuUfL 96 569 A- 119335.1 asAfsaAfgUfuCf aAfauaUfcCfgGf aAfcscsg 590 4502 MusRat 6 AD- 58654.1 A- 119334.2 GfsusUfcCfgG faUfAfUfuUfg AfaCfuUfuUfL 96 570 A- 119342.1 asAfsaAfsgUfsu CfsaAfsauaUfsc CfsgGfsaAfscsc sg 591 4502 MusRat 14 AD- 58121.2 A- 118382.1 UfgCfaGfaUfc AfAfAfcAfcAf aUfuUfcAfL96 571 A- 118383.1 uGfaAfaUfuGfuG fuuuGfaUfcUfgC fasGfsa 592 4945 MusRat 2 AD- 58645.1 A- 119330.1 UfsgsCfaGfaU fcAfAfAfcAfc AfaUfuUfcAfL 96 572 A- 119331.1 usGfsaAfaUfuGf uGfuuuGfaUfcUf gCfasgsa 593 4945 MusRat 6 AD- 58652.1 A- 119330.2 UfsgsCfaGfaU fcAfAfAfcAfc AfaUfuUfcAfL 96 573 A- 119340.1 usGfsaAfsaUfsu GfsuGfsuuuGfsa UfscUfsgCfsasg sa 594 4945 MusRat 14 AD- 58123.2 A- 118320.1 AfaGfcAfaGfa UfAfUfuUfuUf aUfaAfuAfL96 574 A- 118321.1 uAfuUfaUfaAfaA fauaUfcUfuGfcU fusUfsu 595 786 HumRheMus 2 AD- 58643.1 A- 119326.1 AfsasGfcAfaG faUfAfUfuUfu UfaUfaAfuAfL 96 575 A- 119327.1 usAfsuUfaUfaAf aAfauaUfcUfuGf cUfususu 596 786 HumRheMus 6 AD- 58650.1 A- 119326.2 AfsasGfcAfaG faUfAfUfuUfu UfaUfaAfuAfL 96 576 A- 119338.1 usAfsuUfsaUfsa Af s aAf s auaUf s c UfsuGfscUfsusu su 597 786 HumRheMus 14 AD- 58133.2 A- 118386.1 CfaGfaUfcAfa AfCfAfcAfaUf uUfcAfgUfL96 577 A- 118387.1 aCfuGfaAfaUfuG fuguUfuGfaUfcU fgsCfsa 598 4947 MusRat 2 AD- 58646.1 A- 119332.1 CfsasGfaUfcA faAfCfAfcAfa UfuUfcAfgUfL 96 578 A- 119333.1 asCfsuGfaAfaUf uGfuguUfuGfaUf cUfgscsa 599 4947 MusRat 6 AD- 58653.1 A- 119332.2 CfsasGfaUfcA faAfCfAfcAfa UfuUfcAfgUfL 96 579 A- 119341.1 asCfsuGfsaAfsa Uf suGfsuguUfsu GfsaUfscUfsgsc sa 600 4947 MusRat 14

Example 4. Design, synthesis and screening of additional siRNAs in vitro siRNA design.

Nineteen-nucleotide C5 duplexes for both sense and antisense strands were constructed using the human C5 mRNA sequence submitted to GenBank accession no. NM_001735.2. Five hundred sixty-nine duplexes containing no repeats longer than seven nucleotides were initially identified, encompassing essentially the entire 5,480-nucleotide transcript. All 569 duplexes were then evaluated for predicted efficiency according to a linear model that estimates base pairs at each duplex position, and dose and cell line were used for screening. Duplexes were also aligned to all transcripts in the human RefSeq collection using a proprietary brute-force algorithm, and the smallest mismatch values (per strand) to transcripts other than C5 were calculated. Duplexes were synthesized and screened, and then selected from 569, according to the following scheme: Starting from the 5' end of the transcript, a duplex was selected within a window of every 10±2 nucleotides that:

1) had the highest predicted efficacy,

2) had at least one mismatch in both strands to all transcripts other than SERPINC1,

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3) have not yet been synthesized and screened as part of other duplex sets.

If a duplex that met all criteria was not identified in a given window, the window was skipped.

A detailed list of 569 C5 sense and antisense strand sequences is shown in Table 20.

The in vitro efficiency of duplexes containing the sense and antisense sequences listed in Table 20 was determined using the following methods.

Cell culture and transfections.

HepG2 cells (ATCC, Manassas, VA) were grown to near confluence at 37°C under 5% _CO2 in Eagle's minimal essential medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before detaching from the dish by trypsinization. Transfection was performed by adding 14.8 μl of Opti-MEM with 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad, CA, cat. #13778-150) to 5 μl of each of the 164 siRNA duplexes in a separate well in a 96-well plate. The mixture was then incubated at room temperature for 15 min. Eighty microliters of complete culture media without antibiotics, containing ~2.5 x ¹⁰⁴ HepG2 cells, were then added to the siRNA mixture. The cells were incubated for 24 hours before RNA purification. Experiments were performed at 20 nM and included naive cells and cells transfected with AD-1955, with luciferase-targeting siRNA serving as a negative control.

Total RNA isolation using the DYNABEADS mRNA Isolation Kit (Invitrogen, catalog #610-12).

Cells were collected and lysed in 150 μl of lysis/binding buffer, then mixed for 5 min at 700 rpm on a platform shaker (mixing speed was maintained throughout). Ten microliters of magnetic beads and 80 μl of lysis/binding buffer mixture were added to a round-bottomed plate and mixed for 1 min. The magnetic beads were fixed using a magnetic stand, and the supernatant was removed without dislodging the beads. After removing the supernatant, lysed cells were added to the remaining beads and mixed for 5 min. After removing the supernatant, the magnetic beads were washed twice with 150 μl of wash buffer A and mixed for 1 min. The beads were fixed again, and the supernatant was removed. The beads were then washed with 150 μl of wash buffer B, fixed, and the supernatant was removed. The beads were then washed with 150 µl of elution buffer B, fixed, and the supernatant removed. The beads were allowed to dry for 2 min. After drying, 50 µl of elution buffer was added and mixed for 5 min at 70°C. The beads were fixed on a magnet for 5 min. Forty µl of the supernatant containing the isolated RNA was removed and added to another 96-well plate.

cDNA synthesis using the ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Forster City, CA, cat. #4368813).

A master mix of 2 μl 10X buffer, 0.8 μl 25X dNTPs, 2 μl random primers, 1 μl reverse transcriptase, 1 μl RNase inhibitor, and 3.2 μl _H2O per reaction was added to 10 μl total RNA. cDNA was prepared using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, California) using the following steps: 25°C for 10 min, 37°C for 120 min, 85°C for 5 s, and storage at 4°C.

Real-time PCR.

µl of cDNA was added to a master mix mixture containing 0.5 µl of TaqMan probe for human GAPDH (Applied Biosystems, catalog #4326317E), 0.5 µl of TaqMan probe for human SERPINC1 (Applied Biosystems, catalog #Hs00892758_m1), and 5 µl of Lightcycler 480 master mix probe (Roche, catalog #04887301001) in each well of a 384-well plate (Roche, catalog #04887301001). Real-time PCR was performed on an LC480 Real Time PCR instrument (Roche).

To calculate the relative fold change, real-time data were analyzed using the AACt method and normalized to those of assays performed with cells transfected with 20 nM AD-1955.

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Table 20

Additional sequences of the unmodified and antisense strand of C5

Oligonucleotide name Position in NM_001735.2 Semantic sequence SEQ ID NO: Antisense sequence SEQ ID NO: NM_001735.2_321_s 3-21 UAUCCGUGGUUUCCUGCUA 601 UAGCAGGAAACCACGGAUA 1170 NM_001735.2_10- 2 8_s 10-28 GGUUUCCUGCUACCUCCAA 602 UUGGAGGUAGCAGGAAACC 1171 NM-001735.2_22- 4 0_s 22:40 CCUCCAACCAUGGGCCUUU 603 AAAGGCCCAUGGUUGGAGG 1172 NM_001735.2_3351_s 33-51 GGGCCUUUUGGGAAUACUU 604 AAGUAUUCCCAAAAGGCCC 1173 NM_001735.2_4361_s 43-61 GGAAUACUUUGUUUUUUAA 605 UUAAAAAACAAAGUAUUC C 1174 NM_001735.2_4967_s 49-67 CUUUGUUUUUUAAUCUUCC 606 GGAAGAUUAAAAAACAAAG 1175 NM_001735.2_6381_s 63-81 CUUCCUGGGGAAAACCUGG 607 CCAGGUUUUCCCCAGGAAG 1176 NM_001735.2_71- 8 9_s 71-89 GGAAAACCUGGGGACAGGA 608 UCCUGUCCC CAGGUUUUC C 1177 NM_001735.2_8199_s 81-99 GGGACAGGAGCAAACAUAU 609 AUAUGUUUGCUCCUGUCCC 1178 NM_001735.2_91- 10 9_s 91-109 CAAACAUAUGUCAUUUCAG 610 CUGAAAUGACAUAUGUUUG 1179 NM_001735.2_102- 12 0_s 102-120 CAUUUCAGCACCAAAAAUA 611 UAUUUUUGGUGCUGAAAUG 1180 NM_001735.2_109127_s 109-127 GCAC CAAAAAUAUUC C GUG 612 CACGGAAUAUUUUUGGUGC 1181 NM_001735.2_123141_s 123-141 CCGUGUUGGAGCAUCUGAA 613 UUCAGAUGCUCCAACACGG 1182 NM_001735.2_130- 14 8_s 130-148 GGAGCAUCUGAAAAUAUUG 614 CAAUAUUUUCAGAUGCUCC 1183 NM_001735.2_139157_s 139157 GAAAAUAUUGUGAUUCAAG 615 CUUGAAUCACAAUAUUUUC 1184 NM_001735.2_150168_s 150-168 GAUUCAAGUUUAUGGAUAC 616 GUAUC CAUAAACUUGAAUC 1185 NM_001735.2_163181_s 163-181 GGAUACACUGAAGCAUUUG 617 CAAAUGCUUCAGUGUAUCC 1186 NM_001735.2_172190_s 172-190 GAAGCAUUUGAUGCAACAA 618 UUGUUGCAUCAAAUGCUUC 1187 NM_001735.2_183- 2 01_s 183-201 UGCAACAAUCUCUAUUAAA 619 UUUAAUAGAGAUUGUUGCA 1188 NM_001735.2_189207_s 189-207 AAUCUCUAUUAAAAGUUAU 620 AUAACUUUUAAUAGAGAUU 1189 NM_001735.2_201219_s 201-219 AAGUUAUC CUGAUAAAAAA 621 UUUUUUAUCAGGAUAACUU 1190

- 130 044245

NM_001735.2_209227_s 209-227 CUGAUAAAAAAUUUAGUUA 622 UAACUAAAUUUUUUAUCAG 1191 NM_001735.2_221- 2 3 9_s 221-239 UUAGUUACUCCUCAGGCCA 623 UGGCCUGAGGAGUAACUAA 1192 NM_001735.2_230- 2 4 8_s 230-248 CCUCAGGCCAUGUUCAUUU 624 AAAUGAACAUGGCCUGAGG 1193 NM_001735.2_242- 2 60_s 242-260 UUCAUUUAUC CUCAGAGAA 625 UUCUCUGAGGAUAAAUGAA 1194 NM_001735.2_252- 2 7 0_s 252-270 CUCAGAGAAUAAAUUC CAA 626 UUGGAAUUUAUUCUCUGAG 1195 NM_001735.2_259- 2 7 7_s 259-277 AAUAAAUUC CAAAACUCUG 627 CAGAGUUUUGGAAUUUAUU 1196 NM_001735.2_273291_s 273-291 CUCUGCAAUCUUAACAAUA 628 UAUUGUUAAGAUUGCAGAG 1197 NM_001735.2_282- 3 0 0_s 282-300 CUUAACAAUACAAC CAAAA 629 UUUUGGUUGUAUUGUUAAG 1198 NM_001735.2_292310_s 292-310 CAAC CAAAACAAUUGC CUG 630 CAGGCAAUUGUUUUGGUUG 1199 NM_001735.2_301319_s 301-319 CAAUUGCCUGGAGGACAAA 631 UUUGUCCUCCAGGCAAUUG 1200 NM_001735.2_313331_s 313-331 GGACAAAAC C CAGUUUCUU 632 AAGAAACUGGGUUUUGUCC 1201 NM_001735.2_322- 34 0_s 322-340 CCAGUUUCUUAUGUGUAUU 633 AAUACACAUAAGAAACUGG 1202 NM_001735.2_332350_s 332-350 AUGUGUAUUUGGAAGUUGU 634 ACAACUUC CAAAUACACAU 1203 NM_001735.2_342- 3 60_s 342-360 GGAAGUUGUAUCAAAGCAU 635 AUGCUUUGAUACAACUUCC 1204 NM_001735.2_349367_s 349-367 GUAUCAAAGCAUUUUUCAA 636 UUGAAAAAUGCUUUGAUAC 1205 NM_001735.2_361- 37 9_s 361-379 UUUUCAAAAUCAAAAAGAA 637 UUCUUUUUGAUUUUGAAAA 1206 NM_001735.2_371- 3 8 9_s 371-389 CAAAAAGAAUGCCAAUAAC 638 GUUAUUGGCAUUCUUUUUG 1207 NM_001735.2_381- 3 99_s 381-399 GC CAAUAAC CUAUGACAAU 639 AUUGUCAUAGGUUAUUGGC 1208 NM_001735.2_389407_s 389-407 CCUAUGACAAUGGAUUUCU 640 AGAAAUCCAUUGUCAUAGG 1209 NM_001735.2_399417_s 399-417 UGGAUUUCUCUUCAUUCAU 641 AUGAAUGAAGAGAAAUC CA 1210 NM_001735.2_411- 42 9_s 411-429 CAUUCAUACAGACAAAC CU 642 AGGUUUGUCUGUAUGAAUG 1211 NM_001735.2_419437_s 419-437 CAGACAAAC CUGUUUAUAC 643 GUAUAAACAGGUUUGUCUG 1212 NM_001735.2_430- 4 4 8_s 430-448 GUUUAUACUC CAGAC CAGU 644 ACUGGUCUGGAGUAUAAAC 1213 NM_001735.2_441- 4 59_s 441-459 AGAC CAGUCAGUAAAAGUU 645 AACUUUUACUGACUGGUCU 1214

- 131 044245

NM_001735.2_450468_s 450-468 AGUAAAAGUUAGAGUUUAU 646 AUAACUCUAACUUUUACU 1215 NM_001735.2_460- 4 7 8_s 460-478 AGAGUUUAUUC GUUGAAUG 647 CAUUCAACGAAUAAACUCU 1216 NM_001735.2_470- 4 8 8_s 470-488 CGUUGAAUGACGACUUGAA 648 UUCAAGUC GUCAUUCAAC G 1217 NM_001735.2_483501_s 483-501 CUUGAAGCCAGCCAAAAGA 649 UCUUUUGGCUGGCUUCAAG 1218 NM_001735.2_490508_s 490-508 C CAGC CAAAAGAGAAACUG 650 CAGUUUCUCUUUUGGCUGG 1219 NM_001735.2_503521_s 503-521 AAACUGUCUUAACUUUCAU 651 AUGAAAGUUAAGACAGUUU 1220 NM_001735.2_513531_s 513-531 AACUUUCAUAGAUCCUGAA 652 UUCAGGAUCUAUGAAAGUU 1221 NM_001735.2_519537_s 519-537 CAUAGAUCCUGAAGGAUCA 653 UGAUCCUUCAGGAUCUAUG 1222 NM_001735.2_529547_s 529-547 GAAGGAUCAGAAGUUGACA 654 UGUCAACUUCUGAUCCUUC 1223 NM_001735.2_543561_s 543-561 UGACAUGGUAGAAGAAAUU 655 AAUUUCUUCUACCAUGUCA 1224 NM_001735.2_553571_s 553-571 GAAGAAAUUGAUCAUAUUG 656 CAAUAUGAUCAAUUUCUUC 1225 NM_001735.2_562- 58 0_s 562-580 GAUCAUAUUGGAAUUAUCU 657 AGAUAAUUC CAAUAUGAUC 1226 NM_001735.2_571- 58 9_s 571-589 GGAAUUAUCUCUUUUCCUG 658 CAGGAAAAGAGAUAAUUCC 1227 NM_001735.2_579597_s 579-597 CUCUUUUCCUGACUUCAAG 659 CUUGAAGUCAGGAAAAGAG 1228 NM_001735.2_590608_s 590-608 ACUUCAAGAUUCCGUCUAA 660 UUAGACGGAAUCUUGAAGU 1229 NM_001735.2_601619_s 601-619 С C GUCUAAUC CUAGAUAUG 661 CAUAUCUAGGAUUAGACGG 1230 NM_001735.2_610628_s 610-628 CCUAGAUAUGGUAUGUGGA 662 UC CACAUAC CAUAUCUAGG 1231 NM_001735.2_623641_s 623-641 UGUGGACGAUCAAGGCUAA 663 UUAGCCUUGAUCGUCCACA 1232 NM_001735.2_629647_s 629-647 CGAUCAAGGCUAAAUAUAA 664 UUAUAUUUAGC CUUGAUC G 1233 NM_001735.2_642- 6 60_s 642-660 AUAUAAAGAGGACUUUUCA 665 UGAAAAGUC CUCUUUAUAU 1234 NM_001735.2_649667_s 649-667 GAGGACUUUUCAACAACUG 666 CAGUUGUUGAAAAGUCCUC 1235 NM_001735.2_662- 68 0_s 662-680 CAACUGGAACCGCAUAUUU 667 AAAUAUGCGGUUCCAGUUG 1236 NM_001735.2_672690_s 672-690 CGCAUAUUUUGAAGUUAAA 668 UUUAACUUCAAAAUAUGCG 1237 NM_001735.2_683- 7 01_s 683-701 AAGUUAAAGAAUAUGUCUU 669 AAGACAUAUUCUUUAACUU 1238

- 132044245

NM_001735.2_691- 7 0 9_s 691-709 GAAUAUGUCUUGCCACAUU 670 AAUGUGGCAAGACAUAUUC 1239 NM_001735.2_703721_s 703-721 CCACAUUUUUCUGUCUCAA 671 UUGAGACAGAAAAAUGUGG 1240 NM_001735.2_713- 7 31_s 713-731 CUGUCUCAAUCGAGCCAGA 672 UCUGGCUCGAUUGAGACAG 1241 NM_001735.2_719737_s 719-737 C AAUC GAG C C AGAAUAUAA 673 UUAUAUUCUGGCUCGAUUG 1242 NM_001735.2_730- 7 4 8_s 730-748 GAAUAUAAUUUCAUUGGUU 674 AAC CAAUGAAAUUAUAUUC 1243 NM_001735.2_742- 7 60_s 742-760 AUUGGUUACAAGAACUUUA 675 UAAAGUUCUUGUAACCAAU 1244 NM_001735.2_752- 7 7 0_s 752-770 AGAACUUUAAGAAUUUUGA 676 UCAAAAUUCUUAAAGUUCU 1245 NM_001735.2_762- 7 8 0_s 762-780 GAAUUUUGAAAUUACUAUA 677 UAUAGUAAUUUCAAAAUUC 1246 NM_001735.2_769- 7 8 7_s 769-787 GAAAUUACUAUAAAAGCAA 678 UUGCUUUUAUAGUAAUUUC 1247 NM_001735.2_781- 7 99_s 781-799 AAAGCAAGAUAUUUUUAUA 679 UAUAAAAAUAUCUUGCUUU 1248 NM_001735.2_789807_s 789-807 AUAUUUUUAUAAUAAAGUA 680 UACUUUAUUAUAAAAAUAU 1249 NM_001735.2_803821_s 803-821 AAGUAGUCACUGAGGCUGA 681 UCAGCCUCAGUGACUACUU 1250 NM_001735.2_810828_s 810-828 CACUGAGGCUGACGUUUAU 682 AUAAACGUCAGCCUCAGUG 1251 NM_001735.2_822- 8 4 0_s 822-840 CGUUUAUAUCACAUUUGGA 683 UC CAAAUGUGAUAUAAAC G 1252 NM_001735.2_831- 8 4 9_s 831-849 CACAUUUGGAAUAAGAGAA 684 UUCUCUUAUUCCAAAUGUG 1253 NM_001735.2_840858_s 840-858 AAUAAGAGAAGACUUAAAA 685 UUUUAAGUCUUCUCUUAUU 1254 NM_001735.2_852- 8 7 0_s 852-870 CUUAAAAGAUGAUCAAAAA 686 UUUUUGAUCAUCUUUUAAG 1255 NM_001735.2_859- 8 7 7_s 859-877 GAUGAUCAAAAAGAAAUGA 687 UCAUUUCUUUUUGAUCAUC 1256 NM_001735.2_872- 8 90_s 872-890 AAAUGAUGCAAACAGCAAU 688 AUUGCUGUUUGCAUCAUUU 1257 NM_001735.2_883901_s 883-901 ACAGCAAUGCAAAACACAA 689 UUGUGUUUUGCAUUGCUGU 1258 NM_001735.2_893911_s 893-911 AAAACACAAUGUUGAUAAA 690 UUUAUCAACAUUGUGUUUU 1259 NM_001735.2_899917_s 899-917 CAAUGUUGAUAAAUGGAAU 691 AUUC CAUUUAUCAACAUUG 1260 NM_001735.2_913931_s 913-931 GGAAUUGCUCAAGUCACAU 692 AUGUGACUUGAGCAAUUCC 1261 NM_001735.2_919937_s 919-937 GCUCAAGUCACAUUUGAUU 693 AAUCAAAUGUGACUUGAGC 1262

- 133 044245

NM_001735.2_930948_s 930-948 AUUUGAUUCUGAAACAGCA 694 UGCUGUUUCAGAAUCAAAU 1263 NM_001735.2_939957_s 939-957 UGAAACAGCAGUCAAAGAA 695 UUCUUUGACUGCUGUUUCA 1264 NM_001735.2_951- 9 69_s 951-969 CAAAGAACUGUCAUACUAC 696 GUAGUAUGACAGUUCUUUG 1265 NM_001735.2_962- 98 0_s 962-980 CAUACUACAGUUUAGAAGA 697 UCUUCUAAACUGUAGUAUG 1266 NM_001735.2_969987_s 969-987 CAGUUUAGAAGAUUUAAAC 698 GUUUAAAUCUUCUAAACUG 1267 NM_001735.2_9831001_s 983-1001 UAAACAACAAGUAC CUUUA 699 UAAAGGUACUUGUUGUUUA 1268 NM_001735.2_9901008_s 990-1008 CAAGUACCUUUAUAUUGCU 700 AGCAAUAUAAAGGUACUUG 1269 NM_001735.2_10021020_s 1002-1020 UAUUGCUGUAACAGUCAUA 701 UAUGACUGUUACAGCAAUA 1270 NM_001735.2_10111029_s 1011-1029 AACAGUCAUAGAGUCUACA 702 UGUAGACUCUAUGACUGUU 1271 NM_001735.2_10201038_s 1020-1038 AGAGUCUACAGGUGGAUUU 703 AAAUCCACCUGUAGACUCU 1272 NM_001735.2_10331051_s 1033-1051 GGAUUUUCUGAAGAGGCAG 704 CUGCCUCUUCAGAAAAUCC 1273 NM_001735.2_10421060—s 1042-1060 GAAGAGGCAGAAAUACCUG 705 CAGGUAUUUCUGCCUCUUC 1274 NM—001735.2_10501068—s 1050-1068 AGAAAUACCUGGCAUCAAA 706 UUUGAUGCCAGGUAUUUCU 1275 NM—001735.2_10611079—s 1061-1079 GCAUCAAAUAUGUCCUCUC 707 GAGAGGACAUAUUUGAUGC 1276 NM—001735.2_10711089—s 1071-1089 UGUCCUCUCUCCCUACAAA 708 UUUGUAGGGAGAGAGGACA 1277 NM—001735.2_10921110—s 1092-1110 GAAUUUGGUUGCUACUCCU 709 AGGAGUAGCAACCAAAUUC 1278 NM—001735.2_11021120—s 1102-1120 GCUACUCCUCUUUUCCUGA 710 UCAGGAAAAGAGGAGUAGC 1279 NM—001735.2_11091127—s 1109-1127 CUCUUUUCCUGAAGCCUGG 711 CCAGGCUUCAGGAAAAGAG 1280 NM—001735.2_11231141—s 1123-1141 CCUGGGAUUCCAUAUCCCA 712 UGGGAUAUGGAAUCCCAGG 1281 NM—001735.2_11331151—s 1133-1151 CAUAUCCCAUCAAGGUGCA 713 UGCACCUUGAUGGGAUAUG 1282 NM—001735.2_11391157—s 1139-1157 CCAUCAAGGUGCAGGUUAA 714 UUAACCUGCACCUUGAUGG 1283 NM—001735.2_11501168—s 1150-1168 CAGGUUAAAGAUUCGCUUG 715 CAAGCGAAUCUUUAACCUG 1284 NM—001735.2_11611179—s 1161-1179 UUCGCUUGACCAGUUGGUA 716 UACCAACUGGUCAAGCGAA 1285 NM—001735.2_11701188—s 1170-1188 CCAGUUGGUAGGAGGAGUC 717 GACUCCUCCUACCAACUGG 1286

- 134044245

NM_001735.2_11801198_s 1180-1198 GGAGGAGUCCCAGUAACAC 718 GUGUUACUGGGACUCCUCC 1287 NM_001735.2_11901208_s 1190-1208 CAGUAACACUGAAUGCACA 719 UGUGCAUUCAGUGUUACUG 1288 NM_001735.2_12001218—s 1200-1218 GAAUGCACAAACAAUUGAU 720 AUCAAUUGUUUGUGCAUUC 1289 NM—001735.2_12091227—s 1209-1227 AACAAUUGAUGUAAAC GAA 721 UUGGUUUACAUCAAUUGUU 1290 NM—001735.2_12201238—s 1220-1238 UAAAC CAAGAGACAUCUGA 722 UCAGAUGUCUCUUGGUUUA 1291 NM—001735.2_12321250—s 1232-1250 CAUCUGACUUGGAUCCAAG 723 CUUGGAUCCAAGUCAGAUG 1292 NM—001735.2_12431261—s 1243-1261 GAUCCAAGCAAAAGUGUAA 724 UUACACUUUUGCUUGGAUC 1293 NM—001735.2_12511269—s 1251-1269 CAAAAGUGUAACACGUGUU 725 AACAC GUGUUACACUUUUG 1294 NM—001735.2_12601278—s 1260-1278 AACACGUGUUGAUGAUGGA 726 UCCAUCAUCAACACGUGUU 1295 NM—001735.2_12721290—s 1272-1290 UGAUGGAGUAGCUUCCUUU 727 AAAGGAAGCUACUCCAUCA 1296 NM—001735.2_12791297—s 1279-1297 GUAGCUUCCUUUGUGCUUA 728 UAAGCACAAAGGAAGCUAC 1297 NM—001735.2_12931311—s 1293-1311 GCUUAAUCUCCCAUCUGGA 729 UCCAGAUGGGAGAUUAAGC 1298 NM—001735.2_13031321—s 1303-1321 CCAUCUGGAGUGACGGUGC 730 GCACCGUCACUCCAGAUGG 1299 NM—001735.2_13131331—s 1313-1331 UGACGGUGCUGGAGUUUAA 731 UUAAACUC CAGCACCGUCA 1300 NM—001735.2_13201338—s 1320-1338 GCUGGAGUUUAAUGUCAAA 732 UUUGACAUUAAACUCCAGC 1301 NM—001735.2_13321350—s 1332-1350 UGUCAAAACUGAUGCUCCA 733 UGGAGCAUCAGUUUUGACA 1302 NM—001735.2_13421360—s 1342-1360 GAUGCUCCAGAUCUUCCAG 734 CUGGAAGAUCUGGAGCAUC 1303 NM—001735.2_13491367—s 1349-1367 CAGAUCUUC CAGAAGAAAA 735 UUUUCUUCUGGAAGAUCUG 1304 NM—001735.2_13621380—s 1362-1380 AGAAAAUCAGGCCAGGGAA 736 UUCCCUGGCCUGAUUUUCU 1305 NM—001735.2_13711389—s 1371-1389 GGCCAGGGAAGGUUACCGA 737 UCGGUAACCUUCCCUGGCC 1306 NM—001735.2_13821400—s 1382-1400 GUUACCGAGCAAUAGCAUA 738 UAUGCUAUUGCUCGGUAAC 1307 NM—001735.2_13931411—s 1393-1411 AUAGCAUACUCAUCUCUCA 739 UGAGAGAUGAGUAUGCUAU 1308 NM—001735.2_13991417—s 1399-1471 UACUCAUCUCUCAGCCAAA 740 UUUGGCUGAGAGAUGAGUA 1309 NM—001735.2_14121430—s 1412-1430 GC CAAAGUUAC CUUUAUAU 741 AUAUAAAGGUAACUUUGGC 1310

- 135 044245

NM_001735.2_14221440_s 1422-1440 CCUUUAUAUUGAUUGGACU 742 AGUCCAAUCAAUAUAAAGG 1311 NM_001735.2_14321450_s 1432-1450 GAUUGGACUGAUAACCAUA 743 UAUGGUUAUCAGUCCAAUC 1312 NM_001735.2_14391457_s 1439-1457 CUGAUAACCAUAAGGCUUU 744 AAAGCCUUAUGGUUAUCAG 1313 NM_001735.2_14511469_s 1451-1469 AGGCUUUGCUAGUGGGAGA 745 UCUCCCACUAGCAAAGCCU 1314 NM_001735.2_14621480_s 1462-1480 GUGGGAGAACAUCUGAAUA 746 UAUUCAGAUGUUCUCCCAC 1315 NM_001735.2_14711489_s 1471-1489 CAUCUGAAUAUUAUUGUUA 747 UAACAAUAAUAUUCAGAUG 1316 NM_001735.2_14791497_s 1479-1497 UAUUAUUGUUAC С С С CAAA 748 UUUGGGGGUAACAAUAAUA 1317 NM_001735.2_14921510_s 1492-1510 With CAAAAG With CAUAUAUUG 749 CAAUAUAUGGGCUUUUGGG 1318 NM_001735.2_14931511_s 1493-1511 With CAAAAG With CAUAUAUUGA 750 UCAAUAUAUGGGCUUUUGG 1319 NM_001735.2_14941512_s 1494-1512 CAAAAG C C CAUAUAUUGAC 751 GUCAAUAUAUGGGCUUUUG 1320 NM_001735.2_14951513_s 1495-1513 AAAAG C CAUAUAUUGACA 752 UGUCAAUAUAUGGGCUUUU 1321 NM_001735.2_14961514_s 1496-1514 AAAG C CAUAUAUUGACAA 753 UUGUCAAUAUAUGGGCUUU 1322 NM_001735.2_14971515_s 1497-1515 AAG C CAUAUAUUGACAAA 754 UUUGUCAAUAUAUGGGCUU 1323 NM_001735.2_14981516_s 1498-1516 AG С С CAUAUAUUGACAAAA 755 UUUUGUCAAUAUAUGGGCU 1324 NM_001735.2_14991517_s 1499-1517 G C C CAUAUAUUGACAAAAU 756 AUUUUGUCAAUAUAUGGGC 1325 NM_001735.2_15001518_s 1500-1518 C C CAUAUAUUGACAAAAUA 757 UAUUUUGUCAAUAUAUGGG 1326 NM_001735.2_15011519_s 1501-1519 C CAUAUAUUGACAAAAUAA 758 UUAUUUUGUCAAUAUAUGG 1327 NM_001735.2_15021520_s 1502-1520 CAUAUAUUGACAAAAUAAC 759 GUUAUUUUGUCAAUAUAUG 1328 NM_001735.2_15031521_s 1503-1521 AUAUAUUGACAAAAUAACU 760 AGUUAUUUUGUCAAUAUAU 1329 NM_001735.2_15041522_s 1504-1522 UAUAUUGACAAAAUAACUC 761 GAGUUAUUUUGUCAAUAUA 1330 NM_001735.2_15051523_s 1505-1523 AUAUUGACAAAAUAACUCA 762 UGAGUUAUUUUGUCAAUAU 1331 NM_001735.2_15061524_s 1506-1524 UAUUGACAAAAUAACUCAC 763 GUGAGUUAUUUUGUCAAUA 1332 NM_001735.2_15071525_s 1507-1525 AUUGACAAAAUAACUCACU 764 AGUGAGUUAUUUUGUCAAU 1333 NM_001735.2_15081526_s 1508-1526 UUGACAAAAUAACUCACUA 765 UAGUGAGUUAUUUUGUCAA 1334

- 136044245

NM_001735.2_15091527_s 1509-1527 UGACAAAAUAACUCACUAU 766 AUAGUGAGUUAUUUUGUCA 1335 NM_001735.2_15101528_s 1510-1528 GACAAAAUAACUCACUAUA 767 UAUAGUGAGUUAUUUUGUC 1336 NM_001735.2_15131531—s 1513-1531 AAAAUAACUCACUAUAAUU 768 AAUUAUAGUGAGUUAUUUU 1337 NM—001735.2_15141532—s 1514-1532 AAAUAACUCACUAUAAUUA 769 UAAUUAUAGUGAGUUAUUU 1338 NM—001735.2_15151533—s 1515-1533 AAUAACUCACUAUAAUUAC 770 GUAAUUAUAGUGAGUUAUU 1339 NM—001735.2_15161534—s 1516-1534 AUAACUCACUAUAAUUACU 771 AGUAAUUAUAGUGAGUUAU 1340 NM—001735.2_15181536—s 1518-1536 AACUCACUAUAAUUACUUG 772 CAAGUAAUUAUAGUGAGUU 1341 NM—001735.2_15191537—s 1519-1537 ACUCACUAUAAUUACUUGA 773 UCAAGUAAUUAUAGUGAGU 1342 NM—001735.2_15201538—s 1520-1538 CUCACUAUAAUUACUUGAU 774 AUCAAGUAAUUAUAGUGAG 1343 NM—001735.2_15211539—s 1521-1539 UCACUAUAAUUACUUGAUU 775 AAUCAAGUAAUUAUAGUGA 1344 NM—001735.2_15231541—s 1523-1541 ACUAUAAUUACUUGAUUUU 776 AAAAUCAAGUAAUUAUAGU 1345 NM—001735.2_15241542—s 1524-1542 CUAUAAUUACUUGAUUUUA 777 UAAAAUCAAGUAAUUAUAG 1346 NM—001735.2_15251543—s 1525-1543 UAUAAUUACUUGAUUUUAU 778 AUAAAAUCAAGUAAUUAUA 1347 NM—001735.2_15261544—s 1526-1544 AUAAUUACUUGAUUUUAUC 779 GAUAAAAUCAAGUAAUUAU 1348 NM—001735.2_15271545—s 1527-1545 UAAUUACUUGAUUUUAUC C 780 GGAUAAAAUCAAGUAAUUA 1349 NM—001735.2_15281546—s 1528-1546 AAUUACUUGAUUUUAUC CA 781 UGGAUAAAAUCAAGUAAUU 1350 NM—001735.2_15291547—s 1529-1547 AUUACUUGAUUUUAUCCAA 782 UUGGAUAAAAUCAAGUAAU 1351 NM—001735.2_15401558—s 1540-1558 UUAUCCAAGGGCAAAAUUA 783 UAAUUUUGCCCUUGGAUAA 1352 NM—001735.2_15501568—s 1550-1568 GCAAAAUUAUCCACUUUGG 784 CCAAAGUGGAUAAUUUUGC 1353 NM—001735.2_15611579—s 1561-1579 CACUUUGGCACGAGGGAGA 785 UCUCCCUCGUGCCAAAGUG 1354 NM—001735.2_15711589—s 1571-1589 CGAGGGAGAAAUUUUCAGA 786 UCUGAAAAUUUCUCCCUCG 1355 NM—001735.2_15811599—s 1581-1599 AUUUUCAGAUGCAUCUUAU 787 AUAAGAUGCAUCUGAAAAU 1356 NM—001735.2_15911609—s 1591-1609 GCAUCUUAUCAAAGUAUAA 788 UUAUACUUUGAUAAGAUGC 1357 NM—001735.2_16001618—s 1600-1618 CAAAGUAUAAACAUUC GAG 789 CUGGAAUGUUUAUACUUUG 1358

- 137044245

NM_001735.2_16121630_s 1612-1630 AUUC CAGUAACACAGAACA 790 UGUUCUGUGUUACUGGAAU 1359 NM_001735.2_16221640_s 1622-1640 CACAGAACAUGGUUCCUUC 791 GAAGGAACCAUGUUCUGUG 1360 NM_001735.2_16321650_s 1632-1560 GGUUCCUUCAUCCCGACUU 792 AAGUCGGGAUGAAGGAACC 1361 NM_001735.2_16431661_s 1643-1661 CCCGACUUCUGGUCUAUUA 793 UAAUAGACCAGAAGUCGGG 1362 NM_001735.2_16531671_s 1653-1671 GGUCUAUUACAUCGUCACA 794 UGUGAC GAUGUAAUAGAC C 1363 NM_001735.2_16631681_s 1663-1681 AUCGUCACAGGAGAACAGA 795 UCUGUUCUCCUGUGACGAU 1364 NM_001735.2_16701688_s 1670-1688 CAGGAGAACAGACAGCAGA 796 UCUGCUGUCUGUUCUCCUG 1365 NM_001735.2_16821700_s 1682-1700 CAGCAGAAUUAGUGUCUGA 797 UCAGACACUAAUUCUGCUG 1366 NM_001735.2_16931711_s 1693-1711 GUGUCUGAUUCAGUCUGGU 798 ACCAGACUGAAUCAGACAC 1367 NM_001735.2_17031721_s 1703-1721 CAGUCUGGUUAAAUAUUGA 799 UCAAUAUUUAAC CAGACUG 1368 NM_001735.2_17101728_s 1710-1728 GUUAAAUAUUGAAGAAAAA 800 UUUUUCUUCAAUAUUUAAC 1369 NM_001735.2_17221740_s 1722-1740 AGAAAAAUGUGGCAACCAG 801 CUGGUUGCCACAUUUUUCU 1370 NM_001735.2_17331751_s 1733-1751 GCAACCAGCUCCAGGUUCA 802 UGAACCUGGAGCUGGUUGC 1371 NM_001735.2_17401758_s 1740-1758 GCUCCAGGUUCAUCUGUCU 803 AGACAGAUGAACCUGGAGC 1372 NM_001735.2_17511769_s 1751-1769 AUCUGUCUCCUGAUGCAGA 804 UCUGCAUCAGGAGACAGAU 1373 NM_001735.2_17621780_s 1762-1780 GAUGCAGAUGCAUAUUCUC 805 GAGAAUAUGCAUCUGCAUC 1374 NM_001735.2_17711789_s 1771-1789 GCAUAUUCUCCAGGCCAAA 806 UUUGGCCUGGAGAAUAUGC 1375 NM_001735.2_17821800_s 1782-1800 AGGCCAAACUGUGUCUCUU 807 AAGAGACACAGUUUGGCCU 1376 NM_001735.2_17921810_s 1792-1810 GUGUCUCUUAAUAUGGCAA 808 UUGCCAUAUUAAGAGACAC 1377 NM_001735.2_17991817_s 1799-1817 UUAAUAUGGCAACUGGAAU 809 AUUC CAGUUGC CAUAUUAA 1378 NM_001735.2_18091827_s 1809-1827 AACUGGAAUGGAUUCCUGG 810 C CAGGAAUC CAUUC CAGUU 1379 NM_001735.2_18211839_s 1821-1839 UUCCUGGGUGGCAUUAGCA 811 UGCUAAUGCCACCCAGGAA 1380 NM_001735.2_18301848_s 1830-1848 GGCAUUAGCAGCAGUGGAC 812 GUCCACUGCUGCUAAUGCC 1381 NM_001735.2_18421860_s 1842-1860 AGUGGACAGUGCUGUGUAU 813 AUACACAGCACUGUCCACU 1382

- 138044245

NM_001735.2_18521870_s 1852-1870 GCUGUGUAUGGAGUCCAAA 814 UUUGGACUCCAUACACAGC 1383 NM_001735.2_18631881_s 1863-1881 AGUCCAAAGAGGAGCCAAA 815 UUUGGCUCCUCUUUGGACU 1384 NM_001735.2_18701888_s 1870-1888 AGAGGAGCCAAAAAGCCCU 816 AGGGCUUUUUGGCUCCUCU 1385 NM_001735.2_18831901_s 1883-1901 AGCCCUUGGAAAGAGUAUU 817 AAUACUCUUUCCAAGGGCU 1386 NM_001735.2_18931911_s 1893-1911 AAGAGUAUUUCAAUUCUUA 818 UAAGAAUUGAAAUACUCUU 1387 NM_001735.2_19001918_s 1900-1918 UUUCAAUUCUUAGAGAAGA 819 UCUUCUCUAAGAAUUGAAA 1388 NM_001735.2_19121930_s 1912-1930 GAGAAGAGUGAUCUGGGCU 820 AGCCCAGAUCACUCUUCUC 1389 NM_001735.2_19201938_s 1920-1938 UGAUCUGGGCUGUGGGGCA 821 UGCCCCACAGCCCAGAUCA 1390 NM_001735.2_19331951_s 1933-1951 GGGGCAGGUGGUGGCCUCA 822 UGAGGCCACCACCUGCCCC 1391 NM_001735.2_19431961_s 1943-1961 GUGGCCUCAACAAUGCCAA 823 UUGGCAUUGUUGAGGCCAC 1392 NM_001735.2_19501968_s 1950-1968 CAACAAUGCCAAUGUGUUC 824 GAACACAUUGGCAUUGUUG 1393 NM_001735.2_19591977_s 1959-1977 CAAUGUGUUCCACCUAGCU 825 AGCUAGGUGGAACACAUUG 1394 NM_001735.2_19691987_s 1969-1987 CACCUAGCUGGACUUACCU 826 AGGUAAGUCCAGCUAGGUG 1395 NM_001735.2_19791997_s 1979-1997 GACUUACCUUCCUCACUAA 827 UUAGUGAGGAAGGUAAGUC 1396 NM_001735.2_19912009_s 1991-2009 UCACUAAUGCAAAUGCAGA 828 UCUGCAUUUGCAUUAGUGA 1397 NM_001735.2_2001- 2019_s 2001-2019 AAAUGCAGAUGACUCСCAA 829 UUGGGAGUCAUCUGCAUUU 1398 NM_001735.2_20132031_s 2013-2013 CUC C CAAGAAAAUGAUGAA 830 UUCAUCAUUUUCUUGGGAG 1399 NM_001735.2_20322050_s 2032-2050 CCUUGUAAAGAAAUUCUCA 831 UGAGAAUUUCUUUACAAGG 1400 NM_001735.2_20432061_s 2043-2061 AAUUCUCAGGCCAAGAAGA 832 UCUUCUUGGCCUGAGAAUU 1401 NM_001735.2_20532071_s 2053-2071 CCAAGAAGAACGCUGCAAA 833 UUUGCAGCGUUCUUCUUGG 1402 NM_001735.2_20632081_s 2063-2081 CGCUGCAAAAGAAGAUAGA 834 UCUAUCUUCUUUUGCAGCG 1403 NM_001735.2_20702088_s 2070-2088 AAAGAAGAUAGAAGAAAUA 835 UAUUUCUUCUAUCUUCUUU 1404 NM_001735.2_20822100_s 2082-2100 AGAAAUAGCUGCUAAAUAU 836 AUAUUUAGCAGCUAUUUCU 1405 NM_001735.2_2089- 2107_s 2089-2107 GCUGCUAAAUAUAAACAUU 837 AAUGUUUAUAUUUAGCAGC 1406

- 139044245

NM_001735.2_2103- 2121_s 2103-2121 ACAUUCAGUAGUGAAGAAA 838 UUUCUUCACUACUGAAUGU 1407 NM_001735.2_21102128_s 2110-2128 GUAGUGAAGAAAUGUUGUU 839 AACAACAUUUCUUCACUAC 1408 NM_001735.2_21192137_s 2119-2137 AAAUGUUGUUACGAUGGAG 840 CUC CAUC GUAACAACAUUU 1409 NM_001735.2_21302148_s 2130-2148 CGAUGGAGCCUGCGUUAAU 841 AUUAACGCAGGCUCCAUCG 1410 NM_001735.2_21422160_s 2142-2160 C GUUAAUAAUGAUGAAAC C 842 GGUUUCAUCAUUAUUAACG 1411 NM_001735.2_21502168_s 2150-2168 AUGAUGAAACCUGUGAGCA 843 UGCUCACAGGUUUCAUCAU 1412 NM_001735.2_21602178_s 2160-2178 CUGUGAGCAGCGAGCUGCA 844 UGCAGCUCGCUGCUCACAG 1413 NM_001735.2_21702188_s 2170-2188 CGAGCUGCACGGAUUAGUU 845 AACUAAUCCGUGCAGCUCG 1414 NM_001735.2_2180- 2198_s 2180-2198 GGAUUAGUUUAGGGCCAAG 846 CUUGGCCCUAAACUAAUCC 1415 NM_001735.2_21912209_s 2191-2209 GGGCCAAGAUGCAUCAAAG 847 CUUUGAUGCAUCUUGGCCC 1416 NM_001735.2_22022220_s 2202-2220 CAUCAAAGCUUUCACUGAA 848 UUCAGUGAAAGCUUUGAUG 1417 NM_001735.2_22092227_s 2209-2227 GCUUUCACUGAAUGUUGUG 849 CACAACAUUCAGUGAAAGC 1418 NM_001735.2_22192237_s 2219-2237 AAUGUUGUGUCGUCGCAAG 850 CUUGC GAC GACACAACAUU 1419 NM_001735.2_22292247_s 2229-2247 CGUCGCAAGCCAGCUCCGU 851 ACGGAGCUGGCUUGCGACG 1420 NM_001735.2_2241- 2259_s 2241-2259 GCUCCGUGCUAAUAUCUCU 852 AGAGAUAUUAGCACGGAGC 1421 NM_001735.2_22492267_s 2249-2267 CUAAUAUCUCUCAUAAAGA 853 UCUUUAUGAGAGAUAUUAG 1422 NM_001735.2_22632281_s 2263-2281 AAAGACAUGCAAUUGGGAA 854 UUCCCAAUUGCAUGUCUUU 1423 NM_001735.2_22722290_s 2272-2290 CAAUUGGGAAGGCUACACA 855 UGUGUAGCCUUCCCAAUUG 1424 NM_001735.2_2283- 2301_s 2283-2301 GCUACACAUGAAGACCCUG 856 CAGGGUCUUCAUGUGUAGC 1425 NM_001735.2_22892307_s 2289-2307 CAUGAAGACCCUGUUACGA 857 UGGUAACAGGGUCUUCAUG 1426 NM_001735.2_2303- 2321_s 2303-2321 UACCAGUAAGCAAGCCAGA 858 UCUGGCUUGCUUACUGGUA 1427 NM_001735.2_23112329_s 2311-2329 AGCAAGCCAGAAAUUCGGA 859 UCCGAAUUUCUGGCUUGCU 1428 NM_001735.2_23192337_s 2319-2337 AGAAAUUCGGAGUUAUUUU 860 AAAAUAACUCCGAAUUUCU 1429 NM_001735.2_23292347_s 2329-2347 AGUUAUUUUCCAGAAAGCU 861 AGCUUUCUGGAAAAUAACU 1430

- 140044245

NM_001735.2_23392357_s 2339-2357 CAGAAAGCUGGUUGUGGGA 862 UC C GAGAAC CAGCUUUCUG 1431 NM_001735.2_23522370_s 2352-2370 GUGGGAAGUUCAUCUUGUU 863 AACAAGAUGAACUUC C CAC 1432 NM_001735.2_2361- 2379_s 2361-2379 UCAUCUUGUUCCCAGAAGA 864 UCUUCUGGGAACAAGAUGA 1433 NM_001735.2_23722390_s 2372-2390 CCAGAAGAAAACAGUUGCA 865 UGCAACUGUUUUCUUCUGG 1434 NM_001735.2_2383- 2401_s 2383-2401 CAGUUGCAGUUUGCCCUAC 866 GUAGGGCAAACUGCAACUG 1435 NM_001735.2_23892407_s 2389-2407 CAGUUUGCCCUACCUGAUU 867 AAUCAGGUAGGGCAAACUG 1436 NM_001735.2_2401- 2419_s 2401-2419 CCUGAUUCUCUAACCACCU 868 AGGUGGUUAGAGAAUCAGG 1437 NM_001735.2_2413- 2431_s 2413-2431 ACCACCUGGGAAAUUCAAG 869 CUUGAAUUUCCCAGGUGGU 1438 NM_001735.2_24222440_s 2422-2440 GAAAUUCAAGGCGUUGGCA 870 UGCCAACGCCUUGAAUUUC 1439 NM_001735.2_2433- 2451_s 2433-2451 CGUUGGCAUUUCAAACACU 871 AGUGUUUGAAAUGC CAAC G 1440 NM_001735.2_24392457_s 2439-2457 CAUUUCAAACACUGGUAUA 872 UAUAC CAGUGUUUGAAAUG 1441 NM_001735.2_2453- 2471_s 2453-2471 GUAUAUGUGUUGCUGAUAC 873 GUAUCAGCAACACAUAUAC 1442 NM_001735.2_24632481_s 2463-2481 UGCUGAUACUGUCAAGGCA 874 UGCCUUGACAGUAUCAGCA 1443 NM_001735.2_24712489_s 2471-2489 CUGUCAAGGCAAAGGUGUU 875 AACAC CUUUGC CUUGACAG 1444 NM_001735.2_2483- 2501_s 2483-2501 AGGUGUUCAAAGAUGUCUU 876 AAGACAUCUUUGAACACCU 1445 NM_001735.2_24902508_s 2490-2508 CAAAGAUGUCUUCCUGGAA 877 UUCCAGGAAGACAUCUUUG 1446 NM_001735.2_24992517_s 2499-2517 CUUCCUGGAAAUGAAUAUA 878 UAUAUUCAUUUCCAGGAAG 1447 NM_001735.2_25112529_s 2511-2529 GAAUAUAC CAUAUUCUGUU 879 AACAGAAUAUGGUAUAUUC 1448 NM_001735.2_25202538_s 2520-2538 AUAUUCUGUUGUACGAGGA 880 UC CUC GUACAACAGAAUAU 1449 NM_001735.2_2533- 2551_s 2533-2551 C GAG GAGAACAGAUC CAAU 881 AUUGGAUCUGUUCUCCUCG 1450 NM_001735.2_25392557_s 2539-2557 GAACAGAUC CAAUUGAAAG 882 CUUUCAAUUGGAUCUGUUC 1451 NM_001735.2_2553- 2571_s 2553-2571 GAAAGGAACUGUUUACAAC 883 GUUGUAAACAGUUCCUUUC 1452 NM_001735.2_25602578_s 2560-2578 ACUGUUUACAACUAUAGGA 884 UC CUAUAGUUGUAAACAGU 1453 NM_001735.2_25692587_s 2569-2587 AACUAUAGGACUUCUGGGA 885 UCCCAGAAGUCCUAUAGUU 1454

- 141 044245

NM_001735.2_2583- 2601_s 2583-2601 UGGGAUGCAGUUCUGUGUU 886 AACACAGAACUGCAUCCCA 1455 NM_001735.2_25922610_s 2592-2610 GUUCUGUGUUAAAAUGUCU 887 AGACAUUUUAACACAGAAC 1456 NM_001735.2_26002618_s 2600-2618 UUAAAAUGUCUGCUGUGGA 888 UCCACAGCAGACAUUUUAA 1457 NM_001735.2_26122630_s 2612-2630 CUGUGGAGGGAAUCUGCAC 889 GUGCAGAUUC C CUC CACAG 1458 NM_001735.2_26202638_s 2620-2638 GGAAUCUGCACUUCGGAAA 890 UUUC C GAAGUGCAGAUUC C 1459 NM_001735.2_26332651_s 2633-2651 CGGAAAGCCCAGUCAUUGA 891 UCAAUGACUGGGCUUUCCG 1460 NM_001735.2_26412659_s 2641-2659 CCAGUCAUUGAUCAUCAGG 892 CCUGAUGAUCAAUGACUGG 1461 NM_001735.2_26532671_s 2653-2671 CAUCAGGGCACAAAGUCCU 893 AGGACUUUGUGCCCUGAUG 1462 NM_001735.2_26592677_s 2659-2677 GGCACAAAGUCCUCCAAAU 894 AUUUGGAGGACUUUGUGCC 1463 NM_001735.2_26732691_s 2673-2691 CAAAUGUGUGCGCCAGAAA 895 UUUCUGGCGCACACAUUUG 1464 NM_001735.2_26822700_s 2682-2700 GCGCCAGAAAGUAGAGGGC 896 GCCCUCUACUUUCUGGCGC 1465 NM_001735.2_26912709_s 2691-2709 AGUAGAGGGCUCCUCCAGU 897 ACUGGAGGAGCCCUCUACU 1466 NM_001735.2_27022720_s 2702-2720 C CUC CAGUCACUUGGUGAC 898 GUCACCAAGUGACUGGAGG 1467 NM_001735.2_27092727_s 2709-2727 UCACUUGGUGACAUUCACU 899 AGUGAAUGUCACCAAGUGA 1468 NM_001735.2_27202738_s 2720-2738 CAUUCACUGUGCUUCCUCU 900 AGAGGAAGCACAGUGAAUG 1469 NM_001735.2_27392757_s 2739-2757 GGAAAUUGGCCUUCACAAC 901 GUUGUGAAGGCCAAUUUCC 1470 NM_001735.2_27492767_s 2749-2767 CUUCACAACAUCAAUUUUU 902 AAAAAUUGAUGUUGUGAAG 1471 NM_001735.2_2761- 2779_s 2761-2779 AAUUUUUCACUGGAGACUU 903 AAGUCUCCAGUGAAAAAUU 1472 NM_001735.2_27702788_s 2770-2788 CUGGAGACUUGGUUUGGAA 904 UUC CAAAC CAAGUCUC CAG 1473 NM_001735.2_27802798_s 2780-2798 GGUUUGGAAAAGAAAUCUU 905 AAGAUUUCUUUUC CAAAC C 1474 NM_001735.2_27932811_s 2793-2811 AAUCUUAGUAAAAACAUUA 906 UAAUGUUUUUACUAAGAUU 1475 NM_001735.2_28022820_s 2802-2820 AAAAACAUUACGAGUGGUG 907 CACCACUCGUAAUGUUUUU 1476 NM_001735.2_28132831_s 2813-2831 GAGUGGUGCCAGAAGGUGU 908 ACACCUUCUGGCACCACUC 1477 NM_001735.2_28232841_s 2823-2841 AGAAGGUGUCAAAAGGGAA 909 UUC C CUUUUGACAC CUUCU 1478

- 142044245

NM_001735.2_28292847_s 2829-2847 UGUCAAAAGGGAAAGCUAU 910 AUAGCUUUCCCUUUUGACA 1479 NM_001735.2_28432861_s 2843-2861 GCUAUUCUGGUGUUACUUU 911 AAAGUAACACCAGAAUAGC 1480 NM_001735.2_28522870_s 2852-2870 GUGUUACUUUGGAUCCUAG 912 CUAGGAUCCAAAGUAACAC 1481 NM_001735.2_28622880_s 2862-2880 GGAUCCUAGGGGUAUUUAU 913 AUAAAUAC С C CUAGGAUC C 1482 NM_001735.2_28722890_s 2872-2890 GGUAUUUAUGGUACCAUUA 914 UAAUGGUAC CAUAAAUAC C 1483 NM_001735.2_2882- 2900_s 2882-2900 GUAC CAUUAG CAGAC GAAA 915 UUUCGUCUGCUAAUGGUAC 1484 NM_001735.2_2892- 2910_s 2892-2910 CAGAC GAAAGGAGUUC C CA 916 UGGGAACUCCUUUCGUCUG 1485 NM_001735.2_29002918_s 2900-2918 AGGAGUUCCCAUACAGGAU 917 AUCCUGUAUGGGAACUCCU 1486 NM_001735.2_29092927_s 2909-2927 CAUACAG GAUAC C CUUAGA 918 UCUAAGGGUAUCCUGUAUG 1487 NM_001735.2_29222940_s 2922-2940 CUUAGAUUUGGUCССCAAA 919 UUUGGGGACCAAAUCUAAG 1488 NM_001735.2_2933- 2951_s 2933-2951 UC C C CAAAACAGAAAUCAA 920 UUGAUUUCUGUUUUGGGGA 1489 NM_001735.2_2941- 2959_s 2941-2959 ACAGAAAUCAAAAGGAUUU 921 AAAUCCUUUUGAUUUCUGU 1490 NM_001735.2_2951- 2969_s 2951-2969 AAAGGAUUUUGAGUGUAAA 922 UUUACACUCAAAAUC CUUU 1491 NM_001735.2_29622980_s 2962-2980 AGUGUAAAAGGACUGCUUG 923 CAAGCAGUCCUUUUACACU 1492 NM_001735.2_29692987_s 2969-2987 AAGGACUGCUUGUAGGUGA 924 UCAC CUACAAGCAGUC CUU 1493 NM_001735.2_2980- 2998_s 2980-2998 GUAGGUGAGAUCUUGUCUG 925 CAGACAAGAUCUCACCUAC 1494 NM_001735.2_29893007_s 2989-3007 AUCUUGUCUGCAGUUCUAA 926 UUAGAACUGCAGACAAGAU 1495 NM_001735.2_3001- 3019_s 3001-3019 GUUCUAAGUCAGGAAGGCA 927 UGCCUUCCUGACUUAGAAC 1496 NM_001735.2_30133031_s 3013-3031 GAAGGCAUCAAUAUCCUAA 928 UUAGGAUAUUGAUGCCUUC 1497 NM_001735.2_30203038_s 3020-3038 UCAAUAUC CUAAC C CAC CU 929 AGGUGGGUUAGGAUAUUGA 1498 NM_001735.2_30333051_s 3033-3051 CCACCUCCCCAAAGGGAGU 930 ACUCCCUUUGGGGAGGUGG 1499 NM_001735.2_30393057_s 3039-3057 CCCCAAAGGGAGUGCAGAG 931 CUCUGCACUCCCUUUGGGG 1500 NM_001735.2_30503068_s 3050-3068 GUGCAGAGGCGGAGCUGAU 932 AUCAGCUCCGCCUCUGCAC 1501 NM_001735.2_30603078_s 3060-3078 GGAGCUGAUGAGCGUUGUC 933 GACAACGCUCAUCAGCUCC 1502

- 143 044245

NM_001735.2_30723090—s 3072-3090 CGUUGUCCCAGUAUUCUAU 934 AUAGAAUACUGGGACAACG 1503 NM_001735.2_30793097—s 3079-3097 CCAGUAUUCUAUGUUUUUC 935 GAAAAACAUAGAAUACUGG 1504 NM—001735.2_30913109—s 3091-3109 GUUUUUCACUACCUGGAAA 936 UUUCCAGGUAGUGAAAAAC 1505 NM-001735.2_31023120—s 3102-3120 CCUGGAAACAGGAAAUCAU 937 AUGAUUUCCUGUUUCCAGG 1506 NM_001735.2_31223140—s 3122-3140 GGAACAUUUUUCAUUCUGA 938 UCAGAAUGAAAAAUGUUC C 1507 NM_001735.2_31333151—s 3133-3151 CAUUCUGAC C CAUUAAUUG 939 CAAUUAAUGGGUCAGAAUG 1508 NM_001735.2_31423160—s 3142-3160 CCAUUAAUUGAAAAGCAGA 940 UCUGCUUUUCAAUUAAUGG 1509 NM_001735.2_31533171—s 3153-3171 AAAGCAGAAACUGAAGAAA 941 UUUCUUCAGUUUCUGCUUU 1510 NM_001735.2_31613179—s 3161-3179 AACUGAAGAAAAAAUUAAA 942 UUUAAUUUUUUUCAGUU 1511 NM_001735.2_31693187—s 3169-3187 AAAAAAUUAAAAGAAGGGA 943 UCCCUUCUUUUAAUUUUUU 1512 NM_001735.2_31833201—s 3183-3201 AGGGAUGUUGAGCAUUAUG 944 CAUAAUGCUCAACAUCCCU 1513 NM_001735.2_31923210—s 3192-3210 GAGCAUUAUGUCCUACAGA 945 UCUGUAGGACAUAAUGCUC 1514 NM_001735.2_32003218—s 3200-3218 UGUCCUACAGAAAUGCUGA 946 UCAGCAUUUCUGUAGGACA 1515 NM_001735.2_32113229—s 3211-3229 AAUGCUGACUACUCUUACA 947 UGUAAGAGUAGUCAGCAUU 1516 NM_001735.2_32203238—s 3220-3238 UACUCUUACAGUGUGUGGA 948 UCCACACACUGUAAGAGUA 1517 NM_001735.2_32293247—s 3229-3247 AGUGUGUGGAAGGGUGGAA 949 UUC CAC C CUUC CACACACU 1518 NM_001735.2_32403258—s 3240-3258 GGGUGGAAGUGCUAGCACU 950 AGUGCUAGCACUUCCACCC 1519 NM_001735.2_32503268—s 3250-3268 GCUAGCACUUGGUUAACAG 951 CUGUUAACCAAGUGCUAGC 1520 NM_001735.2_32603278—s 3260-3278 GGUUAACAGCUUUUGCUUU 952 AAAGCAAAAGCUGUUAACC 1521 NM_001735.2_32733291—s 3273-3291 UGCUUUAAGAGUACUUGGA 953 UCCAAGUACUCUUAAAGCA 1522 NM_001735.2_32833301—s 3283-3301 GUACUUGGACAAGUAAAUA 954 UAUUUACUUGUC CAAGUAC 1523 NM_001735.2_32923310—s 3292-3317 CAAGUAAAUAAAUAC GUAG 955 CUAC GUAUUUAUUUACUUG 1524 NM_001735.2_32993317—s 3299-3317 AUAAAUACGUAGAGCAGAA 956 UUCUGCUCUACGUAUUUAU 1525 NM_001735.2_33103328—s 3310-3328 GAGCAGAACCAAAAUUCAA 957 UUGAAUUUUGGUUCUGCUC 1526

- 144044245

NM_001735.2_33223340_s 3322-3340 AAUUCAAUUUGUAAUUCUU 958 AAGAAUUACAAAUUGAAUU 1527 NM_001735.2_33323350_s 3332-3350 GUAAUUCUUUAUUGUGGCU 959 AGCCACAAUAAAGAAUUAC 1528 NM_001735.2_33423360—s 3342-3360 AUUGUGGCUAGUUGAGAAU 960 AUUCUCAACUAGCCACAAU 1529 NM—001735.2_33493367—s 3349-3367 CUAGUUGAGAAUUAUCAAU 961 AUUGAUAAUUCUCAACUAG 1530 NM—001735.2_33603378—s 3360-3378 UUAUCAAUUAGAUAAUGGA 962 UC CAUUAUCUAAUUGAUAA 1531 NM—001735.2_33733391—s 3373-3391 AAUGGAUCUUUCAAGGAAA 963 UUUCCUUGAAAGAUCCAUU 1532 NM—001735.2_33803398—s 3380-3398 CUUUCAAGGAAAAUUCACA 964 UGUGAAUUUUCCUUGAAAG 1533 NM—001735.2_33913409—s 3391-3409 AAUUCACAGUAUCAAC CAA 965 UUGGUUGAUACUGUGAAUU 1534 NM—001735.2_33993417—s 3399-3417 GUAUCAAC CAAUAAAAUUA 966 UAAUUUUAUUGGUUGAUAC 1535 NM—001735.2_34113429—s 3411-3429 AAAAUUACAGGGUACCUUG 967 CAAGGUAC C CUGUAAUUUU 1536 NM—001735.2_34193437—s 3419-3437 AGGGUACCUUGCCUGUUGA 968 UCAACAGGCAAGGUACCCU 1537 NM—001735.2_34333451—s 3433-3451 GUUGAAGC C C GAGAGAACA 969 UGUUCUCUCGGGCUUCAAC 1538 NM—001735.2_34413459—s 3441-3559 С C GAGAGAACAG CUUAUAU 970 AUAUAAGCUGUUCUCUCGG 1539 NM—001735.2_34523470—s 3452-3470 GCUUAUAUCUUACAGCCUU 971 AAGGCUGUAAGAUAUAAGC 1540 NM—001735.2_34603478—s 3460-3478 CUUACAGCCUUUACUGUGA 972 UCACAGUAAAGGCUGUAAG 1541 NM—001735.2_34823500—s 3482-3500 GAAUUAGAAAGGCUUUCGA 973 UCGAAAGCCUUUCUAAUUC 1542 NM—001735.2_34923510—s 3492-3510 GGCUUUCGAUAUAUGCCCC 974 GGGGCAUAUAUCGAAAGCC 1543 NM—001735.2_34993517—s 3499-3517 GAUAUAUGCCCCCUGGUGA 975 UCACCAGGGGGCAUAUAUC 1544 NM—001735.2_35133531—s 3513-3531 GGUGAAAAUCGACACAGCU 976 AGCUGUGUC GAUUUUCAC C 1545 NM—001735.2_35223540—s 3522-3540 CGACACAGCUCUAAUUAAA 977 UUUAAUUAGAGCUGUGUCG 1546 NM—001735.2_35293547—s 3529-3547 GCUCUAAUUAAAGCUGACA 978 UGUCAGCUUUAAUUAGAGC 1547 NM—001735.2_35423560—s 3542-3560 CUGACAACUUUCUGCUUGA 979 UCAAGCAGAAAGUUGUCAG 1548 NM—001735.2_35493567—s 3549-3567 CUUUCUGCUUGAAAAUACA 980 UGUAUUUUCAAGCAGAAAG 1549 NM—001735.2_35603578—s 3560-3578 AAAAUACACUGCCAGCCCA 981 UGGGCUGGCAGUGUAUUUU 1550

- 145 044245

NM_001735.2_3573- 3591_s 3573-3591 AGCCCAGAGCACCUUUACA 982 UGUAAAGGUGCUCUGGGCU 1551 NM_001735.2_35813599_s 3581-3599 GCACCUUUACAUUGGCCAU 983 AUGGCCAAUGUAAAGGUGC 1552 NM_001735.2_35893607_s 3589-3607 ACAUUGGCCAUUUCUGCGU 984 ACGCAGAAAUGGCCAAUGU 1553 NM_001735.2_36023620_s 3602-3620 CUGCGUAUGCUCUUUCCCU 985 AGGGAAAGAGCAUACGCAG 1554 NM_001735.2_3613- 3631_s 3613-3631 CUUUCCCUGGGAGAUAAAA 986 UUUUAUCUCCCAGGGAAAG 1555 NM_001735.2_3623- 3641_s 3623-3641 GAGAUAAAACUCAC C GAGA 987 UGUGGGUGAGUUUUAUCUC 1556 NM_001735.2_3631- 3649_s 3631-3649 ACUCAC C CACAGUUUC GUU 988 AACGAAACUGUGGGUGAGU 1557 NM_001735.2_36403658_s 3640-3658 CAGUUUC GUUCAAUUGUUU 989 AAACAAUUGAACGAAACUG 1558 NM_001735.2_36503668_s 3650-3668 CAAUUGUUUCAGCUUUGAA 990 UUCAAAGCUGAAACAAUUG 1559 NM_001735.2_36623680_s 3662-3680 CUUUGAAGAGAGAAGCUUU 991 AAAGCUUCUCUCUUCAAAG 1560 NM_001735.2_36693687_s 3669-3687 GAGAGAAGCUUUGGUUAAA 992 UUUAACCAAAGCUUCUCUC 1561 NM_001735.2_36823700_s 3682-3700 GUUAAAG GUAAUC CAC C CA 993 UGGGUGGAUUACCUUUAAC 1562 NM_001735.2_36913709_s 3691-3709 AAUC CAC C CAUUUAUC GUU 994 AACGAUAAAUGGGUGGAUU 1563 NM_001735.2_36993717_s 3699-3717 CAUUUAUCGUUUUUGGAAA 995 UUUC CAAAAACGAUAAAUG 1564 NM_001735.2_37103728_s 3710-3728 UUUGGAAAGACAAUCUUCA 996 UGAAGAUUGUCUUUCCAAA 1565 NM_001735.2_3721- 3739_s 3721-3739 AAUCUUCAGCAUAAAGACA 997 UGUCUUUAUGCUGAAGAUU 1566 NM_001735.2_37303748_s 3730-3748 CAUAAAGACAGCUCUGUAC 998 GUACAGAGCUGUCUUUAUG 1567 NM_001735.2_37413759_s 3741-3759 CUCUGUACCUAACACUGGU 999 ACCAGUGUUAGGUACAGAG 1568 NM_001735.2_37523770_s 3752-3770 ACACUGGUACGGCACGUAU 1000 AUACGUGCCGUACCAGUGU 1569 NM_001735.2_37623780_s 3762-3780 GGCACGUAUGGUAGAAACA 1001 UGUUUCUACCAUACGUGCC 1570 NM_001735.2_37713789_s 3771-3789 GGUAGAAACAACUGCCUAU 1002 AUAGGCAGUUGUUUCUACC 1571 NM_001735.2_37793797_s 3779-3797 CAACUGCCUAUGCUUUACU 1003 AGUAAAGCAUAGGCAGUUG 1572 NM_001735.2_37913809_s 3791-3809 CUUUACUCACCAGUCUGAA 1004 UUCAGACUGGUGAGUAAAG 1573 NM_001735.2_38033821_s 3803-3821 GUCUGAACUUGAAAGAUAU 1005 AUAUCUUUCAAGUUCAGAC 1574

- 146044245

NM_001735.2_38093827_s 3809-3827 ACUUGAAAGAUAUAAAUUA 1006 UAAUUUAUAUCUUUCAAGU 1575 NM_001735.2_38193837—s 3819-3837 UAUAAAUUAUGUUAAC WITH CA 1007 UGGGUUAACAUAAUUUAUA 1576 NM—001735.2_38293847—s 3829-3847 GUUAAC C CAGUCAUCAAAU 1008 AUUUGAUGACUGGGUUAAC 1577 NM—001735.2_38393857—s 3839-3857 UCAUCAAAUGGCUAUCAGA 1009 UCUGAUAGCCAUUUGAUGA 1578 NM—001735.2_38513869—s 3851-3869 UAUCAGAAGAGCAGAGGUA 1010 UACCUCUGCUCUUCUGAUA 1579 NM—001735.2_38633881—s 3863-3881 AGAGGUAUGGAGGUGGCUU 1011 AAGCCACCUCCAUACCUCU 1580 NM—001735.2_38723890—s 3872-3890 GAGGUGGCUUUUAUUCAAC 1012 GUUGAAUAAAAGC CAC CUC 1581 NM—001735.2_38833901—s 3883-3901 UAUUCAAG C GAG GAGACAA 1013 UUGUGUCCUGGGUUGAAUA 1582 NM—001735.2_38933911—s 3893-3911 AGGACACAAUCAAUGCCAU 1014 AUGGCAUUGAUUGUGUCCU 1583 NM—001735.2_38993917—s 3899-3917 CAAUCAAUGCCAUUGAGGG 1015 CCCUCAAUGGCAUUGAUUG 1584 NM—001735.2_39093927—s 3909-3927 CAUUGAGGGCCUGACGGAA 1016 UUCCGUCAGGCCCUCAAUG 1585 NM—001735.2_39223940—s 3922-3940 ACGGAAUAUUCACUCCUGG 1017 C CAGGAGUGAAUAUUC C GU 1586 NM—001735.2_39303948—s 3930-3948 UUCACUCCUGGUUAAACAA 1018 UUGUUUAACCAGGAGUGAA 1587 NM—001735.2_39393957—s 3939-3957 GGUUAAACAACUCCGCUUG 1019 CAAGCGGAGUUGUUUAACC 1588 NM—001735.2_39513969—s 3951-3969 CCGCUUGAGUAUGGACAUC 1020 GAUGUCCAUACUCAAGCGG 1589 NM—001735.2_39633981—s 3963-3981 GGACAUCGAUGUUUCUUAC 1021 GUAAGAAACAUCGAUGUCC 1590 NM—001735.2_39693987—s 3969-3987 CGAUGUUUCUUACAAGCAU 1022 AUGCUUGUAAGAAACAUCG 1591 NM—001735.2_39813999—s 3981-3999 CAAGCAUAAAGGUGCCUUA 1023 UAAGGCACCUUUAUGCUUG 1592 NM—001735.2_39924010—s 3992-4010 GUGCCUUACAUAAUUAUAA 1024 UUAUAAUUAUGUAAGGCAC 1593 NM—001735.2_39994017—s 3999-4017 ACAUAAUUAUAAAAUGACA 1025 UGUCAUUUUAUAAUUAUGU 1594 NM—001735.2_40094027—s 4009-4027 AAAAUGACAGACAAGAAUU 1026 AAUUCUUGUCUGUCAUUUU 1595 NM—001735.2_40204038—s 4020-4038 CAAGAAUUUCCUUGGGAGG 1027 CCUCCCAAGGAAAUUCUUG 1596 NM—001735.2_40294047—s 4029-4047 CCUUGGGAGGCCAGUAGAG 1028 CUCUACUGGCCUCCCAAGG 1597 NM—001735.2_40414059—s 4041-4059 AGUAGAGGUGCUUCUCAAU 1029 AUUGAGAAGCACCUCUACU 1598

- 147044245

NM_001735.2_40514069—s 4051-4069 CUUCUCAAUGAUGACCUCA 1030 UGAGGUCAUCAUUGAGAAG 1599 NM—001735.2_40624080—s 4062-4080 UGACCUCAUUGUCAGUACA 1031 UGUACUGACAAUGAGGUCA 1600 NM—001735.2_40724090—s 4072-4090 GUCAGUACAGGAUUUGGCA 1032 UGCCAAAUCCUGUACUGAC 1601 NM—001735.2_40804098—s 4080-4098 AGGAUUUGGCAGUGGCUUG 1033 CAAGCCACUGCCAAAUCCU 1602 NM—001735.2_40924110—s 4092-4110 UGGCUUGGCUACAGUACAU 1034 AUGUACUGUAGCCAAGCCA 1603 NM—001735.2_40994117—s 4099-4117 GCUACAGUACAUGUAACAA 1035 UUGUUACAUGUACUGUAGC 1604 NM—001735.2_41134131—s 4113-4131 AACAACUGUAGUUCACAAA 1036 UUUGUGAACUACAGUUGUU 1605 NM—001735.2_41204138—s 4120-4138 GUAGUUCACAAAAC CAGUA 1037 UACUGGUUUUGUGAACUAC 1606 NM—001735.2_41304148—s 4130-4148 AAACCAGUACCUCUGAGGA 1038 UCCUCAGAGGUACUGGUUU 1607 NM—001735.2_41434161—s 4143-4161 UGAGGAAGUUUGCAGCUUU 1039 AAAGCUGCAAACUUCCUCA 1608 NM—001735.2_41534171—s 4153-4171 UGCAGCUUUUAUUUGAAAA 1040 UUUUCAAAUAAAAGCUGCA 1609 NM—001735.2_41634181—s 4163-4181 AUUUGAAAAUC GAUACUCA 1041 UGAGUAUC GAUUUUCAAAU 1610 NM—001735.2_41734191—s 4173-4191 CGAUACUCAGGAUAUUGAA 1042 UUCAAUAUC CUGAGUAUC G 1611 NM—001735.2_41824200—s 4182-4200 GGAUAUUGAAGCAUCCCAC 1043 GUGGGAUGCUUCAAUAUCC 1612 NM—001735.2_41894207—s 4189-4207 GAAGCAUC C CACUACAGAG 1044 CUCUGUAGUGGGAUGCUUC 1613 NM—001735.2_41994217—s 4199-4217 ACUACAGAGGCUACGGAAA 1045 UUUCCGUAGCCUCUGUAGU 1614 NM—001735.2_42124230—s 4212-4230 CGGAAACUCUGAUUACAAA 1046 UUUGUAAUCAGAGUUUCCG 1615 NM—001735.2_42214239—s 4221-4239 UGAUUACAAACGCAUAGUA 1047 UACUAUGCGUUUGUAAUCA 1616 NM—001735.2_42324250—s 4232-4250 GCAUAGUAGCAUGUGCCAG 1048 CUGGCACAUGCUACUAUGC 1617 NM—001735.2_42404258_s 4240-4258 GCAUGUGCCAGCUACAAGC 1049 GCUUGUAGCUGGCACAUGC 1618 NM—001735.2_42514269_s 4251-4269 CUACAAGCCCAGCAGGGAA 1050 UUCCCUGCUGGGCUUGUAG 1619 NM—001735.2_42604278_s 4260-4278 CAGCAGGGAAGAAUCAUCA 1051 UGAUGAUUCUUCCCUGCUG 1620 NM—001735.2_42704288_s 4270-4288 GAAUCAUCAUCUGGAUCCU 1052 AGGAUCCAGAUGAUGAUUC 1621 NM—001735.2_42834301—s 4283-4301 GAUCCUCUCAUGCGGUGAU 1053 AUCACCGCAUGAGAGGAUC 1622

- 148044245

NM_001735.2_42894307_s 4289-4307 CUCAUGCGGUGAUGGACAU 1054 AUGUCCAUCACCGCAUGAG 1623 NM_001735.2_42994317_s 4299-4317 GAUGGACAUCUCCUUGCCU 1055 AGGCAAGGAGAUGUCCAUC 1624 NM_001735.2_43114329_s 4311-4329 CUUGCCUACUGGAAUCAGU 1056 ACUGAUUCCAGUAGGCAAG 1625 NM_001735.2_43224340_s 4322-4340 GAAUCAGUGCAAAUGAAGA 1057 UCUUCAUUUGCACUGAUUC 1626 NM_001735.2_43324350_s 4332-4350 AAAUGAAGAAGACUUAAAA 1058 UUUUAAGUCUUCUUCAUUU 1627 NM_001735.2_43394357_s 4339-4357 GAAGACUUAAAAGC C CUUG 1059 CAAGGGCUUUUAAGUCUUC 1628 NM_001735.2_4353- 4371_s 4353-4371 CCUUGUGGAAGGGGUGGAU 1060 AUG CAC C C CUUC CACAAGG 1629 NM_001735.2_43604378_s 4360-4378 GAAGGGGUGGAUCAACUAU 1061 AUAGUUGAUCCACCCCUUC 1630 NM_001735.2_43704388_s 4370-4388 AUCAACUAUUCACUGAUUA 1062 UAAUCAGUGAAUAGUUGAU 1631 NM_001735.2_43804398_s 4380-4398 CACUGAUUAC CAAAUCAAA 1063 UUUGAUUUGGUAAUCAGUG 1632 NM_001735.2_4393- 4411_s 4393-4411 AUCAAAGAUGGACAUGUUA 1064 UAACAUGUCCAUCUUUGAU 1633 NM_001735.2_44024420_s 4402-4420 GGACAUGUUAUUCUGCAAC 1065 GUUGCAGAAUAACAUGUCC 1634 NM_001735.2_4413- 4431_s 4413-4431 UCUGCAACUGAAUUCGAUU 1066 AAUCGAAUUCAGUUGCAGA 1635 NM_001735.2_44224440_s 4422-4440 GAAUUCGAUUCCCUCCAGU 1067 ACUGGAGGGAAUCGAAUUC 1636 NM_001735.2_44324450_s 4432-4450 С C CUC CAGUGAUUUC CUUU 1068 AAAGGAAAUCACUGGAGGG 1637 NM_001735.2_4441- 4459_s 4441-4459 GAUUUCCUUUGUGUACGAU 1069 AUCGUACACAAAGGAAAUC 1638 NM_001735.2_4453- 4471_s 4453-4471 GUAC GAUUC C GGAUAUUUG 1070 CAAAUAUC C GGAAUC GUAC 1639 NM_001735.2_44624480_s 4462-4480 CGGAUAUUUGAACUCUUUG 1071 CAAAGAGUUCAAAUAUC C G 1640 NM_001735.2_4473- 4491_s 4473-4491 ACUCUUUGAAGUUGGGUUU 1072 AAAC C CAACUUCAAAGAGU 1641 NM_001735.2_44824500_s 4482-4500 AGUUGGGUUUCUCAGUCCU 1073 AGGACUGAGAAACCCAACU 1642 NM_001735.2_44904508_s 4490-4508 UUCUCAGUCCUGCCACUUU 1074 AAAGUGGCAGGACUGAGAA 1643 NM_001735.2_45034521_s 4503-4521 CACUUUCACAGUGUACGAA 1075 UUCGUACACUGUGAAAGUG 1644 NM_001735.2_45094527_s 4509-4527 CACAGUGUAC GAAUAC CAC 1076 GUGGUAUUCGUACACUGUG 1645 NM_001735.2_4523- 4541_s 4523-4541 AC C AC AGAC C AGAUAAAC A 1077 UGUUUAUCUGGUCUGUGGU 1646

- 149044245

NM_001735.2_45314549_s 4531-4549 C CAGAUAAACAGUGUACCA 1078 UGGUACACUGUUUAUCUGG 1647 NM_001735.2_45404558_s 4540-4558 CAGUGUAC CAUGUUUUAUA 1079 UAUAAAACAUGGUACACUG 1648 NM_001735.2_4551- 4569_s 4551-4569 GUUUUAUAGCACUUCCAAU 1080 AUUGGAAGUGCUAUAAAAC 1649 NM_001735.2_45624580_s 4562-4580 CUUC CAAUAUCAAAAUUCA 1081 UGAAUUUUGAUAUUGGAAG 1650 NM_001735.2_45704588_s 4570-4588 AUCAAAAUUCAGAAAGUCU 1082 AGACUUUCUGAAUUUUGAU 1651 NM_001735.2_4581- 4599_s 4581-4599 GAAAGUCUGUGAAGGAGCC 1083 GGCUCCUUCACAGACUUUC 1652 NM_001735.2_45914609_s 4591-4609 GAAGGAGCCGCGUGCAAGU 1084 ACUUGCACGCGGCUCCUUC 1653 NM_001735.2_46014619_s 4601-4619 CGUGCAAGUGUGUAGAAGC 1085 GCUUCUACACACUUGCACG 1654 NM_001735.2_46124630_s 4612-4630 GUAGAAGCUGAUUGUGGGC 1086 GCCCACAAUCAGCUUCUAC 1655 NM_001735.2_46194637_s 4619-4637 CUGAUUGUGGGCAAAUGCA 1087 UGCAUUUGCCCACAAUCAG 1656 NM_001735.2_46294647_s 4629-4647 GCAAAUGCAGGAAGAAUUG 1088 CAAUUCUUCCUGCAUUUGC 1657 NM_001735.2_46394657_s 4639-4657 GAAGAAUUGGAUCUGACAA 1089 UUGUCAGAUCCAAUUCUUC 1658 NM_001735.2_46514669_s 4651-4669 CUGACAAUCUCUGCAGAGA 1090 UCUCUGCAGAGAUUGUCAG 1659 NM_001735.2_46634681_s 4663-4681 GCAGAGACAAGAAAACAAA 1091 UUUGUUUUCUUGUCUCUGC 1660 NM_001735.2_46704688_s 4670-4688 CAAGAAAACAAACAGCAUG 1092 CAUGCUGUUUGUUUUCUUG 1661 NM_001735.2_46814699_s 4681-4699 ACAGCAUGUAAACCAGAGA 1093 UCUCUGGUUUACAUGCUGU 1662 NM_001735.2_46934711_s 4693-4711 CCAGAGAUUGCAUAUGCUU 1094 AAGCAUAUGCAAUCUCUGG 1663 NM_001735.2_47024720_s 4702-4720 GCAUAUGCUUAUAAAGUUA 1095 UAACUUUAUAAGCAUAUGC 1664 NM_001735.2_47104728_s 4710-4728 UUAUAAAGUUAGCAUCACA 1096 UGUGAUGCUAACUUUAUAA 1665 NM_001735.2_47224740_s 4722-4740 CAUCACAUCCAUCACUGUA 1097 UACAGUGAUGGAUGUGAUG 1666 NM_001735.2_4733- 4751_s 4733-4751 UCACUGUAGAAAAUGUUUU 1098 AAAACAUUUUCUACAGUGA 1667 NM_001735.2_47404758_s 4740-4758 AGAAAAUGUUUUUGUCAAG 1099 CUUGACAAAAACAUUUUCU 1668 NM_001735.2_47504768_s 4750-4768 UUUGUCAAGUACAAGGCAA 1100 UUGCCUUGUACUUGACAAA 1669 NM_001735.2_47634781_s 4763-4781 AGGCAACCCUUCUGGAUAU 1101 AUAUCCAGAAGGGUUGCCU 1670

- 150044245

NM_001735.2_47704788_s 4770-4788 CCUUCUGGAUAUCUACAAA 1102 UUUGUAGAUAUCCAGAAGG 1671 NM_001735.2_47794797_s 4779-4797 UAUCUACAAAACUGGGGAA 1103 UUC С C CAGUUUUGUAGAUA 1672 NM_001735.2_47904808_s 4790-4808 CUGGGGAAGCUGUUGCUGA 1104 UCAGCAACAGCUUCCCCAG 1673 NM_001735.2_47994817_s 4799-4817 CUGUUGCUGAGAAAGACUC 1105 GAGUCUUUCUCAGCAACAG 1674 NM_001735.2_48134831_s 4813-4831 GACUCUGAGAUUACCUUCA 1106 UGAAGGUAAUCUCAGAGUC 1675 NM_001735.2_48194837_s 4819-4837 GAGAUUAC CUUCAUUAAAA 1107 UUUUAAUGAAGGUAAUCUC 1676 NM_001735.2_48314849_s 4831-4849 AUUAAAAAGGUAACCUGUA 1108 UACAGGUUACCUUUUUAAU 1677 NM_001735.2_48414859_s 4841-4859 UAACCUGUACUAACGCUGA 1109 UCAGCGUUAGUACAGGUUA 1678 NM_001735.2_48504868_s 4850-4868 CUAACGCUGAGCUGGUAAA 1110 UUUACCAGCUCAGCGUUAG 1679 NM_001735.2_48634881_s 4863-4881 GGUAAAAGGAAGACAGUAC 1111 GUACUGUCUUCCUUUUACC 1680 NM_001735.2_48714889_s 4871-4889 GAAGACAGUACUUAAUUAU 1112 AUAAUUAAGUACUGUCUUC 1681 NM_001735.2_4881- 4899_s 4881-4899 CUUAAUUAUGGGUAAAGAA 1113 UUCUUUAC C CAUAAUUAAG 1682 NM_001735.2_4893- 4911_s 4893-4911 UAAAGAAGC C CUC CAGAUA 1114 UAUCUGGAGGGCUUCUUUA 1683 NM_001735.2_49024920_s 4902-4920 C CUC CAGAUAAAAUACAAU 1115 AUUGUAUUUUAUCUGGAGG 1684 NM_001735.2_49124930_s 4912-4930 AAAUACAAUUUCAGUUUCA 1116 UGAAACUGAAAUUGUAUUU 1685 NM_001735.2_4923- 4941_s 4923-4941 CAGUUUCAGGUACAUCUAC 1117 GUAGAUGUACCUGAAACUG 1686 NM_001735.2_4931- 4949_s 4931-4949 GGUACAUCUAC C CUUUAGA 1118 UCUAAAGGGUAGAUGUACC 1687 NM_001735.2_49424960_s 4942-4960 CCUUUAGAUUCCUUGACCU 1119 AGGUCAAGGAAUCUAAAGG 1688 NM_001735.2_49524970_s 4952-4970 CCUUGACCUGGAUUGAAUA 1120 UAUUCAAUCCAGGUCAAGG 1689 NM_001735.2_4961- 4979_s 4961-4979 GGAUUGAAUACUGGCCUAG 1121 CUAGGCCAGUAUUCAAUCC 1690 NM_001735.2_49714989_s 4971-4989 CUGGCCUAGAGACACAACA 1122 UGUUGUGUCUCUAGGCCAG 1691 NM_001735.2_49794997_s 4979-4997 GAGACACAACAUGUUCAUC 1123 GAUGAACAUGUUGUGUCUC 1692 NM_001735.2_49915009_s 4991-5009 GUUCAUCGUGUCAAGCAUU 1124 AAUGCUUGACACGAUGAAC 1693 NM_001735.2_50005018_s 5000-5018 GUCAAGCAUUUUUAGCUAA 1125 UUAGCUAAAAAUGCUUGAC 1694

- 151 044245

NM_001735.2_50135031_s 5013-5031 AGCUAAUUUAGAUGAAUUU 1126 AAAUUCAUCUAAAUUAGCU 1695 NM_001735.2_50225040_s 5022-5040 AGAUGAAUUUGC C GAAGAU 1127 AUCUUCGGCAAAUUCAUCU 1696 NM_001735.2_50335051_s 5033-5051 C C GAAGAUAUCUUUUUAAA 1128 UUUAAAAAGAUAUCUUCGG 1697 NM_001735.2_50435061_s 5043-5061 CUUUUUAAAUGGAUGCUAA 1129 UUAGCAUCCAUUUAAAAAG 1698 NM_001735.2_50535071_s 5053-5071 GGAUGCUAAAAUUCCUGAA 1130 UUCAGGAAUUUUAGCAUCC 1699 NM_001735.2_50595077_s 5059-5077 UAAAAUUC CUGAAGUUCAG 1131 CUGAACUUCAGGAAUUUUA 1700 NM_001735.2_50715089_s 5071-5089 AGUUCAGCUGCAUACAGUU 1132 AACUGUAUGCAGCUGAACU 1701 NM_001735.2_50805098_s 5080-5098 GCAUACAGUUUGCACUUAU 1133 AUAAGUGCAAACUGUAUGC 1702 NM_001735.2_50935111_s 5093-5111 ACUUAUGGACUCCUGUUGU 1134 ACAACAGGAGUCCAUAAGU 1703 NM_001735.2_5099- 5117_s 5099-5117 GGACUCCUGUUGUUGAAGU 1135 ACUUCAACAACAGGAGUCC 1704 NM_001735.2_51095127_s 5109-5127 UGUUGAAGUUC GUUUUUUU 1136 AAAAAAAC GAACUUCAACA 1705 NM_001735.2_51225140_s 5122-5140 UUUUUUGUUUUCUUCUUUU 1137 AAAAGAAGAAAACAAAAAA 1706 NM_001735.2_51325150_s 5132-5150 UCUUCUUUUUUUAAACAUU 1138 AAUGUUUAAAAAAAGAAGA 1707 NM_001735.2_51395157_s 5139-5157 UUUUUAAACAUUCAUAGCU 1139 AGCUAUGAAUGUUUAAAAA 1708 NM_001735.2_51525170_s 5152-5170 AUAGCUGGUCUUAUUUGUA 1140 UACAAAUAAGACCAGCUAU 1709 NM_001735.2_51595177_s 5159-5177 GUCUUAUUUGUAAAGCUCA 1141 UGAGCUUUACAAAUAAGAC 1710 NM_001735.2_51705188_s 5170-5188 AAAGCUCACUUUACUUAGA 1142 UCUAAGUAAAGUGAGCUUU 1711 NM_001735.2_51825200_s 5182-5200 ACUUAGAAUUAGUGGCACU 1143 AGUGCCACUAAUUCUAAGU 1712 NM_001735.2_51925210_s 5192-5210 AGUGGCACUUGCUUUUAUU 1144 AAUAAAAGCAAGUGCCACU 1713 NM_001735.2_52025220_s 5202-5220 GCUUUUAUUAGAGAAUGAU 1145 AUCAUUCUCUAAUAAAAGC 1714 NM_001735.2_52125230_s 5212-5230 GAGAAUGAUUUCAAAUGCU 1146 AGCAUUUGAAAUCAUUCUC 1715 NM_001735.2_52205238_s 5220-5238 UUUCAAAUGCUGUAACUUU 1147 AAAGUUACAGCAUUUGAAA 1716 NM_001735.2_5231- 5249_s 5231-5249 GUAACUUUCUGAAAUAACA 1148 UGUUAUUUCAGAAAGUUAC 1717 NM_001735.2_5241- 5259_s 5241-5259 GAAAUAACAUGGCCUUGGA 1149 UCCAAGGCCAUGUUAUUUC 1718

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NM_001735.2_5253- 5271_s 5253-5271 CCUUGGAGGGCAUGAAGAC 1150 GUCUUCAUGCCCUCCAAGG 1719 NM_001735.2_52595277_s 5259-5277 AGGGCAUGAAGACAGAUAC 1151 GUAUCUGUCUUCAUGCCCU 1720 NM_001735.2_5273- 5291_s 5273-5291 GAUACUC CUC CAAGGUUAU 1152 AUAACCUUGGAGGAGUAUC 1721 NM_001735.2_52795297_s 5279-5297 CCUCCAAGGUUAUUGGACA 1153 UGUCCAAUAACCUUGGAGG 1722 NM_001735.2_5293- 5311_s 5293-5311 GGACACCGGAAACAAUAAA 1154 UUUAUUGUUUCCGGUGUCC 1723 NM_001735.2_5301- 5319_s 5301-5319 GAAACAAUAAAUUGGAACA 1155 UGUUCCAAUUUAUUGUUUC 1724 NM_001735.2_53115329_s 5311-5329 AUUGGAACACCUCCUCAAA 1156 UUUGAGGAGGUGUUCCAAU 1725 NM_001735.2_53225340_s 5322-5340 UCCUCAAACCUACCACUCA 1157 UGAGUGGUAGGUUUGAGGA 1726 NM_001735.2_5331- 5349_s 5331-5349 CUACCACUCAGGAAUGUUU 1158 AAACAUUCCUGAGUGGUAG 1727 NM_001735.2_5343- 5361_s 5343-5361 AAUGUUUGCUGGGGCCGAA 1159 UUCGGCCCCAGCAAACAUU 1728 NM_001735.2_53495367_s 5349-5367 UGCUGGGGCCGAAAGAACA 1160 UGUUCUUUCGGCCCCAGCA 1729 NM_001735.2_53605378_s 5360-5378 AAAGAACAGUCCAUUGAAA 1161 UUUCAAUGGACUGUUCUUU 1730 NM_001735.2_53715389_s 5371-5389 CAUUGAAAGGGAGUAUUAC 1162 GUAAUACUCCCUUUCAAUG 1731 NM_001735.2_53805398_s 5380-5398 GGAGUAUUACAAAAACAUG 1163 CAUGUUUUUGUAAUACUCC 1732 NM_001735.2_53915409_s 5391-5409 AAAACAUGGCCUUUGCUUG 1164 CAAGCAAAGGCCAUGUUUU 1733 NM_001735.2_53995417_s 5399-5417 GCCUUUGCUUGAAAGAAAA 1165 UUUUCUUUCAAGCAAAGGC 1734 NM_001735.2_54095427_s 5409-5427 GAAAGAAAAUACCAAGGAA 1166 UUCCUUGGUAUUUUCUUUC 1735 NM_001735.2_54205438_s 5420-5438 CCAAGGAACAGGAAACUGA 1167 UCAGUUUCCUGUUCCUUGG 1736 NM_001735.2_5433- 5451_s 5433-5451 AACUGAUCAUUAAAGCCUG 1168 CAGGCUUUAAUGAUCAGUU 1737 NM_001735.2_5441- 5459_s 5441-5459 AUUAAAGCCUGAGUUUGCU 1169 AGCAAACUCAGGCUUUAAU 1738

Example 5. C5 silencing in vivo.

Groups of three female cynomolgus macaques were treated subcutaneously in the scapula and mid-dorsa with C5-siRNA AD-58641 at a dose of 2.5 mg/kg or 5 mg/kg, or with vehicle as a control. Two rounds of the dosing regimen were administered with eight doses in each round, every third day. Serum C5 was collected and assessed using a C5-specific ELISA assay (Abeam) at the indicated time points (Fig. 13). C5 levels were normalized to the mean of three pre-treatment samples. Samples collected pre-treatment and on day 23 (24 h after the last dose in the first treatment round) were subjected to serum biochemistry, hematology, and coagulation analysis.

Analysis of serum C5 protein levels compared to pretreatment serum C5 protein levels showed that the 5 mg/kg AD-58641 regimen resulted in up to a 98% reduction in serum C5 protein levels (Fig. 12). Mean serum C5 levels were reduced by 97% at the nadir, indicating that the majority of circulating C5 originates from the liver. A dose-dependent effect and durable knockdown of serum C5 protein levels were observed with subcutaneous administration of AD-58641. No changes in hematology, serum biochemistry, or coagulation parameters were detected 24 hours after the first round of the regimen.

Serum hemolytic activity was also analyzed using the sensitized sheep red blood cell assay to measure classical pathway activity. The percentage of hemolytic activity was calculated as the ratio of maximum hemolysis to background hemolysis in control samples.

The mean hemolysis index +/- SEM for three animals was calculated and analyzed (Fig. 13). Hemolysis was reduced to 94% in the 5 mg/kg treatment regimen, with a mean inhibition value of 92% at the nadir. This reduction in hemolysis was maintained for more than two weeks after the last dose.

Example 6. Screening of additional siRNAs in vitro.

The sequences of the sense and antisense strands presented in Table 20 were modified at the 3' end with a short deoxythymine nucleotide sequence (dT) (Table 21). The in vitro efficiency of duplexes containing the sense and antisense sequences presented in Table 21 was determined using the following methods.

Cell culture and transfections.

Hep3B cells (ATCC, Manassas, VA) were grown to near confluence at 37°C under 5% _CO2 in EMEM (ATCC) supplemented with 10% FBS (ATCC) before detaching from the dish by trypsinization. Transfection was accomplished by adding 5 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad, CA, catalog #13778-150) to 5 μl of siRNA duplexes per well in a 384-well plate and incubated at room temperature for 15 min. 40 μl of complete growth media containing ~5 x ¹⁰³ Hep3B cells were then added to the siRNA mixture. Cells were incubated for 24 h before RNA purification. Experiments were performed at a final duplex concentration of 10 nM.

Total RNA isolation using the DYNABEADS mRNA Isolation Kit (Invitrogen, catalog #610-12).

RNA extraction was performed using a Biotek EL 405 semiautomated washer. Briefly, cells were lysed in 75 μl of lysis/binding buffer containing 2 μl of Dynabeads and then mixed for 10 min at setting 7 on an electromagnetic shaker (Union Scientific). Magnetic beads were fixed using a magnetic stand, and the supernatant was removed. After removing the supernatant, the magnetic beads were washed with 90 μl of wash buffer A, then with 90 μl of wash buffer B. The beads were washed twice with 100 μl of elution buffer, which was then aspirated, and cDNA was obtained directly from bead-bound RNA in a 384-well plate.

cDNA synthesis using the ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Forster City, CA, cat. #4368813).

A master mix of 2 µl 10X buffer, 0.8 µl 25X dNTPs, 2 µl random primers, 1 µl reverse transcriptase, 1 µl RNase inhibitor, and 3.2 µl H2O per reaction was added directly to the bead-bound RNA in 384-well plates used for RNA extraction. The plates were then shaken on an electromagnetic shaker for 10 min and then incubated at 37°C for 2 h. Following this incubation, the plates were shaken in an incubator at 80°C for 7 min to inactivate the enzyme and elute RNA/cDNA from the beads.

Real-time PCR.

µl of cDNA was added to a master mix mixture containing 0.5 µl of TaqMan probe for GAPDH (Applied Biosystems, catalog #4326317E), 0.5 µl of TaqMan probe for C5 (Applied Biosystems, catalog #Hs00156197_M1), and 0.5 µl of Lightcycler 480 master mix probe (Roche, catalog #04887301001) in each well of a 384-well plate (Roche, catalog #04887301001). Real-time PCR was performed on a Roche LC480 Real Time PCR system (Roche). Each duplex was tested in at least two independent transfections, and each transfection was assessed in duplicate.

To calculate the relative fold change, real-time data were analyzed using the AACt method and normalized to those of assays performed with cells transfected with 10 nM AD-1955 or mock-transfected cells.

Table 22 shows the results of a single-dose assay in Hep3B cells transfected with the indicated dT-modified mRNAs. Data are expressed as a percentage of the remaining transcript relative to untreated cells.

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Table 21 dT-modified C5 mRNAs

Duplex ID Semantic sequence SEQ ID NO: Position in NM_001735.2 Antisense sequence SEQ ID NO: AD-61779.2 UAUCCGUGGUUUCCUGCUAdTdT 1739 3-21 UAGCAGGAAACCACGGAUAdTdT 2306 AD-61785.2 GGUUUCCUGCUACCUCCAAdTdT 1740 10-28 UUGGAGGUAGCAGGAAACCdTdT 2307 AD-61791.2 CCUCCAACCAUGGGCCUUUdTdT 1741 22:40 AAAGGCCCAUGGUUGGAGGdTdT 2308 AD-61797.2 GGGCCUUUUGGGAAUACUUdTdT 1742 33-51 AAGUAUUCCCAAAAGGCCCdTdT 2309 AD-61803.2 GGAAUACUUUGUUUUUUAAdTdT 1743 43-61 UUAAAAAACAAAGUAUUCCdTdT 2310 AD-61809.2 CUUUGUUUUUUAAUCUUCCdTdT 1744 49-67 GGAAGAUUAAAAAACAAAGdT dT 2311 AD-61815.2 CUUCCUGGGGAAAACCUGGdTdT 1745 63-81 CCAGGUUUUCCCCAGGAAGdTdT 2312 AD-61821.2 GGAAAACCUGGGGACAGGAdTdT 1746 71-89 UCCUGUCCCCAGGUUUUCCdTdT 2313 AD-61780.2 GGGACAGGAGCAAACAUAUdTdT 1747 81-99 AUAUGUUUGCUCCUGUCCCdTdT 2314 AD-61786.2 CAAACAUAUGUCAUUUCAGdTdT 1748 91-109 CUGAAAUGACAUAUGUUUGdTdT 2315 AD-61792.2 CAUUUCAGCACCAAAAAUAdTdT 1749 102-120 UAUUUUUGGUGCUGAAAUGdTdT 2316 AD-61798.2 GCACCAAAAAUAUUCCGUGdTdT 1750 109-127 CACGGAAUAUUUUUGGUGCdTdT 2317 AD-61804.2 CCGUGUUGGAGCAUCUGAAdTdT 1751 123-141 UUCAGAUGCUCCAACACGGdTdT 2318 AD-61810.2 GGAGCAUCUGAAAAUAUUGdTdT 1752 130-148 CAAUAUUUUCAGAUGCUCCdTdT 2319 AD-61816.2 GAAAAUAUUGUGAUUCAAGdT dT 1753 139-157 CUUGAAUCACAAUAUUUUCdTdT 2320 AD-61822.2 GAUUCAAGUUUAUGGAUACdTdT 1754 150-168 GUAUCCAUAAACUUGAAUCdTdT 2321 AD-61781.2 GGAUACACUGAAGCAUUUGdTdT 1755 163-181 CAAAUGCUUCAGUGUAUCCdTdT 2322 AD-61787.2 GAAGCAUUUGAUGCAACAAdTdT 1756 172-190 UUGUUGCAUCAAAUGCUUCdTdT 2323 AD-61793.2 UGCAACAAUCUCUAUUAAAdTdT 1757 183-201 UUUAAUAGAGAUUGUUGCAdT dT 2324 AD-61799.2 ^UCUCUAUUAAAAGUUAUdT dT 1758 189-207 AUAACUUUUAAUAGAGAUUdT dT 2325 AD-61805.2 ^GUUAUCCUGAUAAAAAAdTdT 1759 201-219 UUUUUUAUCAGGAUAACUUdT dT 2326 AD-61811.2 CUGAUAAAAAAUUUAGUUAdT dT 1760 209-227 UAACUAAAUUUUUUAUCAGdT dT 2327 AD-61817.2 UUAGUUACUCCUCAGGCCAdTdT 1761 221-239 UGGCCUGAGGAGUAACUAAdTdT 2328 AD-61823.2 CCUCAGGCCAUGUUCAUUUdTdT 1762 230-248 AAAUGAACAUGGCCUGAGGdTdT 2329 AD-61782.2 UUCAUUUAUCCUCAGAGAAdTdT 1763 242-260 UUCUCUGAGGAUAAAUGAAdTdT 2330 AD-61788.2 CUCAGAGAAUAAAUUCCAAdTdT 1764 252-270 UUGGAAUUUAUUCUCUGAGdTdT 2331 AD-61794.2 4AUAAAUUCCAAAACUCUGdTdT 1765 259-277 CAGAGUUUUGGAAUUUAUUdT dT 2332 AD-61800.2 CUCUGCAAUCUUAACAAUAdTdT 1766 273-291 UAUUGUUAAGAUUGCAGAGdTdT 2333 AD-61806.2 CUUAACAAUACAACCAAAAdTdT 1767 282-300 UUUUGGUUGUAUUGUUAAGdTdT 2334 AD-61812.2 CAACCAAAACAAUUGCCUGdTdT 1768 292-310 CAGGCAAUUGUUUUGGUUGdTdT 2335 AD-61818.2 CAAUUGCCUGGAGGACAAAdTdT 1769 301-319 UUUGUCCUCCAGGCAAUUGdTdT 2336 AD-61824.2 GGACAAAACCCAGUUUCUUdTdT 1770 313-331 AAGAAACUGGGUUUUGUCCdTdT 2337 AD-61783.2 CCAGUUUCUUAUGUGUAUUdTdT 1771 322-340 AAUACACAUAAGAAACUGGdTdT 2338 AD-61789.2 AJGUGUAUUUGGAAGUUGUdTdT 1772 332-350 ACAACUUCCAAAUACACAUdTdT 2339 AD-61795.2 GGAAGUUGUAUCAAAGCAUdTdT 1773 342-360 AUGCUUUGAUACAACUUCCdTdT 2340 AD-61801.2 GUAUCAAAGCAUUUUUCAAdTdT 1774 349-367 UUGAAAAAUGCUUUGAUACdTdT 2341 AD-61807.2 DUUUCAAAAUCAAAAAGAAdT dT 1775 361-379 UUCUUUUUGAUUUUGAAAAdTdT 2342 AD-61813.2 CAAAAAGAAUGCCAAUAACdTdT 1776 371-389 GUUAUUGGCAUUCUUUUUGdTdT 2343 AD-61819.2 GCCAAUAACCUAUGACAAUdTdT 1777 381-399 AUUGUCAUAGGUUAUUGGCdTdT 2344 AD-61825.2 CCUAUGACAAUGGAUUUCUdTdT 1778 389-407 AGAAAUCCAUUGUCAUAGGdTdT 2345

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AD-61784.2 UGGAUUUCUCUUCAUUCAUdTdT 1779 399-417 AUGAAUGAAGAGAAAUCCAdTdT 2346 AD-61790.2 CAUUCAUACAGACAAACCUdTdT 1780 411-429 AGGUUUGUCUGUAUGAAUGdTdT 2347 AD-61796.2 CAGACAAACCUGUUUAUACdTdT 1781 419-437 GUAUAAACAGGUUUGUCUGdTdT 2348 AD-61802.2 GUUUAUACUCCAGACCAGUdTdT 1782 430-448 ACUGGUCUGGAGUAUAAACdTdT 2349 AD-61808.2 AGACCAGUCAGUAAAAGUUdTdT 1783 441-459 AACUUUUACUGACUGGUCUdTdT 2350 AD-61814.2 AGUAAAAGUUAGAGUUUAUdT dT 1784 450-468 AUAAACUCUAACUUUUACUdTdT 2351 AD-61820.2 ASAGUUUAUUCGUUGAAUGdTdT 1785 460-478 CAUUCAACGAAUAAACUCUdTdT 2352 AD-61826.2 CGUUGAAUGACGACUUGAAdTdT 1786 470-488 UUCAAGUCGUCAUUCAACGdTdT 2353 AD-61832.2 CUUGAAGCCAGCCAAAAGAdTdT 1787 483-501 UCUUUUGGCUGGCUUCAAGdTdT 2354 AD-61838.2 CCAGCCAAAAGAGAAACUGdTdT 1788 490-508 CAGUUUCUCUUUUGGCUGGdTdT 2355 AD-61844.2 4AACUGUCUUhACUUUCAUdTdT 1789 503-521 AUGAAAGUUAAGACAGUUUdT dT 2356 AD-61850.2 4ACUUUCAUAGAUCCUGAAdTdT 1790 513-531 UUCAGGAUCUAUGAAAGUUdTdT 2357 AD-61856.2 CAUAGAUCCUGAAGGAUCAdTdT 1791 519-537 UGAUCCUUCAGGAUCUAUGdTdT 2358 AD-61862.2 GAAGGAUCAGAAGUUGACAdTdT 1792 529-547 UGUCAACUUCUGAUCCUUCdTdT 2359 AD-61868.2 UGACAUGGUAGAAGAAAUUdTdT 1793 543-561 AAUUUCUUCUACCAUGUCAdTdT 2360 AD-61827.2 GAAGAAAUUGAUCAUAUUGdT dT 1794 553-571 CAAUAUGAUCAAUUUCUUCdTdT 2361 AD-61833.2 GAUCAUAUUGGAAUUAUCUdT dT 1795 562-580 AGAUAAUUCCAAUAUGAUCdTdT 2362 AD-61839.2 GGAAUUAUCUCUUUUCCUGdTdT 1796 571-589 CAGGAAAAGAGAUAAUUCCdTdT 2363 AD-61845.2 CUCUUUUCCUGACUUCAAGdTdT 1797 579-597 CUUGAAGUCAGGAAAAGAGdTdT 2364 AD-61851.2 ACUUCAAGAUUCCGUCUAAdTdT 1798 590-608 UUAGACGGAAUCUUGAAGUdTdT 2365 AD-61857.2 CCGUCUAAUCCUAGAUAUGdTdT 1799 601-619 CAUAUCUAGGAUUAGACGGdTdT 2366 AD-61863.2 CCUAGAUAUGGUAUGUGGAdTdT 1800 610-628 UCCACAUACCAUAUCUAGGdTdT 2367 AD-61869.2 UGUGGACGAUCAAGGCUAAdTdT 1801 623-641 UUAGCCUUGAUCGUCCACAdTdT 2368 AD-61828.2 CGAUCAAGGCUAAAUAUAAdTdT 1802 629-647 UUAUAUUUAGCCUUGAUCGdTdT 2369 AD-61834.2 AJAUAAAGAGGACUUUUCAdT dT 1803 642-660 UGAAAAGUCCUCUUUAUAUdTdT 2370 AD-61840.2 GAGGACUUUUCAACAACUGdTdT 1804 649-667 CAGUUGUUGAAAAGUCCUCdTdT 2371 AD-61846.2 CAACUGGAACCGCAUAUUUdTdT 1805 662-680 AAAUAUGCGGUUCCAGUUGdTdT 2372 AD-61852.2 CGCAUAUUUUGAAGUUAAAdTdT 1806 672-690 UUUAACUUCAAAAUAUGCGdTdT 2373 AD-61858.2 AASUUAAAGAAUAUGUCUUdTdT 1807 683-701 AAGACAUAUUCUUUAACUUdT dT 2374 AD-61864.2 GAAUAUGUCUUGCCACAUUdTdT 1808 691-709 AAUGUGGCAAGACAUAUUCdTdT 2375 AD-61870.2 CCACAUUUUUCUGUCUCAAdTdT 1809 703-721 UUGAGACAGAAAAAUGUGGdTdT 2376 AD-61829.2 CUGUCUCAAUCGAGCCAGAdTdT 1810 713-731 UCUGGCUCGAUUGAGACAGdTdT 2377 AD-61835.2 CAAUCGAGCCAGAAUAUAAdTdT 1811 719-737 UUAUAUUCUGGCUCGAUUGdTdT 2378 AD-61841.2 GAAUAUAAUUUCAUUGGUUdT dT 1812 730-748 AACCAAUGAAAUUAUAUUCdTdT 2379 AD-61847.2 AJUGGUUACAAGAACUUUAdTdT 1813 742-760 UAAAGUUCUUGUAACCAAUdTdT 2380 AD-61853.2 AGAACUUUAAGAAUUUUGAdT dT 1814 752-770 UCAAAAUUCUUAAAGUUCUdTdT 2381 AD-61859.2 GAAUUUUGAAAUUACUAUAdT dT 1815 762-780 UAUAGUAAUUUCAAAAUUCdTdT 2382 AD-61865.2 GAAAUUACUAUAAAAGCAAdT dT 1816 769-787 UUGCUUUUAUAGUAAUUUCdTdT 2383 AD-61871.2 /WkGCAAGAUAUUUUUAUAdT dT 1817 781-799 UAUAAAAAUAUCUUGCUUUdT dT 2384 AD-61830.2 AJAUUUUUAUAAUAAAGUAdT dT 1818 789-807 UACUUUAUUAUAAAAAUAUdT dT 2385 AD-61836.2 ?\AGUAGUCACUGAGGCUGAdTdT 1819 803-821 UCAGCCUCAGUGACUACUUdTdT 2386 AD-61842.2 CACUGAGGCUGACGUUUAUdTdT 1820 810-828 AUAAACGUCAGCCUCAGUGdTdT 2387 AD-61848.2 CGUUUAUAUCACAUUUGGAdTdT 1821 822-840 UCCAAAUGUGAUAUAAACGdTdT 2388 AD-61854.2 CACAUUUGGAAUAAGAGAAdT dT 1822 831-849 UUCUCUUAUUCCAAAUGUGdTdT 2389

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AD-61860.2 4AUAAGAGAAGACUUAAAAdT dT 1823 840-858 UUUUAAGUCUUCUCUUAUUdTdT 2390 AD-61866.2 GUUAAAAGAUGAUCAAAAAdT dT 1824 852-870 UUUUUGAUCAUCUUUUAAGdTdT 2391 AD-61872.2 GAUGAUCAAAAAGAAAUGAdT dT 1825 859-877 UCAUUUCUUUUUGAUCAUCdTdT 2392 AD-61831.2 4AAUGAUGCAAACAGCAAUdTdT 1826 872-890 AUUGCUGUUUGCAUCAUUUdTdT 2393 AD-61837.2 AGAGCAAUGCAAAACACAAdT dT 1827 883-901 UUGUGUUUUGCAUUGCUGUdTdT 2394 AD-61843.2 /WkACACAAUGUUGAUAAAdT dT 1828 893-911 UUUAUCAACAUUGUGUUUUdTdT 2395 AD-61849.2 CAAUGUUGAUAAAUGGAAUdTdT 1829 899-917 AUUCCAUUUAUCAACAUUGdTdT 2396 AD-61855.2 GGAAUUGCUCAAGUCACAUdTdT 1830 913-931 AUGUGACUUGAGCAAUUCCdTdT 2397 AD-61861.2 GCUCAAGUCACAUUUGAUUdTdT 1831 919-937 AAUCAAAUGUGACUUGAGCdTdT 2398 AD-61867.2 AJUUGAUUCUGAAACAGCAdTdT 1832 930-948 UGCUGUUUCAGAAUCAAAUdTdT 2399 AD-620 62.1 UGAAACAGCAGUCAAAGAAdTdT 1833 939-957 UUCUUUGACUGCUGUUUCAdTdT 2400 AD-62068.1 GAAAGAACUGUCAUACUACdTdT 1834 951-969 GUAGUAUGACAGUUCUUUGdTdT 2401 AD-62074.1 GAUACUACAGUUUAGAAGAdT dT 1835 962-980 UCUUCUAAACUGUAGUAUGdTdT 2402 AD-6208 0.1 GAGUUUAGAAGAUUUAAACdTdT 1836 969-987 GUUUAAAUCUUCUAAACUGdTdT 2403 AD-620 86.1 UAAACAACAAGUACCUUUAdTdT 1837 983-1001 UAAAGGUACUUGUUGUUUAdTdT 2404 AD-62092.1 GAAGUACCUUUAUAUUGCUdTdT 1838 990-1008 AGCAAUAUAAAGGUACUUGdTdT 2405 AD-62098.1 UAUUGCUGUAACAGUCAUAdTdT 1839 1002-1020 UAUGACUGUUACAGCAAUAdT dT 2406 AD-62104.1 4ACAGUCAUAGAGUCUACAdTdT 1840 1011-1029 UGUAGACUCUAUGACUGUUdTdT 2407 AD-62063.1 AGAGUCUACAGGUGGAUUUdTdT 1841 1020-1038 AAAUCCACCUGUAGACUCUdTdT 2408 AD-62069.1 GGAUUUUCUGAAGAGGCAGdTdT 1842 1033-1051 CUGCCUCUUCAGAAAAUCCdTdT 2409 AD-62075.1 GAAGAGGCAGAAAUACCUGdTdT 1843 1042-1060 CAGGUAUUUCUGCCUCUUCdTdT 2410 AD-62081.1 AGAAAUACCUGGCAUCAAAdTdT 1844 1050-1068 UUUGAUGCCAGGUAUUUCUdTdT 2411 AD-62087.1 GCAUCAAAUAUGUCCUCUCdTdT 1845 1061-1079 GAGAGGACAUAUUUGAUGCdTdT 2412 AD-62093.1 UGUCCUCUCUCCCUACAAAdTdT 1846 1071-1089 UUUGUAGGGAGAGAGGACAdTdT 2413 AD-62099.1 GAAUUUGGUUGCUACUCCUdTdT 1847 1092-1110 AGGAGUAGCAACCAAAUUCdTdT 2414 AD-62105.1 GCUACUCCUCUUUUCCUGAdTdT 1848 1102-1120 UCAGGAAAAGAGGAGUAGCdTdT 2415 AD-62064.1 GUCUUUUCCUGAAGCCUGGdTdT 1849 1109-1127 CCAGGCUUCAGGAAAAGAGdTdT 2416 AD-62070.1 GCUGGGAUUCCAUAUCCCAdTdT 1850 1123-1141 UGGGAUAUGGAAUCCCAGGdTdT 2417 AD-62076.1 GAUAUCCCAUCAAGGUGCAdTdT 1851 1133-1151 UGCACCUUGAUGGGAUAUGdTdT 2418 AD-62082.1 GCAUCAAGGUGCAGGUUAAdTdT 1852 1139-1157 UUAACCUGCACCUUGAUGGdTdT 2419 AD-62088.1 GAGGUUAAAGAUUCGCUUGdTdT 1853 1150-1168 CAAGCGAAUCUUUAACCUGdTdT 2420 AD-62094.1 UUCGCUUGACCAGUUGGUAdTdT 1854 1161-1179 UACCAACUGGUCAAGCGAAdTdT 2421 AD-62100.1 GCAGUUGGUAGGAGGAGUCdTdT 1855 1170-1188 GACUCCUCCUACCAACUGGdTdT 2422 AD-62106.1 GGAGGAGUCCCAGUAACACdTdT 1856 1180-1198 GUGUUACUGGGACUCCUCCdTdT 2423 AD-62065.1 GAGUAACACUGAAUGCACAdTdT 1857 1190-1208 UGUGCAUUCAGUGUUACUGdTdT 2424 AD-62071.1 GAAUGCACAAACAAUUGAUdTdT 1858 1200-1218 AUCAAUUGUUUGUGCAUUCdTdT 2425 AD-62077.1 4ACAAUUGAUGUAAACCAAdTdT 1859 1209-1227 UUGGUUUACAUCAAUUGUUdTdT 2426 AD-62083.1 UAAACCAAGAGACAUCUGAdTdT 1860 1220-1238 UCAGAUGUCUCUUGGUUUAdTdT 2427 AD-62089.1 GAUCUGACUUGGAUCCAAGdTdT 1861 1232-1250 CUUGGAUCCAAGUCAGAUGdTdT 2428 AD-62095.1 GAUCCAAGCAAAAGUGUAAdTdT 1862 1243-1261 UUACACUUUUGCUUGGAUCdTdT 2429 AD-62101.1 GAAAAGUGUAACACGUGUUdTdT 1863 1251-1269 AACACGUGUUACACUUUUGdTdT 2430 AD-62107.1 4ACACGUGUUGAUGAUGGAdTdT 1864 1260-1278 UCCAUCAUCAACACGUGUUdTdT 2431 AD-620 66.1 DGAUGGAGUAGCUUCCUUUdTdT 1865 1272-1290 AAAGGAAGCUACUCCAUCAdTdT 2432 AD-62072.1 GUAGCUUCCUUUGUGCUUAdTdT 1866 1279-1297 UAAGCACAAAGGAAGCUACdTdT 2433

- 157044245

AD-62078.1 GCUUAAUCUCCCAUCUGGAdTdT 1867 1293-1311 UCCAGAUGGGAGAUUAAGCdTdT 2434 AD-62084.1 GCAUCUGGAGUGACGGUGCdTdT 1868 1303-1321 GCACCGUCACUCCAGAUGGdTdT 2435 AD-62090.1 UGACGGUGCUGGAGUUUAAdTdT 1869 1313-1331 UUAAACUCCAGCACCGUCAdTdT 2436 AD-62096.1 GCUGGAGUUUAAUGUCAAAdTdT 1870 1320-1338 UUUGACAUUAAACUCCAGCdTdT 2437 AD-62102.1 UGUCAAAACUGAUGCUCCAdTdT 1871 1332-1350 UGGAGCAUCAGUUUUGACAdTdT 2438 AD-62108.1 GAUGCUCCAGAUCUUCCAGdTdT 1872 1342-1360 CUGGAAGAUCUGGAGCAUCdTdT 2439 AD-62067.1 CAGAUCUUCCAGAAGAAAAdTdT 1873 1349-1367 UUUUCUUCUGGAAGAUCUGdTdT 2440 AD-62073.1 AGAAAAUCAGGCCAGGGAAdTdT 1874 1362-1380 UUCCCUGGCCUGAUUUUCUdTdT 2441 AD-62079.1 GGCCAGGGAAGGUUACCGAdTdT 1875 1371-1389 UCGGUAACCUUCCCUGGCCdTdT 2442 AD-62085.1 GUUACCGAGCAAUAGCAUAdTdT 1876 1382-1400 UAUGCUAUUGCUCGGUAACdTdT 2443 AD-62091.1 AJAGCAUACUCAUCUCUCAdTdT 1877 1393-1411 UGAGAGAUGAGUAUGCUAUdT dT 2444 AD-62097.1 UACUCAUCUCUCAGCCAAAdTdT 1878 1399-1417 UUUGGCUGAGAGAUGAGUAdTdT 2445 AD-62103.1 GCCAAAGUUACCUUUAUAUdTdT 1879 1412-1430 AUAUAAAGGUAACUUUGGCdTdT 2446 AD-62109.1 GCUUUAUAUUGAUUGGACUdTdT 1880 1422-1440 AGUCCAAUCAAUAUAAAGGdTdT 2447 AD-62115.1 GAUUGGACUGAUAACCAUAdTdT 1881 1432-1450 UAUGGUUAUCAGUCCAAUCdTdT 2448 AD-62121.1 GUGAUAACCAUAAGGCUUUdTdT 1882 1439-1457 AAAGCCUUAUGGUUAUCAGdTdT 2449 AD-62127.1 AGGCUUUGCUAGUGGGAGAdTdT 1883 1451-1469 UCUCCCACUAGCAAAGCCUdTdT 2450 AD-62133.1 GUGGGAGAACAUCUGAAUAdTdT 1884 1462-1480 UAUUCAGAUGUUCUCCCACdTdT 2451 AD-62139.1 CAUCUGAAUAUUAUUGUUAdT dT 1885 1471-1489 UAACAAUAAUAUUCAGAUGdT dT 2452 AD-62145.1 UAUUAUUGUUACCCCCAAAdTdT 1886 1479-1497 UUUGGGGGUAACAAUAAUAdTdT 2453 AD-62151.1 CCCAAAAGCCCAUAUAUUGdTdT 1887 1492-1510 CAAUAUAUGGGCUUUUGGGdTdT 2454 AD-62110.1 CCAAAAGCCCAUAUAUUGAdTdT 1888 1493-1511 UCAAUAUAUGGGCUUUUGGdTdT 2455 AD-62116.1 GAAAAGCCCAUAUAUUGACdTdT 1889 1494-1512 GUCAAUAUAUGGGCUUUUGdTdT 2456 AD-62122.1 4AAAGCCCAUAUAUUGACAdTdT 1890 1495-1513 UGUCAAUAUAUGGGCUUUUdTdT 2457 AD-62128.1 4AAGCCCAUAUAUUGACAAdTdT 1891 1496-1514 UUGUCAAUAUAUGGGCUUUdTdT 2458 AD-62134.1 4AGCCCAUAUAUUGACAAAdTdT 1892 1497-1515 UUUGUCAAUAUAUGGGCUUdTdT 2459 AD-6214 0.1 AGCCCAUAUAUUGACAAAAdTdT 1893 1498-1516 UUUUGUCAAUAUAUGGGCUdTdT 2460 AD-62146.1 GCCCAUAUAUUGACAAAAUdTdT 1894 1499-1517 AUUUUGUCAAUAUAUGGGCdTdT 2461 AD-62152.1 CCCAUAUAUUGACAAAAUAdTdT 1895 1500-1518 UAUUUUGUCAAUAUAUGGGdTdT 2462 AD-62111.1 CCAUAUAUUGACAAAAUAAdTdT 1896 1501-1519 UUAUUUUGUCAAUAUAUGGdTdT 2463 AD-62117.1 GAUAUAUUGACAAAAUAACdTdT 1897 1502-1520 GUUAUUUUGUCAAUAUAUGdT dT 2464 AD-62123.1 AJAUAUUGACAAAAUAACUdT dT 1898 1503-1521 AGUUAUUUUGUCAAUAUAUdT dT 2465 AD-62129.1 UAUAUUGACAAAAUAACUCdTdT 1899 1504-1522 GAGUUAUUUUGUCAAUAUAdT dT 2466 AD-62135.1 AJAUUGACAAAAUAACUCAdT dT 1900 1505-1523 UGAGUUAUUUUGUCAAUAUdT dT 2467 AD-62141.1 UAUUGACAAAAUAACUCACdTdT 1901 1506-1524 GUGAGUUAUUUUGUCAAUAdT dT 2468 AD-62147.1 AJUGACAAAAUAACUCACUdT dT 1902 1507-1525 AGUGAGUUAUUUUGUCAAUdTdT 2469 AD-62153.1 UUGACAAAAUAACUCACUAdT dT 1903 1508-1526 UAGUGAGUUAUUUUGUCAAdTdT 2470 AD-62112.1 UGACAAAAUAACUCACUAUdT dT 1904 1509-1527 AUAGUGAGUUAUUUUGUCAdT dT 2471 AD-62118.1 GACAAAAUAACUCACUAUAdT dT 1905 1510-1528 UAUAGUGAGUUAUUUUGUCdTdT 2472 AD-62124.1 ikAAAUAACUCACUAUAAUUdT dT 1906 1513-1531 AAUUAUAGUGAGUUAUUUUdT dT 2473 AD-62130.1 AA№AACUCACUAUAAUUAdT dT 1907 1514-1532 UAAUUAUAGUGAGUUAUUUdT dT 2474 AD-62136.1 4AUAACUCACUAUAAUUACdTdT 1908 1515-1533 GUAAUUAUAGUGAGUUAUUdT dT 2475 AD-62142.1 AUAACUCACUAUAAUUACUdT dT 1909 1516-1534 AGUAAUUAUAGUGAGUUAUdT dT 2476 AD-62148.1 AACUCACUAUAAUUACUUGdTdT 1910 1518-1536 CAAGUAAUUAUAGUGAGUUdT dT 2477

- 158044245

AD-62154.1 AGUCACUAUAAUUACUUGAdT dT 1911 1519-1537 UCAAGUAAUUAUAGUGAGUdT dT 2478 AD-62113.1 CUCACUAUAAUUACUUGAUdT dT 1912 1520-1538 AUCAAGUAAUUAUAGUGAGdT dT 2479 AD-62119.1 UCACUAUAAUUACUUGAUUdT dT 1913 1521-1539 AAUCAAGUAAUUAUAGUGAdT dT 2480 AD-62125.1 AGUAUAAUUACUUGAUUUUdT dT 1914 1523-1541 AAAAUCAAGUAAUUAUAGUdT dT 2481 AD-62131.1 CUAUAAUUACUUGAUUUUAdT dT 1915 1524-1542 UAAAAUCAAGUAAUUAUAGdT dT 2482 AD-62137.1 UAUAAUUACUUGAUUUUAUdT dT 1916 1525-1543 AUAAAAUCAAGUAAUUAUAdT dT 2483 AD-62143.1 AJAAUUACUUGAUUUUAUCdTdT 1917 1526-1544 GAUAAAAUCAAGUAAUUAUdT dT 2484 AD-62149.1 UAAUUACUUGAUUUUAUCCdTdT 1918 1527-1545 GGAUAAAAUCAAGUAAUUAdTdT 2485 AD-62155.1 4AUUACUUGAUUUUAUCCAdTdT 1919 1528-1546 UGGAUAAAAUCAAGUAAUUdT dT 2486 AD-62114.1 AJUACUUGAUUUUAUCCAAdTdT 1920 1529-1547 UUGGAUAAAAUCAAGUAAUdT dT 2487 AD-62120.1 UUAUCCAAGGGCAAAAUUAdTdT 1921 1540-1558 UAAUUUUGCCCUUGGAUAAdTdT 2488 AD-62126.1 GCAAAAUUAUCCACUUUGGdTdT 1922 1550-1568 CCAAAGUGGAUAAUUUUGCdTdT 2489 AD-62132.1 CACUUUGGCACGAGGGAGAdTdT 1923 1561-1579 UCUCCCUCGUGCCAAAGUGdTdT 2490 AD-62138.1 GGAGGGAGAAAUUUUCAGAdTdT 1924 1571-1589 UCUGAAAAUUUCUCCCUCGdTdT 2491 AD-62144.1 AJUUUCAGAUGCAUCUUAUdTdT 1925 1581-1599 AUAAGAUGCAUCUGAAAAUdT dT 2492 AD-62150.1 GCAUCUUAUCAAAGUAUAAdT dT 1926 1591-1609 UUAUACUUUGAUAAGAUGCdTdT 2493 AD-62156.1 CAAAGUAUAAACAUUCCAGdTdT 1927 1600-1618 CUGGAAUGUUUAUACUUUGdTdT 2494 AD-62162.1 AJUCCAGUAACACAGAACAdTdT 1928 1612-1630 UGUUCUGUGUUACUGGAAUdTdT 2495 AD-62168.1 CACAGAACAUGGUUCCUUCdTdT 1929 1622-1640 GAAGGAACCAUGUUCUGUGdTdT 2496 AD-62174.1 GGUUCCUUCAUCCCGACUUdTdT 1930 1632-1650 AAGUCGGGAUGAAGGAACCdTdT 2497 AD-62180.1 CCCGACUUCUGGUCUAUUAdTdT 1931 1643-1661 UAAUAGACCAGAAGUCGGGdTdT 2498 AD-62186.1 GGUCUAUUACAUCGUCACAdTdT 1932 1653-1671 UGUGACGAUGUAAUAGACCdTdT 2499 AD-62192.1 AJCGUCACAGGAGAACAGAdTdT 1933 1663-1681 UCUGUUCUCCUGUGACGAUdTdT 2500 AD-62198.1 CAGGAGAACAGACAGCAGAdTdT 1934 1670-1688 UCUGCUGUCUGUUCUCCUGdTdT 2501 AD-62157.1 CAGCAGAAUUAGUGUCUGAdTdT 1935 1682-1700 UCAGACACUAAUUCUGCUGdTdT 2502 AD-62163.1 GUGUCUGAUUCAGUCUGGUdTdT 1936 1693-1711 ACCAGACUGAAUCAGACACdTdT 2503 AD-62169.1 CAGUCUGGUUAAAUAUUGAdTdT 1937 1703-1721 UCAAUAUUUAACCAGACUGdTdT 2504 AD-62175.1 GUUAAAUAUUGAAGAAAAAdT dT 1938 1710-1728 UUUUUCUUCAAUAUUUAACdTdT 2505 AD-62181.1 /IGAAAAAUGUGGCAACCAGdTdT 1939 1722-1740 CUGGUUGCCACAUUUUUCUdTdT 2506 AD-62187.1 GCAACCAGCUCCAGGUUCAdTdT 1940 1733-1751 UGAACCUGGAGCUGGUUGCdTdT 2507 AD-62193.1 GCUCCAGGUUCAUCUGUCUdTdT 1941 1740-1758 AGACAGAUGAACCUGGAGCdTdT 2508 AD-62199.1 AJCUGUCUCCUGAUGCAGAdTdT 1942 1751-1769 UCUGCAUCAGGAGACAGAUdTdT 2509 AD-62158.1 GAUGCAGAUGCAUAUUCUCdTdT 1943 1762-1780 GAGAAUAUGCAUCUGCAUCdTdT 2510 AD-62164.1 GCAUAUUCUCCAGGCCAAAdTdT 1944 1771-1789 UUUGGCCUGGAGAAUAUGCdTdT 2511 AD-62170.1 AGGCCAAACUGUGUCUCUUdTdT 1945 1782-1800 AAGAGACACAGUUUGGCCUdTdT 2512 AD-62176.1 GUGUCUCUUAAUAUGGCAAdTdT 1946 1792-1810 UUGCCAUAUUAAGAGACACdTdT 2513 AD-62182.1 UUAAUAUGGCAACUGGAAUdTdT 1947 1799-1817 AUUCCAGUUGCCAUAUUAAdTdT 2514 AD-62188.1 4ACUGGAAUGGAUUCCUGGdTdT 1948 1809-1827 CCAGGAAUCCAUUCCAGUUdTdT 2515 AD-62194.1 UUCCUGGGUGGCAUUAGCAdTdT 1949 1821-1839 UGCUAAUGCCACCCAGGAAdTdT 2516 AD-62200.1 GGCAUUAGCAGCAGUGGACdTdT 1950 1830-1848 GUCCACUGCUGCUAAUGCCdTdT 2517 AD-62159.1 AGUGGACAGUGCUGUGUAUdTdT 1951 1842-1860 AUACACAGCACUGUCCACUdTdT 2518 AD-62165.1 GCUGUGUAUGGAGUCCAAAdTdT 1952 1852-1870 UUUGGACUCCAUACACAGCdTdT 2519 AD-62171.1 AOUCCAAAGAGGAGCCAAAdTdT 1953 1863-1881 UUUGGCUCCUCUUUGGACUdTdT 2520 AD-62177.1 AGAGGAGCCAAAAAGCCCUdTdT 1954 1870-1888 AGGGCUUUUUGGCUCCUCUdTdT 2521

- 159044245

AD-62183.1 AGCCCUUGGAAAGAGUAUUdTdT 1955 1883-1901 AAUACUCUUUCCAAGGGCUdTdT 2522 AD-62189.1 4AGAGUAUUUCAAUUCUUAdTdT 1956 1893-1911 UAAGAAUUGAAAUACUCUUdTdT 2523 AD-62195.1 UUUCAAUUCUUAGAGAAGAdT dT 1957 1900-1918 UCUUCUCUAAGAAUUGAAAdTdT 2524 AD-62201.1 GAGAAGAGUGAUCUGGGCUdTdT 1958 1912-1930 AGCCCAGAUCACUCUUCUCdTdT 2525 AD-62160.1 UGAUCUGGGCUGUGGGGCAdTdT 1959 1920-1938 UGCCCCACAGCCCAGAUCAdTdT 2526 AD-62166.1 GGGGCAGGUGGUGGCCUCAdTdT 1960 1933-1951 UGAGGCCACCACCUGCCCCdTdT 2527 AD-62172.1 GUGGCCUCAACAAUGCCAAdTdT 1961 1943-1961 UUGGCAUUGUUGAGGCCACdTdT 2528 AD-62178.1 CAACAAUGCCAAUGUGUUCdTdT 1962 1950-1968 GAACACAUUGGCAUUGUUGdTdT 2529 AD-62184.1 GAAUGUGUUCCACCUAGCUdTdT 1963 1959-1977 AGCUAGGUGGAACACAUUGdTdT 2530 AD-62190.1 GACCUAGCUGGACUUACCUdTdT 1964 1969-1987 AGGUAAGUCCAGCUAGGUGdTdT 2531 AD-62196.1 GACUUACCUUCCUCACUAAdTdT 1965 1979-1997 UUAGUGAGGAAGGUAAGUCdTdT 2532 AD-62202.1 UCACUAAUGCAAAUGCAGAdTdT 1966 1991-2009 UCUGCAUUUGCAUUAGUGAdTdT 2533 AD-62161.1 4AAUGCAGAUGACUCCCAAdTdT 1967 2001-2019 UUGGGAGUCAUCUGCAUUUdTdT 2534 AD-62167.1 GUCCCAAGAAAAUGAUGAAdTdT 1968 2013-2031 UUCAUCAUUUUCUUGGGAGdTdT 2535 AD-62173.1 GCUUGUAAAGAAAUUCUCAdTdT 1969 2032-2050 UGAGAAUUUCUUUACAAGGdTdT 2536 AD-62179.1 4AUUCUCAGGCCAAGAAGAdTdT 1970 2043-2061 UCUUCUUGGCCUGAGAAUUdTdT 2537 AD-62185.1 GCAAGAAGAACGCUGCAAAdTdT 1971 2053-2071 UUUGCAGCGUUCUUCUUGGdTdT 2538 AD-62191.1 GGCUGCAAAAGAAGAUAGAdTdT 1972 2063-2081 UCUAUCUUCUUUUGCAGCGdTdT 2539 AD-62197.1 AAYGAAGAUAGAAGAAAUAdT dT 1973 2070-2088 UAUUUCUUCUAUCUUCUUUdTdT 2540 AD-62203.1 AGAAAUAGCUGCUAAAUAUdTdT 1974 2082-2100 AUAUUUAGCAGCUAUUUCUdTdT 2541 AD-62209.1 GCUGCUAAAUAUAAACAUUdTdT 1975 2089-2107 AAUGUUUAUAUUUAGCAGCdTdT 2542 AD-62215.1 ACAUUCAGUAGUGAAGAAAdT dT 1976 2103-2121 UUUCUUCACUACUGAAUGUdTdT 2543 AD-62221.1 GUAGUGAAGAAAUGUUGUUdTdT 1977 2110-2128 AACAACAUUUCUUCACUACdTdT 2544 AD-62227.1 4AAUGUUGUUACGAUGGAGdTdT 1978 2119-2137 CUCCAUCGUAACAACAUUUdTdT 2545 AD-62233.1 GGAUGGAGCCUGCGUUAAUdTdT 1979 2130-2148 AUUAACGCAGGCUCCAUCGdTdT 2546 AD-62239.1 GGUUAAUAAUGAUGAAACCdTdT 1980 2142-2160 GGUUUCAUCAUUAUUAACGdTdT 2547 AD-62245.1 AUGAUGAAACCUGUGAGCAdTdT 1981 2150-2168 UGCUCACAGGUUUCAUCAUdTdT 2548 AD-62204.1 GUGUGAGCAGCGAGCUGCAdTdT 1982 2160-2178 UGCAGCUCGCUGCUCACAGdTdT 2549 AD-62210.1 CGAGCUGCACGGAUUAGUUdTdT 1983 2170-2188 AACUAAUCCGUGCAGCUCGdTdT 2550 AD-62216.1 GGAUUAGUUUAGGGCCAAGdTdT 1984 2180-2198 CUUGGCCCUAAACUAAUCCdTdT 2551 AD-62222.1 GGGCCAAGAUGCAUCAAAGdTdT 1985 2191-2209 CUUUGAUGCAUCUUGGCCCdTdT 2552 AD-62228.1 GAUCAAAGCUUUCACUGAAdTdT 1986 2202-2220 UUCAGUGAAAGCUUUGAUGdTdT 2553 AD-62234.1 GCUUUCACUGAAUGUUGUGdTdT 1987 2209-2227 CACAACAUUCAGUGAAAGCdTdT 2554 AD-62240.1 4AUGUUGUGUCGUCGCAAGdTdT 1988 2219-2237 CUUGCGACGACACAACAUUdTdT 2555 AD-62246.1 GGUCGCAAGCCAGCUCCGUdTdT 1989 2229-2247 ACGGAGCUGGCUUGCGACGdTdT 2556 AD-62205.1 GCUCCGUGCUAAUAUCUCUdTdT 1990 2241-2259 AGAGAUAUUAGCACGGAGCdTdT 2557 AD-62211.1 GUAAUAUCUCUCAUAAAGAdT dT 1991 2249-2267 UCUUUAUGAGAGAUAUUAGdT dT 2558 AD-62217.1 4AAGACAUGCAAUUGGGAAdTdT 1992 2263-2281 UUCCCAAUUGCAUGUCUUUdTdT 2559 AD-62223.1 GAAUUGGGAAGGCUACACAdTdT 1993 2272-2290 UGUGUAGCCUUCCCAAUUGdTdT 2560 AD-62229.1 GCUACACAUGAAGACCCUGdTdT 1994 2283-2301 CAGGGUCUUCAUGUGUAGCdTdT 2561 AD-62235.1 CAUGAAGACCCUGUUACCAdTdT 1995 2289-2307 UGGUAACAGGGUCUUCAUGdTdT 2562 AD-62241.1 UACCAGUAAGCAAGCCAGAdTdT 1996 2303-2321 UCUGGCUUGCUUACUGGUAdTdT 2563 AD-62247.1 AGCAAGCCAGAAAUUCGGAdTdT 1997 2311-2329 UCCGAAUUUCUGGCUUGCUdTdT 2564 AD-62206.1 AGAAAUUCGGAGUUAUUUUdTdT 1998 2319-2337 AAAAUAACUCCGAAUUUCUdTdT 2565

- 160044245

AD-62212.1 hGUUAUUUUCCAGAAAGCUdTdT 1999 2329-2347 AGCUUUCUGGAAAAUAACUdTdT 2566 AD-62218.1 GAGAAAGCUGGUUGUGGGAdTdT 2000 2339-2357 UCCCACAACCAGCUUUCUGdTdT 2567 AD-62224.1 GUGGGAAGUUCAUCUUGUUdTdT 2001 2352-2370 AACAAGAUGAACUUCCCACdTdT 2568 AD-62230.1 UCAUCUUGUUCCCAGAAGAdTdT 2002 2361-2379 UCUUCUGGGAACAAGAUGAdTdT 2569 AD-62236.1 GCAGAAGAAAACAGUUGCAdTdT 2003 2372-2390 UGCAACUGUUUUCUUCUGGdTdT 2570 AD-62242.1 GAGUUGCAGUUUGCCCUACdTdT 2004 2383-2401 GUAGGGCAAACUGCAACUGdTdT 2571 AD-62248.1 CAGUUUGCCCUACCUGAUUdTdT 2005 2389-2407 AAUCAGGUAGGGCAAACUGdTdT 2572 AD-62207.1 CCUGAUUCUCUAACCACCUdTdT 2006 2401-2419 AGGUGGUUAGAGAAUCAGGdTdT 2573 AD-62213.1 hCCACCUGGGAAAUUCAAGdTdT 2007 2413-2431 CUUGAAUUUCCCAGGUGGUdTdT 2574 AD-62219.1 GAAAUUCAAGGCGUUGGCAdTdT 2008 2422-2440 UGCCAACGCCUUGAAUUUCdTdT 2575 AD-62225.1 GGUUGGCAUUUCAAACACUdTdT 2009 2433-2451 AGUGUUUGAAAUGCCAACGdTdT 2576 AD-62231.1 GAUUUCAAACACUGGUAUAdTdT 2010 2439-2457 UAUACCAGUGUUUGAAAUGdTdT 2577 AD-62237.1 GUAUAUGUGUUGCUGAUACdTdT 2011 2453-2471 GUAUCAGCAACACAUAUACdTdT 2578 AD-62243.1 UGCUGAUACUGUCAAGGCAdTdT 2012 2463-2481 UGCCUUGACAGUAUCAGCAdTdT 2579 AD-62249.1 GUGUCAAGGCAAAGGUGUUdTdT 2013 2471-2489 AACACCUUUGCCUUGACAGdTdT 2580 AD-62208.1 hGGUGUUCAAAGAUGUCUUdTdT 2014 2483-2501 AAGACAUCUUUGAACACCUdTdT 2581 AD-62214.1 GAAAGAUGUCUUCCUGGAAdTdT 2015 2490-2508 UUCCAGGAAGACAUCUUUGdTdT 2582 AD-62220.1 GUUCCUGGAAAUGAAUAUAdTdT 2016 2499-2517 UAUAUUCAUUUCCAGGAAGdTdT 2583 AD-62226.1 GAAUAUACCAUAUUCUGUUdTdT 2017 2511-2529 AACAGAAUAUGGUAUAUUCdTdT 2584 AD-62232.1 AJAUUCUGUUGUACGAGGAdTdT 2018 2520-2538 UCCUCGUACAACAGAAUAUdTdT 2585 AD-62238.1 CGAGGAGAACAGAUCCAAUdTdT 2019 2533-2551 AUUGGAUCUGUUCUCCUCGdTdT 2586 AD-62244.1 GAACAGAUCCAAUUGAAAGdTdT 2020 2539-2557 CUUUCAAUUGGAUCUGUUCdTdT 2587 AD-61874.1 GAAAGGAACUGUUUACAACdTdT 2021 2553-2571 GUUGUAAACAGUUCCUUUCdTdT 2588 AD-61880.1 hCUGUUUACAACUAUAGGAdTdT 2022 2560-2578 UCCUAUAGUUGUAAACAGUdTdT 2589 AD-6188 6.1 hACUAUAGGACUUCUGGGAdTdT 2023 2569-2587 UCCCAGAAGUCCUAUAGUUdTdT 2590 AD-61892.1 UGGGAUGCAGUUCUGUGUUdTdT 2024 2583-2601 AACACAGAACUGCAUCCCAdTdT 2591 AD-61898.1 GUUCUGUGUUAAAAUGUCUdTdT 2025 2592-2610 AGACAUUUUAACACAGAACdTdT 2592 AD-61904.1 UUAAAAUGUCUGCUGUGGAdTdT 2026 2600-2618 UCCACAGCAGACAUUUUAAdTdT 2593 AD-61910.1 CUGUGGAGGGAAUCUGCACdTdT 2027 2612-2630 GUGCAGAUUCCCUCCACAGdTdT 2594 AD-61916.1 GGAAUCUGCACUUCGGAAAdTdT 2028 2620-2638 UUUCCGAAGUGCAGAUUCCdTdT 2595 AD-61875.1 GGGAAAGCCCAGUCAUUGAdTdT 2029 2633-2651 UCAAUGACUGGGCUUUCCGdTdT 2596 AD-61881.1 GCAGUCAUUGAUCAUCAGGdTdT 2030 2641-2659 CCUGAUGAUCAAUGACUGGdTdT 2597 AD-61887.1 GAUCAGGGCACAAAGUCCUdTdT 2031 2653-2671 AGGACUUUGUGCCCUGAUGdTdT 2598 AD-61893.1 GGCACAAAGUCCUCCAAAUdTdT 2032 2659-2677 AUUUGGAGGACUUUGUGCCdTdT 2599 AD-61899.1 GAAAUGUGUGCGCCAGAAAdTdT 2033 2673-2691 UUUCUGGCGCACACAUUUGdTdT 2600 AD-61905.1 GCGCCAGAAAGUAGAGGGCdTdT 2034 2682-2700 GCCCUCUACUUUCUGGCGCdTdT 2601 AD-61911.1 hGUAGAGGGCUCCUCCAGUdTdT 2035 2691-2709 ACUGGAGGAGCCCUCUACUdTdT 2602 AD-61917.1 GCUCCAGUCACUUGGUGACdTdT 2036 2702-2720 GUCACCAAGUGACUGGAGGdTdT 2603 AD-61876.1 UCACUUGGUGACAUUCACUdTdT 2037 2709-2727 AGUGAAUGUCACCAAGUGAdTdT 2604 AD-61882.1 GAUUCACUGUGCUUCCUCUdTdT 2038 2720-2738 AGAGGAAGCACAGUGAAUGdTdT 2605 AD-61888.1 GGAAAUUGGCCUUCACAACdTdT 2039 2739-2757 GUUGUGAAGGCCAAUUUCCdTdT 2606 AD-61894.1 GUUCACAACAUCAAUUUUUdTdT 2040 2749-2767 AAAAAUUGAUGUUGUGAAGdTdT 2607 AD-61900.1 hAUUUUUCACUGGAGACUUdTdT 2041 2761-2779 AAGUCUCCAGUGAAAAAUUdTdT 2608 AD-61906.1 CUGGAGACUUGGUUUGGAAdTdT 2042 2770-2788 UUCCAAACCAAGUCUCCAGdTdT 2609

- 161 044245

AD-61912.1 GGUUUGGAAAAGAAAUCUUdTdT 2043 2780-2798 AAGAUUUCUUUUCCAAACCdTdT 2610 AD-61918.1 4AUCUUAGUAAAAACAUUAdT dT 2044 2793-2811 UAAUGUUUUUACUAAGAUUdT dT 2611 AD-61877.1 4AAAACAUUACGAGUGGUGdTdT 2045 2802-2820 CACCACUCGUAAUGUUUUUdTdT 2612 AD-61883.1 GAGUGGUGCCAGAAGGUGUdTdT 2046 2813-2831 ACACCUUCUGGCACCACUCdTdT 2613 AD-61889.1 AGAAGGUGUCAAAAGGGAAdT dT 2047 2823-2841 UUC C CUUUUGACAC CUUCUdT dT 2614 AD-61895.1 UGUCAAAAGGGAAAGCUAUdTdT 2048 2829-2847 AUAGCUUUCCCUUUUGACAdTdT 2615 AD-61901.1 GCUAUUCUGGUGUUACUUUdTdT 2049 2843-2861 AAAGUAACACCAGAAUAGCdTdT 2616 AD-61907.1 GUGUUACUUUGGAUCCUAGdTdT 2050 2852-2870 CUAGGAUCCAAAGUAACACdTdT 2617 AD-61913.1 GGAUCCUAGGGGUAUUUAUdTdT 2051 2862-2880 AUAAAUACCCCUAGGAUCCdTdT 2618 AD-61919.1 GGUAUUUAUGGUACCAUUAdTdT 2052 2872-2890 UAAUGGUACCAUAAAUACCdTdT 2619 AD-61878.1 GUACCAUUAGCAGACGAAAdTdT 2053 2882-2900 UUUCGUCUGCUAAUGGUACdTdT 2620 AD-61884.1 GAGACGAAAGGAGUUCCCAdTdT 2054 2892-2910 UGGGAACUCCUUUCGUCUGdTdT 2621 AD-61890.1 AGGAGUUCCCAUACAGGAUdTdT 2055 2900-2918 AUCCUGUAUGGGAACUCCUdTdT 2622 AD-61896.1 GAUACAGGAUACCCUUAGAdTdT 2056 2909-2927 UCUAAGGGUAUCCUGUAUGdTdT 2623 AD-61902.1 GUUAGAUUUGGUCCCCAAAdTdT 2057 2922-2940 UUUGGGGACCAAAUCUAAGdTdT 2624 AD-61908.1 DCCCCCAAAACAGAAAUCAAdTdT 2058 2933-2951 UUGAUUUCUGUUUUGGGGAdTdT 2625 AD-61914.1 ACAGAAAUCAAAAGGAUUUdT dT 2059 2941-2959 AAAUCCUUUUGAUUUCUGUdTdT 2626 AD-61920.1 4AAGGAUUUUGAGUGUAAAdTdT 2060 2951-2969 UUUACACUCAAAAUCCUUUdTdT 2627 AD-61879.1 AGUGUAAAAGGACUGCUUGdTdT 2061 2962-2980 CAAGCAGUCCUUUUACACUdTdT 2628 AD-61885.1 4AGGACUGCUUGUAGGUGAdTdT 2062 2969-2987 UCACCUACAAGCAGUCCUUdTdT 2629 AD-61891.1 GUAGGUGAGAUCUUGUCUGdTdT 2063 2980-2998 CAGACAAGAUCUCACCUACdTdT 2630 AD-61897.1 AJCUUGUCUGCAGUUCUAAdTdT 2064 2989-3007 UUAGAACUGCAGACAAGAUdTdT 2631 AD-61903.1 GUUCUAAGUCAGGAAGGCAdTdT 2065 3001-3019 UGCCUUCCUGACUUAGAACdTdT 2632 AD-61909.1 GAAGGCAUCAAUAUCCUAAdTdT 2066 3013-3031 UUAGGAUAUUGAUGCCUUCdTdT 2633 AD-61915.1 UCAAUAUCCUAACСCACCUdTdT 2067 3020-3038 AGGUGGGUUAGGAUAUUGAdTdT 2634 AD-61921.1 GCACCUCCCCAAAGGGAGUdTdT 2068 3033-3051 ACUCCCUUUGGGGAGGUGGdTdT 2635 AD-61927.1 GCCCAAAGGGAGUGCAGAGdTdT 2069 3039-3057 CUCUGCACUCCCUUUGGGGdTdT 2636 AD-61933.1 GUGCAGAGGCGGAGCUGAUdTdT 2070 3050-3068 AUCAGCUCCGCCUCUGCACdTdT 2637 AD-61939.1 GGAGCUGAUGAGCGUUGUCdTdT 2071 3060-3078 GACAACGCUCAUCAGCUCCdTdT 2638 AD-61945.1 CGUUGUCCCAGUAUUCUAUdTdT 2072 3072-3090 AUAGAAUACUGGGACAACGdTdT 2639 AD-61951.1 GCAGUAUUCUAUGUUUUUCdTdT 2073 3079-3097 GAAAAACAUAGAAUACUGGdT dT 2640 AD-61957.1 GUUUUUCACUACCUGGAAAdTdT 2074 3091-3109 UUUCCAGGUAGUGAAAAACdTdT 2641 AD-61963.1 GCUGGAAACAGGAAAUCAUdTdT 2075 3102-3120 AUGAUUUCCUGUUUCCAGGdTdT 2642 AD-61922.1 GGAACAUUUUUCAUUCUGAdTdT 2076 3122-3140 UCAGAAUGAAAAAUGUUCCdTdT 2643 AD-61928.1 GAUUCUGACCCAUUAAUUGdTdT 2077 3133-3151 CAAUUAAUGGGUCAGAAUGdTdT 2644 AD-61934.1 GCAUUAAUUGAAAAGCAGAdTdT 2078 3142-3160 UCUGCUUUUCAAUUAAUGGdTdT 2645 AD-61940.1 ZkAAGCAGAAACUGAAGAAAdTdT 2079 3153-3171 UUUCUUCAGUUUCUGCUUUdTdT 2646 AD-61946.1 ikACUGAAGAAAAAAUUAAAdT dT 2080 3161-3179 UUUAAUUUUUUCUUCAGUUdTdT 2647 AD-61952.1 ZkAAAAAUUAAAAGAAGGGAdT dT 2081 3169-3187 UCCCUUCUUUUAAUUUUUUdTdT 2648 AD-61958.1 AGGGAUGUUGAGCAUUAUGdTdT 2082 3183-3201 CAUAAUGCUCAACAUCCCUdTdT 2649 AD-61964.1 GAGCAUUAUGUCCUACAGAdTdT 2083 3192-3210 UCUGUAGGACAUAAUGCUCdTdT 2650 AD-61923.1 UGUCCUACAGAAAUGCUGAdTdT 2084 3200-3218 UCAGCAUUUCUGUAGGACAdTdT 2651 AD-61929.1 /UWGCUGACUACUCUUACAdTdT 2085 3211-3229 UGUAAGAGUAGUCAGCAUUdTdT 2652 AD-61935.1 DACUCUUACAGUGUGUGGAdTdT 2086 3220-3238 UCCACACACUGUAAGAGUAdTdT 2653

- 162044245

AD-61941.1 AGUGUGUGGAAGGGUGGAAdTdT 2087 3229-3247 UUC CAC C CUUC CACACACUdT dT 2654 AD-61947.1 GGGUGGAAGUGCUAGCACUdTdT 2088 3240-3258 AGUGCUAGCACUUCCACCCdTdT 2655 AD-61953.1 GCUAGCACUUGGUUAACAGdTdT 2089 3250-3268 CUGUUAACCAAGUGCUAGCdTdT 2656 AD-61959.1 GGUUAACAGCUUUUGCUUUdTdT 2090 3260-3278 AAAGCAAAAGCUGUUAACCdTdT 2657 AD-61965.1 UGCUUUAAGAGUACUUGGAdTdT 2091 3273-3291 UCCAAGUACUCUUAAAGCAdTdT 2658 AD-61924.1 GUACUUGGACAAGUAAAUAdT dT 2092 3283-3301 UAUUUACUUGUCCAAGUACdTdT 2659 AD-61930.1 CAAGUAAAUAAAUACGUAGdTdT 2093 3292-3310 CUACGUAUUUAUUUACUUGdTdT 2660 AD-61936.1 AJAAAUACGUAGAGCAGAAdTdT 2094 3299-3317 UUCUGCUCUACGUAUUUAUdTdT 2661 AD-61942.1 GAGCAGAACCAAAAUUCAAdTdT 2095 3310-3328 UUGAAUUUUGGUUCUGCUCdTdT 2662 AD-61948.1 hAUUCAAUUUGUAAUUCUUdTdT 2096 3322-3340 AAGAAUUACAAAUUGAAUUdT dT 2663 AD-61954.1 GUAAUUCUUUAUUGUGGCUdTdT 2097 3332-3350 AGCCACAAUAAAGAAUUACdTdT 2664 AD-61960.1 AJUGUGGCUAGUUGAGAAUdTdT 2098 3342-3360 AUUCUCAACUAGCCACAAUdTdT 2665 AD-61966.1 CUAGUUGAGAAUUAUCAAUdT dT 2099 3349-3367 AUUGAUAAUUCUCAACUAGdTdT 2666 AD-61925.1 UUAUCAAUUAGAUAAUGGAdT dT 2100 3360-3378 UCCAUUAUCUAAUUGAUAAdTdT 2667 AD-61931.1 hAUGGAUCUUUCAAGGAAAdTdT 2101 3373-3391 UUUCCUUGAAAGAUCCAUUdTdT 2668 AD-61937.1 CUUUCAAGGAAAAUUCACAdTdT 2102 3380-3398 UGUGAAUUUUCCUUGAAAGdTdT 2669 AD-61943.1 hAUUCACAGUAUCAACCAAdTdT 2103 3391-3409 UUGGUUGAUACUGUGAAUUdTdT 2670 AD-61949.1 GUAUCAACCAAUAAAAUUAdTdT 2104 3399-3417 UAAUUUUAUUGGUUGAUACdTdT 2671 AD-61955.1 AAAAJUACAGGGUACCUUGdTdT 2105 3411-3429 CAAGGUACCCUGUAAUUUUdTdT 2672 AD-61961.1 AGGGUACCUUGCCUGUUGAdTdT 2106 3419-3437 UCAACAGGCAAGGUACCCUdTdT 2673 AD-61967.1 GUUGAAGCCCGAGAGAACAdTdT 2107 3433-3451 UGUUCUCUCGGGCUUCAACdTdT 2674 AD-61926.1 CCGAGAGAACAGCUUAUAUdTdT 2108 3441-3459 AUAUAAGCUGUUCUCUCGGdTdT 2675 AD-61932.1 GCUUAUAUCUUACAGCCUUdTdT 2109 3452-3470 AAGGCUGUAAGAUAUAAGCdTdT 2676 AD-61938.1 CUUACAGCCUUUACUGUGAdTdT 2110 3460-3478 UCACAGUAAAGGCUGUAAGdTdT 2677 AD-61944.1 GAAUUAGAAAGGCUUUCGAdTdT 2111 3482-3500 UCGAAAGCCUUUCUAAUUCdTdT 2678 AD-61950.1 GGCUUUCGAUAUAUGCCCCdTdT 2112 3492-3510 GGGGCAUAUAUCGAAAGCCdTdT 2679 AD-61956.1 GAUAUAUGCCCCCUGGUGAdTdT 2113 3499-3517 UCACCAGGGGGCAUAUAUCdTdT 2680 AD-61962.1 GGUGAAAAUCGACACAGCUdTdT 2114 3513-3531 AGCUGUGUCGAUUUUCACCdTdT 2681 AD-61968.1 CGACACAGCUCUAAUUAAAdTdT 2115 3522-3540 UUUAAUUAGAGCUGUGUCGdTdT 2682 AD-61974.1 GCUCUAAUUAAAGCUGACAdTdT 2116 3529-3547 UGUCAGCUUUAAUUAGAGCdTdT 2683 AD-61980.1 CUGACAACUUUCUGCUUGAdTdT 2117 3542-3560 UCAAGCAGAAAGUUGUCAGdTdT 2684 AD-61986.1 CUUUCUGCUUGAAAAUACAdTdT 2118 3549-3567 UGUAUUUUCAAGCAGAAAGdTdT 2685 AD-61992.1 hAAAUACACUGCCAGCCCAdTdT 2119 3560-3578 UGGGCUGGCAGUGUAUUUUdTdT 2686 AD-61998.1 AGCCCAGAGCACCUUUACAdTdT 2120 3573-3591 UGUAAAGGUGCUCUGGGCUdTdT 2687 AD-62004.1 GCACCUUUACAUUGGCCAUdTdT 2121 3581-3599 AUGGCCAAUGUAAAGGUGCdTdT 2688 AD-62010.1 ACAUUGGCCAUUUCUGCGUdTdT 2122 3589-3607 ACGCAGAAAUGGCCAAUGUdTdT 2689 AD-61969.1 CUGCGUAUGCUCUUUCCCUdTdT 2123 3602-3620 AGGGAAAGAGCAUACGCAGdTdT 2690 AD-61975.1 CUUUCCCUGGGAGAUAAAAdTdT 2124 3613-3631 UUUUAUCUCCCAGGGAAAGdTdT 2691 AD-61981.1 GAGAUAAAACUCACCCACAdTdT 2125 3623-3641 UGUGGGUGAGUUUUAUCUCdTdT 2692 AD-61987.1 ACUCAC C CACAGUUUC GUUdT dT 2126 3631-3649 AACGAAACUGUGGGUGAGUdTdT 2693 AD-61993.1 CAGUUUCGUUCAAUUGUUUdTdT 2127 3640-3658 AAACAAUUGAACGAAACUGdTdT 2694 AD-61999.1 CAAUUGUUUCAGCUUUGAAdTdT 2128 3650-3668 UUCAAAGCUGAAACAAUUGdTdT 2695 AD-62005.1 CUUUGAAGAGAGAAGCUUUdTdT 2129 3662-3680 AAAGCUUCUCUCUUCAAAGdTdT 2696 AD-62011 . 1 GAGAGAAGCUUUGGUUAAAdTdT 2130 3669-3687 UUUAACCAAAGCUUCUCUCdTdT 2697

-163044245

AD-61970.1 GUUAAAGGUAAUCCACCCAdTdT 2131 3682-3700 UGGGUGGAUUACCUUUAACdTdT 2698 AD-61976.1 4AUCCASSCCAUUUAUCGUUdTdT 2132 3691-3709 AACGAUAAAUGGGUGGAUUdTdT 2699 AD-61982.1 GAUUUAUCGUUUUUGGAAAdTdT 2133 3699-3717 UUUCCAAAAACGAUAAAUGdTdT 2700 AD-61988.1 UUUGGAAAGACAAUCUUCAdTdT 2134 3710-3728 UGAAGAUUGUCUUUCCAAAdTdT 2701 AD-61994.1 4AUCUUCAGCAUAAAGACAdTdT 2135 3721-3739 UGUCUUUAUGCUGAAGAUUdTdT 2702 AD-62006.1 GUCUGUACCUAACACUGGUdTdT 2136 3741-3759 ACCAGUGUUAGGUACAGAGdTdT 2703 AD-62012.1 ^CACUGGUACGGCACGUAUdTdT 2137 3752-3770 AUACGUGCCGUACCAGUGUdTdT 2704 AD-61971.1 GGCACGUAUGGUAGAAACAdTdT 2138 3762-3780 UGUUUCUACCAUACGUGCCdTdT 2705 AD-61977.1 GGUAGAAACAACUGCCUAUdTdT 2139 3771-3789 AUAGGCAGUUGUUUCUACCdTdT 2706 AD-61983.1 GAACUGCCUAUGCUUUACUdTdT 2140 3779-3797 AGUAAAGCAUAGGCAGUUGdTdT 2707 AD-61989.1 GUUUACUCACCAGUCUGAAdTdT 2141 3791-3809 UUCAGACUGGUGAGUAAAGdTdT 2708 AD-61995.1 GUCUGAACUUGAAAGAUAUdTdT 2142 3803-3821 AUAUCUUUCAAGUUCAGACdTdT 2709 AD-62001.1 AGUUGAAAGAUAUAAAUUAdT dT 2143 3809-3827 UAAUUUAUAUCUUUCAAGUdT dT 2710 AD-62007.1 UAUAAAUUAUGUUAACCCAdTdT 2144 3819-3837 UGGGUUAACAUAAUUUAUAdT dT 2711 AD-62013.1 GUUAACCCAGUCAUCAAAUdTdT 2145 3829-3847 AUUUGAUGACUGGGUUAACdTdT 2712 AD-61972.1 UCAUCAAAUGGCUAUCAGAdTdT 2146 3839-3857 UCUGAUAGCCAUUUGAUGAdTdT 2713 AD-61978.1 UAUCAGAAGAGCAGAGGUAdT dT 2147 3851-3869 UACCUCUGCUCUUCUGAUAdTdT 2714 AD-61984.1 AGAGGUAUGGAGGUGGCUUdTdT 2148 3863-3881 AAGCCACCUCCAUACCUCUdTdT 2715 AD-61990.1 GAGGUGGCUUUUAUUCAACdTdT 2149 3872-3890 GUUGAAUAAAAGCCACCUCdTdT 2716 AD-61996.1 UAUUCAACCCAGGACACAAdTdT 2150 3883-3901 UUGUGUCCUGGGUUGAAUAdTdT 2717 AD-62002.1 AOGACACAAUCAAUGCCAUdTdT 2151 3893-3911 AUGGCAUUGAUUGUGUCCUdTdT 2718 AD-62008.1 CAAUCAAUGCCAUUGAGGGdTdT 2152 3899-3917 CCCUCAAUGGCAUUGAUUGdTdT 2719 AD-62014.1 GAUUGAGGGCCUGACGGAAdTdT 2153 3909-3927 UUCCGUCAGGCCCUCAAUGdTdT 2720 AD-61973.1 ACGGAAUAUUCACUCCUGGdTdT 2154 3922-3940 CCAGGAGUGAAUAUUCCGUdTdT 2721 AD-61979.1 UUCACUCCUGGUUAAACAAdTdT 2155 3930-3948 UUGUUUAACCAGGAGUGAAdTdT 2722 AD-61985.1 GGUUAAACAACUCCGCUUGdTdT 2156 3939-3957 CAAGCGGAGUUGUUUAACCdTdT 2723 AD-61991.1 GCGCUUGAGUAUGGACAUCdTdT 2157 3951-3969 GAUGUCCAUACUCAAGCGGdTdT 2724 AD-61997.1 GGACAUCGAUGUUUCUUACdTdT 2158 3963-3981 GUAAGAAACAUCGAUGUCCdTdT 2725 AD-62003.1 CGAUGUUUCUUACAAGCAUdTdT 2159 3969-3987 AUGCUUGUAAGAAACAUCGdTdT 2726 AD-62009.1 CAAGCAUAAAGGUGCCUUAdTdT 2160 3981-3999 UAAGGCACCUUUAUGCUUGdTdT 2727 AD-62056.1 GUGCCUUACAUAAUUAUAAdTdT 2161 3992-4010 UUAUAAUUAUGUAAGGCACdTdT 2728 AD-62015.1 AGAUAAUUAUAAAAUGACAdT dT 2162 3999-4017 UGUCAUUUUAUAAUUAUGUdT dT 2729 AD-62021.1 4AAAUGACAGACAAGAAUUdTdT 2163 4009-4027 AAUUCUUGUCUGUCAUUUUdTdT 2730 AD-62027.1 GAAGAAUUUCCUUGGGAGGdTdT 2164 4020-4038 CCUCCCAAGGAAAUUCUUGdTdT 2731 AD-62033.1 GCUUGGGAGGCCAGUAGAGdTdT 2165 4029-4047 CUCUACUGGCCUCCCAAGGdTdT 2732 AD-62039.1 AGUAGAGGUGCUUCUCAAUdTdT 2166 4041-4059 AUUGAGAAGCACCUCUACUdTdT 2733 AD-62045.1 GUUCUCAAUGAUGACCUCAdTdT 2167 4051-4069 UGAGGUCAUCAUUGAGAAGdTdT 2734 AD-62051.1 UGACCUCAUUGUCAGUACAdTdT 2168 4062-4080 UGUACUGACAAUGAGGUCAdTdT 2735 AD-62057.1 GUCAGUACAGGAUUUGGCAdTdT 2169 4072-4090 UGCCAAAUCCUGUACUGACdTdT 2736 AD-62016.1 AGGAUUUGGCAGUGGCUUGdTdT 2170 4080-4098 CAAGCCACUGCCAAAUCCUdTdT 2737 AD-62022.1 UGGCUUGGCUACAGUACAUdTdT 2171 4092-4110 AUGUACUGUAGCCAAGCCAdTdT 2738 AD-62028.1 GCUACAGUACAUGUAACAAdTdT 2172 4099-4117 UUGUUACAUGUACUGUAGCdTdT 2739 AD-62034.1 ?\ACAACUGUAGUUCACAAAdTdT 2173 4113-4131 UUUGUGAACUACAGUUGUUdTdT 2740 AD-62040.1 GUAGUUCACAAAACCAGUAdTdT 2174 4120-4138 UACUGGUUUUGUGAACUACdTdT 2741

- 164044245

AD-62046.1 4AACCAGUACCUCUGAGGAdTdT 2175 4130-4148 UCCUCAGAGGUACUGGUUUdTdT 2742 AD-62052.1 UGAGGAAGUUUGCAGCUUUdTdT 2176 4143-4161 AAAGCUGCAAACUUCCUCAdTdT 2743 AD-62058.1 UGCAGCUUUUAUUUGAAAAdTdT 2177 4153-4171 UUUUCAAAUAAAAGCUGCAdTdT 2744 AD-62017.1 AJUUGAAAAUCGAUACUCAdTdT 2178 4163-4181 UGAGUAUCGAUUUUCAAAUdTdT 2745 AD-62023.1 CGAUACUCAGGAUAUUGAAdTdT 2179 4173-4191 UUCAAUAUCCUGAGUAUCGdTdT 2746 AD-62029.1 GGAUAUUGAAGCAUCCCACdTdT 2180 4182-4200 GUGGGAUGCUUCAAUAUCCdTdT 2747 AD-62035.1 GAAGCAUCCCACUACAGAGdTdT 2181 4189-4207 CUCUGUAGUGGGAUGCUUCdTdT 2748 AD-62041.1 AGUACAGAGGCUACGGAAAdTdT 2182 4199-4217 UUUCCGUAGCCUCUGUAGUdTdT 2749 AD-62047.1 CGGAAACUCUGAUUACAAAdTdT 2183 4212-4230 UUUGUAAUCAGAGUUUCCGdTdT 2750 AD-62053.1 UGAUUACAAACGCAUAGUAdTdT 2184 4221-4239 UACUAUGCGUUUGUAAUCAdTdT 2751 AD-62059.1 GCAUAGUAGCAUGUGCCAGdTdT 2185 4232-4250 CUGGCACAUGCUACUAUGCdTdT 2752 AD-62018.1 GCAUGUGCCAGCUACAAGCdTdT 2186 4240-4258 GCUUGUAGCUGGCACAUGCdTdT 2753 AD-62024.1 CUACAAGCCCAGCAGGGAAdTdT 2187 4251-4269 UUCCCUGCUGGGCUUGUAGdTdT 2754 AD-62030.1 CAGCAGGGAAGAAUCAUCAdTdT 2188 4260-4278 UGAUGAUUCUUCCCUGCUGdTdT 2755 AD-62036.1 GAAUCAUCAUCUGGAUCCUdTdT 2189 4270-4288 AGGAUCCAGAUGAUGAUUCdTdT 2756 AD-62042.1 GAUCCUCUCAUGCGGUGAUdTdT 2190 4283-4301 AUCACCGCAUGAGAGGAUCdTdT 2757 AD-62048.1 CUCAUGCGGUGAUGGACAUdTdT 2191 4289-4307 AUGUCCAUCACCGCAUGAGdTdT 2758 AD-62054.1 GAUGGACAUCUCCUUGCCUdTdT 2192 4299-4317 AGGCAAGGAGAUGUCCAUCdTdT 2759 AD-62060.1 CUUGCCUACUGGAAUCAGUdTdT 2193 4311-4329 ACUGAUUCCAGUAGGCAAGdTdT 2760 AD-62019.1 GAAUCAGUGCAAAUGAAGAdTdT 2194 4322-4340 UCUUCAUUUGCACUGAUUCdTdT 2761 AD-62025.1 ?\AAUGAAGAAGACUUAAAAdT dT 2195 4332-4350 UUUUAAGUCUUCUUCAUUUdTdT 2762 AD-62031.1 GAAGACUUAAAAGCCCUUGdTdT 2196 4339-4357 CAAGGGCUUUUAAGUCUUCdTdT 2763 AD-62037.1 CCUUGUGGAAGGGGUGGAUdTdT 2197 4353-4371 AUCCACCCCUUCCACAAGGdTdT 2764 AD-62043.1 GAAGGGGUGGAUCAACUAUdTdT 2198 4360-4378 AUAGUUGAUCCACCCCUUCdTdT 2765 AD-62049.1 AJCAACUAUUCACUGAUUAdT dT 2199 4370-4388 UAAUCAGUGAAUAGUUGAUdT dT 2766 AD-62055.1 CACUGAUUACCAAAUCAAAdTdT 2200 4380-4398 UUUGAUUUGGUAAUCAGUGdTdT 2767 AD-62061.1 AJCAAAGAUGGACAUGUUAdT dT 2201 4393-4411 UAACAUGUCCAUCUUUGAUdTdT 2768 AD-62020.1 GGACAUGUUAUUCUGCAACdTdT 2202 4402-4420 GUUGCAGAAUAACAUGUCCdTdT 2769 AD-62026.1 UCUGCAACUGAAUUCGAUUdTdT 2203 4413-4431 AAUCGAAUUCAGUUGCAGAdTdT 2770 AD-62032.1 GAAUUCGAUUCCCUCCAGUdTdT 2204 4422-4440 ACUGGAGGGAAUCGAAUUCdTdT 2771 AD-62038.1 CCCUCCAGUGAUUUCCUUUdTdT 2205 4432-4450 AAAGGAAAUCACUGGAGGGdTdT 2772 AD-62044.1 GAUUUCCUUUGUGUACGAUdTdT 2206 4441-4459 AUCGUACACAAAGGAAAUCdTdT 2773 AD-62050.1 GUACGAUUCCGGAUAUUUGdTdT 2207 4453-4471 CAAAUAUCCGGAAUCGUACdTdT 2774 AD-62320.1 CGGAUAUUUGAACUCUUUGdTdT 2208 4462-4480 CAAAGAGUUCAAAUAUCCGdTdT 2775 AD-62326.1 ACUCUUUGAAGUUGGGUUUdTdT 2209 4473-4491 AAACCCAACUUCAAAGAGUdTdT 2776 AD-62332.1 AGUUGGGUUUCUCAGUCCUdTdT 2210 4482-4500 AGGACUGAGAAACCCAACUdTdT 2777 AD-62338.1 UUCUCAGUCCUGCCACUUUdTdT 2211 4490-4508 AAAGUGGCAGGACUGAGAAdTdT 2778 AD-62344.1 CACUUUCACAGUGUACGAAdTdT 2212 4503-4521 UUCGUACACUGUGAAAGUGdTdT 2779 AD-62350.1 CACAGUGUACGAAUACCACdTdT 2213 4509-4527 GUGGUAUUCGUACACUGUGdTdT 2780 AD-62356.1 ACCACAGACCAGAUAAACAdTdT 2214 4523-4541 UGUUUAUCUGGUCUGUGGUdTdT 2781 AD-62362.1 CCAGAUAAACAGUGUACCAdTdT 2215 4531-4549 UGGUACACUGUUUAUCUGGdTdT 2782 AD-62321.1 CAGUGUACCAUGUUUUAUAdTdT 2216 4540-4558 UAUAAAACAUGGUACACUGdT dT 2783 AD-62327.1 GUUUUAUAGCACUUCCAAUdTdT 2217 4551-4569 AUUGGAAGUGCUAUAAAACdTdT 2784 AD-62333.1 CUUCCAAUAUCAAAAUUCAdTdT 2218 4562-4580 UGAAUUUUGAUAUUGGAAGdTdT 2785

- 165044245

AD-62339.1 AJCAAAAUUCAGAAAGUCUdTdT 2219 4570-4588 AGACUUUCUGAAUUUUGAUdTdT 2786 AD-62345.1 GAAAGUCUGUGAAGGAGCCdTdT 2220 4581-4599 GGCUCCUUCACAGACUUUCdTdT 2787 AD-62351.1 GAAGGAGCCGCGUGCAAGUdTdT 2221 4591-4609 ACUUGCACGCGGCUCCUUCdTdT 2788 AD-62357.1 GGUGCAAGUGUGUAGAAGCdTdT 2222 4601-4619 GCUUCUACACACUUGCACGdTdT 2789 AD-62363.1 GUAGAAGCUGAUUGUGGGCdTdT 2223 4612-4630 GCCCACAAUCAGCUUCUACdTdT 2790 AD-62322.1 GUGAUUGUGGGCAAAUGCAdTdT 2224 4619-4637 UGCAUUUGCCCACAAUCAGdTdT 2791 AD-62328.1 GCAAAUGCAGGAAGAAUUGdTdT 2225 4629-4647 CAAUUCUUCCUGCAUUUGCdTdT 2792 AD-62334.1 GAAGAAUUGGAUCUGACAAdTdT 2226 4639-4657 UUGUCAGAUCCAAUUCUUCdTdT 2793 AD-62340.1 GUGACAAUCUCUGCAGAGAdTdT 2227 4651-4669 UCUCUGCAGAGAUUGUCAGdTdT 2794 AD-62346.1 GCAGAGACAAGAAAACAAAdT dT 2228 4663-4681 UUUGUUUUCUUGUCUCUGCdTdT 2795 AD-62352.1 GAAGAAAACAAACAGCAUGdTdT 2229 4670-4688 CAUGCUGUUUGUUUUCUUGdTdT 2796 AD-62358.1 ACAGCAUGUAAACCAGAGAdTdT 2230 4681-4699 UCUCUGGUUUACAUGCUGUdTdT 2797 AD-62364.1 GCAGAGAUUGCAUAUGCUUdTdT 2231 4693-4711 AAGCAUAUGCAAUCUCUGGdTdT 2798 AD-62323.1 GCAUAUGCUUAUAAAGUUAdT dT 2232 4702-4720 UAACUUUAUAAGCAUAUGCdTdT 2799 AD-62329.1 UUAUAAAGUUAGCAUCACAdT dT 2233 4710-4728 UGUGAUGCUAACUUUAUAAdTdT 2800 AD-62335.1 GAUCACAUCCAUCACUGUAdTdT 2234 4722-4740 UACAGUGAUGGAUGUGAUGdTdT 2801 AD-62341.1 UCACUGUAGAAAAUGUUUUdTdT 2235 4733-4751 AAAACAUUUUCUACAGUGAdT dT 2802 AD-62347.1 AGAAAAUGUUUUUGUCAAGdTdT 2236 4740-4758 CUUGACAAAAACAUUUUCUdTdT 2803 AD-62353.1 DUUGUCAAGUACAAGGCAAdTdT 2237 4750-4768 UUGCCUUGUACUUGACAAAdTdT 2804 AD-62359.1 AGGCAACCCUUCUGGAUAUdTdT 2238 4763-4781 AUAUCCAGAAGGGUUGCCUdTdT 2805 AD-62365.1 CCUUCUGGAUAUCUACAAAdTdT 2239 4770-4788 UUUGUAGAUAUCCAGAAGGdTdT 2806 AD-62324.1 DAUCUACAAAACUGGGGAAdTdT 2240 4779-4797 UUC С C CAGUUUUGUAGAUAdT dT 2807 AD-62330.1 GUGGGGAAGCUGUUGCUGAdTdT 2241 4790-4808 UCAGCAACAGCUUCCCCAGdTdT 2808 AD-62336.1 GUGUUGCUGAGAAAGACUCdTdT 2242 4799-4817 GAGUCUUUCUCAGCAACAGdTdT 2809 AD-62342.1 GACUCUGAGAUUACCUUCAdTdT 2243 4813-4831 UGAAGGUAAUCUCAGAGUCdTdT 2810 AD-62348.1 GAGAUUACCUUCAUUAAAAdTdT 2244 4819-4837 UUUUAAUGAAGGUAAUCUCdTdT 2811 AD-62354.1 AJUAAAAAGGUAACCUGUAdTdT 2245 4831-4849 UACAGGUUACCUUUUUAAUdTdT 2812 AD-62360.1 UAACCUGUACUAACGCUGAdTdT 2246 4841-4859 UCAGCGUUAGUACAGGUUAdTdT 2813 AD-62366.1 CUAACGCUGAGCUGGUAAAdTdT 2247 4850-4868 UUUACCAGCUCAGCGUUAGdTdT 2814 AD-62325.1 GGUAAAAGGAAGACAGUACdTdT 2248 4863-4881 GUACUGUCUUCCUUUUACCdTdT 2815 AD-62331.1 GAAGACAGUACUUAAUUAUdT dT 2249 4871-4889 AUAAUUAAGUACUGUCUUCdTdT 2816 AD-62337.1 GUUAAUUAUGGGUAAAGAAdTdT 2250 4881-4899 UUCUUUACCCAUAAUUAAGdTdT 2817 AD-62343.1 UAAAGAAGCCCUCCAGAUAdTdT 2251 4893-4911 UAUCUGGAGGGCUUCUUUAdTdT 2818 AD-62349.1 GCUCCAGAUAAAAUACAAUdTdT 2252 4902-4920 AUUGUAUUUUAUCUGGAGGdTdT 2819 AD-62355.1 4AAUACAAUUUCAGUUUCAdTdT 2253 4912-4930 UGAAACUGAAAUUGUAUUUdTdT 2820 AD-62361.1 GAGUUUCAGGUACAUCUACdTdT 2254 4923-4941 GUAGAUGUACCUGAAACUGdTdT 2821 AD-62367.1 GGUACAUCUACCCUUUAGAdTdT 2255 4931-4949 UCUAAAGGGUAGAUGUACCdTdT 2822 AD-62373.1 GCUUUAGAUUCCUUGACCUdTdT 2256 4942-4960 AGGUCAAGGAAUCUAAAGGdTdT 2823 AD-62379.1 GCUUGACCUGGAUUGAAUAdTdT 2257 4952-4970 UAUUCAAUCCAGGUCAAGGdTdT 2824 AD-62385.1 GGAUUGAAUACUGGCCUAGdTdT 2258 4961-4979 CUAGGCCAGUAUUCAAUCCdTdT 2825 AD-62391.1 CUGGCCUAGAGACACAACAdTdT 2259 4971-4989 UGUUGUGUCUCUAGGCCAGdTdT 2826 AD-62397.1 GAGACACAACAUGUUCAUCdTdT 2260 4979-4997 GAUGAACAUGUUGUGUCUCdTdT 2827 AD-62403.1 GUUCAUCGUGUCAAGCAUUdTdT 2261 4991-5009 AAUGCUUGACACGAUGAACdTdT 2828 AD-62409.1 GUCAAGCAUUUUUAGCUAAdTdT 2262 5000-5018 UUAGCUAAAAAUGCUUGACdTdT 2829

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AD-62368.1 A.GCUAAUUUAGAUGAAUUUdT dT 2263 5013-5031 AAAUUCAUCUAAAUUAGCUdTdT 2830 AD-62374.1 AGAUGAAUUUGCCGAAGAUdTdT 2264 5022-5040 AUCUUCGGCAAAUUCAUCUdTdT 2831 AD-62380.1 GCGAAGAUAUCUUUUUAAAdTdT 2265 5033-5051 UUUAAAAAGAUAUCUUCGGdTdT 2832 AD-62386.1 GUUUUUAAAUGGAUGCUAAdTdT 2266 5043-5061 UUAGCAUCCAUUUAAAAAGdTdT 2833 AD-62392.1 GGAUGCUAAAAUUCCUGAAdTdT 2267 5053-5071 UUCAGGAAUUUUAGCAUCCdTdT 2834 AD-62398.1 UAAAAUUCCUGAAGUUCAGdTdT 2268 5059-5077 CUGAACUUCAGGAAUUUUAdTdT 2835 AD-62404.1 AGUUCAGCUGCAUACAGUUdTdT 2269 5071-5089 AACUGUAUGCAGCUGAACUdTdT 2836 AD-62410.1 GCAUACAGUUUGCACUUAUdTdT 2270 5080-5098 AUAAGUGCAAACUGUAUGCdTdT 2837 AD-62369.1 ACUUAUGGACUCCUGUUGUdTdT 2271 5093-5111 ACAACAGGAGUCCAUAAGUdTdT 2838 AD-62375.1 GGACUCCUGUUGUUGAAGUdTdT 2272 5099-5117 ACUUCAACAACAGGAGUCCdTdT 2839 AD-62381.1 UGUUGAAGUUCGUUUUUUUdTdT 2273 5109-5127 AAAAAAACGAACUUCAACAdTdT 2840 AD-62387.1 UUUUUUGUUUUCUUCUUUUdTdT 2274 5122-5140 AAAAGAAGAAAACAAAAAAdT dT 2841 AD-62393.1 UCUUCUUUUUUUAAACAUUdTdT 2275 5132-5150 AAUGUUUAAAAAAAGAAGAdT dT 2842 AD-62399.1 UUUUUAAACAUUCAUAGCUdTdT 2276 5139-5157 AGCUAUGAAUGUUUAAAAAdT dT 2843 AD-62405.1 AJAGCUGGUCUUAUUUGUAdTdT 2277 5152-5170 UACAAAUAAGACCAGCUAUdTdT 2844 AD-62411.1 GUCUUAUUUGUAAAGCUCAdTdT 2278 5159-5177 UGAGCUUUACAAAUAAGACdTdT 2845 AD-62370.1 4AAGCUCACUUUACUUAGAdTdT 2279 5170-5188 UCUAAGUAAAGUGAGCUUUdTdT 2846 AD-62376.1 ACUUAGAAUUAGUGGCACUdTdT 2280 5182-5200 AGUGCCACUAAUUCUAAGUdTdT 2847 AD-62382.1 AGUGGCACUUGCUUUUAUUdTdT 2281 5192-5210 AAUAAAAGCAAGUGCCACUdTdT 2848 AD-62388.1 GCUUUUAUUAGAGAAUGAUdT dT 2282 5202-5220 AUCAUUCUCUAAUAAAAGCdTdT 2849 AD-62394.1 GAGAAUGAUUUCAAAUGCUdTdT 2283 5212-5230 AGCAUUUGAAAUCAUUCUCdTdT 2850 AD-62400.1 DUUCAAAUGCUGUAACUUUdTdT 2284 5220-5238 AAAGUUACAGCAUUUGAAAdTdT 2851 AD-62406.1 GUAACUUUCUGAAAUAACAdTdT 2285 5231-5249 UGUUAUUUCAGAAAGUUACdTdT 2852 AD-62412.1 GAAAUAACAUGGCCUUGGAdTdT 2286 5241-5259 UCCAAGGCCAUGUUAUUUCdTdT 2853 AD-62371.1 GCUUGGAGGGCAUGAAGACdTdT 2287 5253-5271 GUCUUCAUGCCCUCCAAGGdTdT 2854 AD-62377.1 AGGGCAUGAAGACAGAUACdTdT 2288 5259-5277 GUAUCUGUCUUCAUGCCCUdTdT 2855 AD-62383.1 GAUACUCCUCCAAGGUUAUdTdT 2289 5273-5291 AUAACCUUGGAGGAGUAUCdTdT 2856 AD-62389.1 GCUCCAAGGUUAUUGGACAdTdT 2290 5279-5297 UGUCCAAUAACCUUGGAGGdTdT 2857 AD-62395.1 GGACACCGGAAACAAUAAAdTdT 2291 5293-5311 UUUAUUGUUUCCGGUGUCCdTdT 2858 AD-62401.1 GAAACAAUAAAUUGGAACAdT dT 2292 5301-5319 UGUUCCAAUUUAUUGUUUCdTdT 2859 AD-62407.1 AJUGGAACACCUCCUCAAAdTdT 2293 5311-5329 UUUGAGGAGGUGUUCCAAUdTdT 2860 AD-62413.1 UCCUCAAACCUACCACUCAdTdT 2294 5322-5340 UGAGUGGUAGGUUUGAGGAdTdT 2861 AD-62372.1 GUACCACUCAGGAAUGUUUdTdT 2295 5331-5349 AAACAUUCCUGAGUGGUAGdTdT 2862 AD-62378.1 4AUGUUUGCUGGGGCCGAAdTdT 2296 5343-5361 UUCGGCCCCAGCAAACAUUdTdT 2863 AD-62384.1 UGCUGGGGCCGAAAGAACAdTdT 2297 5349-5367 UGUUCUUUCGGCCCCAGCAdTdT 2864 AD-62390.1 4AAGAACAGUCCAUUGAAAdTdT 2298 5360-5378 UUUCAAUGGACUGUUCUUUdTdT 2865 AD-62396.1 GAUUGAAAGGGAGUAUUACdTdT 2299 5371-5389 GUAAUACUCCCUUUCAAUGdTdT 2866 AD-62402.1 GGAGUAUUACAAAAACAUGdT dT 2300 5380-5398 CAUGUUUUUGUAAUACUCCdTdT 2867 AD-62408.1 4AAACAUGGCCUUUGCUUGdTdT 2301 5391-5409 CAAGCAAAGGCCAUGUUUUdTdT 2868 AD-62414.1 GCCUUUGCUUGAAAGAAAAdTdT 2302 5399-5417 UUUUCUUUCAAGCAAAGGCdTdT 2869 AD-62415.1 GAAAGAAAAUACCAAGGAAdTdT 2303 5409-5427 UUCCUUGGUAUUUUCUUUCdTdT 2870 AD-62416.1 GCAAGGAACAGGAAACUGAdTdT 2304 5420-5438 UCAGUUUCCUGUUCCUUGGdTdT 2871 AD-62417.1 4ACUGAUCAUUAAAGCCUGdTdT 2305 5433-5451 CAGGCUUUAAUGAUCAGUUdTdT 2872

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Table 22

Single-dose C5 (10 mM) assay in Hep3B cells with dT-modified mRNA

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Example 7. Screening of additional siRNAs in vivo.

Based on the AD-58643 sequence, four additional sense and three antisense sequences were synthesized and used to generate twelve 21/25mer compounds (Table 23). In general, the antisense strands of these compounds were extended by dTdT, and the duplexes contained fewer fluoro-modified nucleotides.

C57BL/6 mice (n=3 per group) were injected subcutaneously with 1 mg/kg of these GalNAc-conjugated duplexes. Serum was collected on day 0 after pre-selection, and C5 protein levels were quantified by ELISA on day 5. C5 protein levels were normalized to the day 0 level in pre-selected serum.

Table 14 shows the results of a single dose in vivo assay with the indicated mRNAs. Data are expressed as the percentage of remaining C5 protein relative to levels in prescreened serum. These mRNAs with improved potency compared to the parent compound included AD-62510, AD-62643, AD-62645, AD-62646, AD-62650, and AD-62651. These mRNAs also demonstrated similar potency (IC ₅₀ approximately 23-59 pM).

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The efficacy of these mRNAs was tested in C57BL/6 mice using a single-dose protocol. Mice were injected subcutaneously with AD-62510, AD-62643, AD-62645, AD-62646, AD-62650, and

AD-62651 at a dose of 0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg, or 2.5 mg/kg. Serum was collected on days 0 and 5 and analyzed for C5 protein levels using ELISA. C5 levels were normalized to day 0 levels in pre-collected sera.

Figure 15 shows that a dose-response relationship was observed with all mRNAs tested and that a single administration of all these mRNAs resulted in C5 protein silencing similar to or improved compared to AD-58641.

The in vivo silencing duration of AD-62510, AD-62643, AD-62645, AD-62646, AD-62650, and AD62651 was determined by administering a single 1.0 mg/kg dose to C57BL/6 mice and measuring the amount of C5 protein detectable on days 6, 13, 20, 27, and 34 using ELISA. C5 levels were normalized to day 0 levels in pre-selected sera.

As shown in Fig. 16, each of the tested mRNAs exhibited the same recovery kinetics as AD-62643, with a trend toward better silencing, but within the assay error.

AD-62510, AD-62643, AD-62645, AD-62646, AD-62650, and AD-62651 were further tested for efficacy and the cumulative effect of mRNA was assessed in rats using a repeated-dose protocol. Wild-type Sprague Dawley rats were injected subcutaneously with each mRNA at 5.0 mg/kg/dose on days 0, 4, and 7. Serum was collected on days 0, 4, 7, 11, 14, 18, 25, 28, and 32. Serum hemolytic activity was quantified as described above.

The results presented in Fig. 17 show that all of the tested mRNAs provide effective and durable reduction of hemolytic activity and similar restoration of hemolysis as observed with AD-58641 treatment.

Table 23 Sequences of the modified sense and antisense strands of GalNAc-conjugated C5 dsRNA

Duplex ID Semantic ID Semantic (from 5' to 3') SEQ ID NO: AS ID Antisense (5' to 3') SEQ ID NO: AD-58643 A-119326.1 Af sasGfcAfaGfaUfAfUfuUfuUfa UfaAfuAfL96 2873 A- 119327.1 usAf suU f aUf aAf aAf auaU f cUfuG f cUf ususu 2886 AD-62642 A-125167.7 asasGfcAfaGfaUfAfUfuUfuuAfu Af auaL96 2874 A- 125139.1 usAfsuuaUfaAfaAfauaUfcUfuGfcuus usudTdT 2887 AD-62510 A-125167.7 asasGfcAfaGfaUfAfUfuUfuuAfu Af auaL96 2875 A- 125173.2 usAfsUfuAfuAfAfaAfauaUfcUfuGfcu ususudTdT 2888 AD-62643 A-125167.7 asasGfcAfaGfaUfAfUfuUfuuAfu Af auaL96 2876 A- 125647.1 usAfsUfuAfuaAfaAfauaUfcUfuGfcuu susudTdT 2889 AD-62644 A-125157.17 asasGfcAfaGfaUfAfUfuUfuuAfu aAfuaL96 2877 A- 125139.1 usAfsuuaUfaAfaAfauaUfcUfuGfcuus usudTdT 2890 AD-62645 A-125157.17 asasGfcAfaGfaUfAfUfuUfuuAfu aAfuaL96 2878 A- 125173.2 usAfsUfuAfuAfAfaAfauaUfcUfuGfcu ususudTdT 2891 AD-62646 A-125157.17 asasGfcAfaGfaUfAfUfuUfuuAfu aAfuaL96 2879 A- 125647.1 usAfsUfuAfuaAfaAfauaUfcUfuGfcuu susudTdT 2892 AD-62647 A-125134.1 asasgcaagauaUfuuuua(Tgn)aau aL96 2880 A- 125139.1 usAfsuuaUfaAfaAfauaUfcUfuGfcuus usudTdT 2893 AD-62648 A-125134.1 asasgcaagauaUfuuuua(Tgn)aau aL96 2881 A- 125173.2 usAfsUfuAfuAfAfaAfauaUfcUfuGfcu ususudTdT 2894 AD-62649 A-125134.1 asasgcaagauaUfuuuua(Tgn)aau aL96 2882 A- 125647.1 usAfsUfuAfuaAfaAfauaUfcUfuGfcuu susudTdT 2895 AD-62428 A-125127.2 asasgcaagaUfaUfuuuuauaaual® 6 2883 A- 125139.1 usAfsuuaUfaAfaAfauaUfcUfuGfcuus usudTdT 2896 AD-62650 A-125127.2 asasgcaagaUfaUfuuuuauaaual9 6 2884 A- 125173.2 usAfsUfuAfuAfAfaAfauaUfcUfuGfcu ususudTdT 2897 AD-62651 A-125127.2 asasgcaagaUfaUfuuuuauaaual9 6 2885 A- 125647.1 usAfsUfuAfuaAfaAfauaUfcUfuGfcuu susudTdT 2898

Claims (64)

1. An agent that is a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C5, wherein said dsRNA contains a sense strand and an antisense strand forming a double-stranded region, where the sense strand and the antisense strand are each independently 19-25 nucleotides in length , the sense strand contains at least 19 contiguous nucleotides from the nucleotide sequence 5'-AAGCAAGAUAUUUUUAUAAUA-3' (SEQ ID NO:62) and the antisense strand contains at least 19 contiguous nucleotides from the nucleotide sequence 5'-UAUUAUAAAAAUAUCUUGCUUUU-3') ( SEQ ID NO:113), all nucleotides of said sense strand and all nucleotides of said antisense strand include a nucleotide modification and at least one strand is conjugated to a ligand. 2. The dsRNA agent according to claim 1, wherein at least one of said nucleotide modifications is selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide modification, a 2'-O-methyl-nucleotide modification , 2'-fluoro-nucleotide modification, 2'-deoxynucleotide modification, closed nucleotide modification, abasic nucleotide modification, 2'-amino-nucleotide modification, 2'-alkyl-nucleotide modification, morpholinonucleotide modification, nucleotide modification of phosphoramidate containing a non-natural base origin, a nucleotide containing a modification of the 5'-phosphorothioate group, and a modification of the terminal nucleotide associated with a cholesteryl derivative or dodecanoic acid bisdecylamide group. 3. The dsRNA agent according to claim 2, wherein said modified nucleotides comprise a modification of a short sequence of 3'-terminal deoxy-thymine nucleotides (dT). 4. The dsRNA agent according to any one of claims 1 to 3, wherein at least one strand contains a 3' overhang of at least 1 nucleotide. 5. The dsRNA agent according to any one of claims 1 to 3, wherein at least one strand contains a 3' overhang of at least 2 nucleotides. 6. The dsRNA agent according to any one of claims 1 to 5, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent. 7. The dsRNA agent according to any one of claims 1 to 6, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative. 8. The dsRNA agent according to claim 7, wherein the ligand is 9. The dsRNA agent according to claim 7, wherein the dsRNA agent is conjugated to a ligand as shown in the following diagram: and where X represents O or S. 10. The dsRNA agent according to claim 9, where X represents O. - 187 044245 11. The dsRNA agent according to any one of claims 1 to 10, wherein said dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. 12. The dsRNA agent of claim 11, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand. 13. The dsRNA agent according to claim 12, wherein said strand is an antisense strand. 14. The dsRNA agent of claim 12, wherein said strand is a sense strand. 15. The dsRNA agent of claim 11, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand. 16. The dsRNA agent according to claim 15, wherein said strand is an antisense strand. 17. The dsRNA agent of claim 15, wherein said strand is a sense strand. 18. The dsRNA agent according to claim 11, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at both the 5' and 3' ends of one strand. 19. The dsRNA agent according to claim 18, wherein said strand is an antisense strand. 20. An agent that is a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of complement component C5, where said dsRNA agent contains a sense strand and an antisense strand forming a double-stranded region, where the sense strand and the antisense strand are each independently 19-25 nucleotides in length , the sense strand contains at least 19 contiguous nucleotides from the nucleotide sequence 5'-AAGCAAGAUAUUUUUAUAAUA-3' (SEQ ID NO:62), and the antisense strand contains at least 19 contiguous nucleotides from the nucleotide sequence 5'UAUUAUAAAAAUAUCUUGCUUUU-3' (SEQ ID NO:113), all nucleotides of the specified sense strand contain a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, the specified sense strand contains two phosphorothioate internucleotide bonds at the 5' end, all nucleotides of said antisense chain contain a modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification and a deoxy-thymine nucleotide modification, said antisense chain contains two phosphorothioate internucleotide bonds at the 5' end and two phosphorothioate internucleotide linkages at the 3' end and the sense strand is conjugated to one or more GalNAc derivatives attached via a monovalent, divalent or trivalent linker at the 3' end. 21. The dsRNA agent of claim 20, wherein the one or more GalNAc derivatives are attached via a trivalent branched linker. 22. The dsRNA agent according to claim 20 or 21, where the ligand is - 188 044245 23. The dsRNA agent of claim 22, wherein the RNAi agent is conjugated to a ligand as shown in the following diagram: 24. The dsRNA agent according to any one of claims 1 to 23, wherein the double-stranded region is 19 to 25 nucleotide pairs in length. 25. The dsRNA agent according to claim 24, where the double-stranded region is 19-23 nucleotide pairs in length. 26. The dsRNA agent according to claim 24, wherein the double-stranded region is 19-21 nucleotide pairs in length. 27. The dsRNA agent according to claim 24, where the double-stranded region is 21-23 nucleotide pairs in length. 28. The dsRNA agent according to any one of claims 1 to 27, wherein each strand is independently 21 to 23 nucleotides in length. 29. The dsRNA agent according to any one of claims 1 to 28, wherein the sense strand comprises the nucleotide sequence 5'-AAGCAAGAUAUUUUUAUAAUA-3' (SEQ ID NO:62). 30. The dsRNA agent according to any one of claims 1 to 29, wherein the antisense strand contains the nucleotide sequence 5'-UAUUAUAAAAAUAUCUUGCUUUU-3' (SEQ ID NO:113). 31. The dsRNA agent according to any one of claims 1 to 30, wherein the sense strand contains the nucleotide sequence 5'-AAGCAAGAUAUUUUUAUAAUA-3' (SEQ ID NO:62) and the antisense strand contains the nucleotide sequence 5'-UAUUAUAAAAAUAUCUUGCUUUU-3' (SEQ ID NO :113). 32. The dsRNA agent according to claim 31, where the sense strand contains 5'-asasGfcAfaGfaUfAfUfuUfuuAfuAfaua3' (SEQ ID NO:2876) and the antisense strand contains 5'-usAfsUfuAfuaAfaAfauaUfcUfuGfcuususudTdT-3' (SEQ ID NO:2889), where a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C and U; Af, Gf, Cf and Uf represent the 2'-fluorine of A, G, C and U; dT is a deoxy-thymine nucleotide and s is a phosphorothioate bond. - 189 044245 33. The dsRNA agent according to any one of claims 1 to 31, wherein the sense and antisense strands contain nucleotide sequences selected from the group consisting of 5' - AfaGfcAfaGfaUfAfUfuUfuUfaUfaAfuAf - 3' (SEQ ID NO:122) and 5' - uAfuUfaUfaAfaAfauaUfcUfuGfcUfusUfsu - 3' (SEQ ID NO:173) r 5' - AfsasGfcAfaGfaUfAfUfuUfuUfaUfaAfuAf - 3' (SEQ ID NO:164) And 5' - usAfsuUfaUfaAfaAfauaUfcUfuGfcUfususu - 3' (SEQ ID NO:215) 5' - asasGfcAfaGfaUfAfUfuUfuuAfuAfaua - 3' (SEQ ID NO:2875) and 5' - usAfsUfuAfuAfAfaAfauaUfcUfuGfcuususudTdT - 3' (SEQ ID NO:2 8 8 8) ; 5' - asasGfcAfaGfaUfAfUfuUfuuAfuAfaua - 3' (SEQ ID NO: 2876) and 5' - usAfsUfuAfuaAfaAfauaUfcUfuGfcuususudTdT - 3' (SEQ ID NO:2 8 8 9) ; 5' - asasGfcAfaGfaUfAfUfuUfuuAfuaAfua - 3' (SEQ ID NO:2878) and 5' - usAfsUfuAfuAfAfaAfauaUfcUfuGfcuususudTdT - 3' (SEQ ID NO:2891); 5' - asasGfcAfaGfaUfAfUfuUfuuAfuaAfua - 3' (SEQ ID NO:2879) and 5' - usAfsUfuAfuaAfaAfauaUfcUfuGfcuususudTdT - 3' (SEQ ID NO:2892); 5' - asasgcaagaUfaUfuuuuauaaua - 3' (SEQ ID NO:2884) and 5' - usAfsUfuAfuAfAfaAfauaUfcUfuGfcuususudTdT - 3' (SEQ ID NO:2897); And 5' - asasgcaagaUfaUfuuuuauaaua - 3' (SEQ ID NO:2885) and 5' - usAfsUfuAfuaAfaAfauaUfcUfuGfcuususudTdT - 3' (SEQ ID NO:2898), where a, g, c and u represent 2'-O-methyl (2'-OMe) A, G, C and U; Af, Gf, Cf and Uf represent the 2'-fluorine of A, G, C and U; dT is a deoxy-thymine nucleotide and s is a phosphorothioate bond. 34. The dsRNA agent of claim 33, wherein the sense strand consists of the nucleotide sequence 5'asasGfcAfaGfaUfAfUfuUfuuAfuAfaua-3' (SEQ ID NO:2876) and the antisense strand consists of the nucleotide sequence 5'-usAfsUfuAfuaAfaAfauaUfcUfuGfcuususudTdT-3' (SEQ ID NO:28 89) , a, g, c and u represent 2'-O-methyl (2'-OMe) A, G, C and U; Af, Gf, Cf and Uf represent the 2'-fluorine of A, G, C and U; dT is a deoxy-thymine nucleotide and s is a phosphorothioate bond; And - 190 044245 the ligand is conjugated at the 3' end of the sense strand, as shown in the following diagram: 35. A cell containing a dsRNA agent according to any one of claims 1 to 34. 36. Pharmaceutical composition for inhibiting the expression of the complement component C5 gene, containing a dsRNA agent according to any one of claims 1 to 34. 37. The pharmaceutical composition according to claim 36, wherein the RNAi agent is contained in a non-buffered solution. 38. The pharmaceutical composition according to claim 37, wherein said non-buffered solution is saline or water. 39. The pharmaceutical composition according to claim 38, wherein said RNAi agent is present with a buffer solution. 40. The pharmaceutical composition according to claim 39, wherein said buffer solution contains acetate, citrate, prolamine, carbonate or phosphate or any combination thereof. 41. The pharmaceutical composition according to claim 39, wherein said buffer solution is phosphate buffered saline (PBS). 42. A method for inhibiting the expression of complement component C5 in a cell, including: (a) contacting the cell with a dsRNA agent according to any one of claims 1 to 34 or a pharmaceutical composition according to any one of claims 36 to 41 and (b) maintaining the cell obtained in step (a) for a time sufficient to destroy the transcript mRNA of the C5 complement component gene, thereby inhibiting the expression of the C5 complement component gene in the cell. 43. A method of treating a subject suffering from a disease or disorder in which reduction in the expression of complement component C5 will have a beneficial effect, the method comprising administering to the subject a therapeutically effective amount of a dsRNA agent according to any one of claims 1 to 34 or a pharmaceutical composition according to any one of 36-41, thereby treating said subject. 44. A method of preventing at least one symptom in a subject suffering from a disease or disorder that would benefit from reducing the expression of complement component C5, the method comprising administering to the subject a prophylactically effective amount of a dsRNA agent according to any one of claims 1 to 34 or a pharmaceutical composition according to any one of claims 36 to 41, thereby preventing at least one symptom in a subject suffering from a disease in which reduction of C5 expression would have a beneficial effect. 45. The method of claim 43 or 44, wherein administration of dsRNA to a subject results in a decrease in intravascular hemolysis, stabilization of hemoglobin levels and/or a decrease in C5 protein. 46. The method according to claim 43 or 44, where the disorder is a disease associated with complement component C5. 47. The method of claim 46, wherein the complement component C5 disease is selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA), antiphospholipid antibody syndrome , lupus nephritis, ischemia-reperfusion injury, typical or infectious hemolytic uremic syndrome (tHUS), dense sediment disease (DDD), neuromyelitis optica (NMO), multifocal motor neuropathy (MMN), multiple sclerosis (MS); macular degeneration (eg, age-related macular degeneration (AMD)); syndrome including hemolysis, increased activity of liver enzymes and decreased platelet count (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous fetal loss; pauci-immune vasculitis; epidermolysis bullosa; recurrent fetal loss; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis bullous pemphigoid associated with Shiga-like toxin E. coli hemolytic-uremic syndrome, C3 nephropathy, vasculitis associated with antibodies to the cytoplasm of neutrophils; graft rejection reactions caused by humoral and vascular mechanisms, graft dysfunction, myocardial infarction, allogeneic transplantation, sepsis, coronary artery disease, dermatomyositis, Graves' disease, atheroma - 191 044245 sclerosis, Alzheimer's disease, sepsis associated with systemic inflammatory response, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos' disease , antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disorder, peripheral vascular disorder, renovascular disorder, mesenteric/enteric vascular disorder, vasculitis, Henoch-Schönlein nephritis, systemic lupus erythematosus-associated vasculitis, vasculitis , associated with rheumatoid arthritis, immune complex vasculitis, Takayasu disease, dilated cardiomyopathy, diabetic angiopathy, Kawasaki disease (arteritis), venous gas embolism (VGE) and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barré syndrome and percutaneous transluminal coronary angioplasty (PTCA). 48. The method of claim 47, wherein the complement component C5 disease is paroxysmal nocturnal hemoglobinuria (PNH). 49. The method of claim 47, wherein the complement component C5 disease is atypical hemolytic uremic syndrome (aHUS). 50. The method according to claim 43 or 44, where the subject is a human. 51. The method according to any one of claims 43-50, further comprising administering to the subject an antibody to complement component C5 or an antigen-binding fragment thereof. 52. The method of any one of claims 43 to 51, wherein the dsRNA agent is administered at a dosage of from about 0.01 to about 10 mg/kg or from about 0.5 to about 50 mg/kg. 53. The method of claim 52, wherein the dsRNA agent is administered at a dosage of from about 10 to about 30 mg/kg. 54. The method of claim 52, wherein the dsRNA agent is administered to the subject once a week. 55. The method of claim 52, wherein the dsRNA agent is administered to the subject twice a week. 56. The method of claim 52, wherein the dsRNA agent is administered to the subject twice a month. 57. The method according to any one of claims 43-56, wherein the dsRNA agent is administered to the subject subcutaneously. 58. The method of claim 43 or 44, further comprising measuring hemoglobin and/or LDH levels in said subject. 59. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C5, where the dsRNA contains a sense strand containing the nucleotide sequence 5'-asasGfcAfaGfaUfAfUfuUfiiuAfuAfaua-3' (SEQ ID NO:2876) and an antisense strand containing the nucleotide sequence 5'-usAfsUfuAfuaAfaAfauaUfcUfuGfcuususudTdT-3' (SEQ ID NO:2889), where a, g, c and u represent 2'-O-methyl (2'-OMe) A, G, C and U; Af, Gf, Cf and Uf represent the 2'-fluorine of A, G, C and U; dT is a deoxy-thymine nucleotide and s is a phosphorothioate bond. 60. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of complement component C5, where the dsRNA contains a sense strand and an antisense strand, where the sense strand contains the nucleotide sequence 5'asasGfcAfaGfaUfAfUfuUfuuAfuAfaua-3' (SEQ ID NO:2876) and the antisense the chain contains the nucleotide sequence 5'-usAfsUfuAfuaAfaAfauaUfcUfuGfcuususudTdT-3' (SEQ ID NO:2889), where a, g, c and u represent 2'-O-methyl (2'-OMe) A, G, C and U; Af, Gf, Cf and Uf represent the 2'-fluorine of A, G, C and U; dT is a deoxy-thymine nucleotide and s is a phosphorothioate bond; The ligand is conjugated at the 3' end of the sense strand, as shown in the following diagram: where X represents O. - 192 044245 61. Pharmaceutical composition for inhibiting complement component gene expression C5, containing the dsRNA agent according to claim 60. 62. A method of treating a subject suffering from a disease or disorder in which reducing the expression of complement component C5 will have a beneficial effect, the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 60 or the pharmaceutical composition of claim 61 to treat those the most specified subject. 63. A method of preventing at least one symptom in a subject suffering from a disease or disorder that would benefit from reducing the expression of complement component C5, the method comprising administering to the subject a prophylactically effective amount of the dsRNA agent of claim 60 or the pharmaceutical composition of claim 60. claim 61, thereby preventing at least one symptom in a subject suffering from a disease that would benefit from reducing C5 expression. 64. The method of claim 62 or 63, wherein the dsRNA agent or pharmaceutical composition is for subcutaneous administration.