HK40025376A - Double-stranded rna molecule targeting ckip-1 and use thereof - Google Patents

Description

Double-stranded RNA molecule targeting CKIP-1 and application thereof Technical Field

The invention relates to the field of biomedicine, in particular to double-stranded RNA molecules targeting CKIP-1 and application thereof, and particularly relates to application of the double-stranded RNA molecules in treating inflammatory diseases, such as arthritis, particularly rheumatoid arthritis.

Background

CKIP-1 (casein kinase interacting protein 1) is a bone formation inhibitory gene that specifically regulates bone formation rather than bone resorption. CKIP-1 is highly expressed in bone tissue in patients with osteoporosis. Targeted inhibition of CKIP-1 expression has been demonstrated to be useful in the treatment of osteoporosis or other pathological bone destruction. However, the prior art does not relate to inflammation with CKIP-1.

In addition, I L-6 blockers have been approved for clinical use, such as tollizumab, in large randomized, double blind clinical trials, tolzumab has a superior therapeutic effect in patients who are non-responsive to TNF- α mab.

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease characterized by polyarticular synovial inflammatory lesions. Synovitis persists with repeated attacks, which can lead to cartilage and bone destruction in joints, joint dysfunction, and even disability. Rheumatoid arthritis occurs in high incidence among adults, with an incidence of about 20-40% in every 10 million adult populations. Research shows that 70-75% of patients with rheumatoid arthritis can have bone destruction within 3 years of disease onset, 10% of patients have serious dysfunction within 2 years of disease onset, and about 50% of patients lose labor capacity after suffering from the disease for 10 years, so that serious economic burden is caused to the patients and the society. At present, the medicines for treating RA mainly comprise non-steroidal anti-inflammatory drugs, hormones, antirheumatic drugs and the like, which are mainly used for relieving pain and inflammation and cannot well prevent the destruction of joints and bones. Some of the new biological agents that have emerged in recent years are capable of alleviating and inhibiting the occurrence of bone destruction, but are not capable of repairing existing bone damage. At present, RA treatment medicines which can relieve inflammation and promote bone repair are lacking clinically.

Brief description of the invention

In a first aspect, the present invention provides a double stranded rna (dsrna) molecule comprising a sense strand and an antisense strand selected from the group consisting of:

1) the sense strand shown in SEQ ID NO. 63 and the antisense strand shown in SEQ ID NO. 64;

2) the sense strand shown in SEQ ID NO. 71 and the antisense strand shown in SEQ ID NO. 72;

3) the sense strand shown as SEQ ID NO. 83 and the antisense strand shown as SEQ ID NO. 84; and

4) the sense strand shown in SEQ ID NO. 161 and the antisense strand shown in SEQ ID NO. 162.

In some embodiments, the sense strand and/or antisense strand additionally has a overhang (overlap) of at least one nucleotide at the 3' end. In some embodiments, the sense strand and/or antisense strand additionally has a 2 nucleotide overhang at the 3' end, preferably the overhang is TT.

In some embodiments, wherein the sense strand and antisense strand comprise 1 or 2 nucleotide substitutions within 6, 5, 4, 3, or 2 nucleotides of the 5 'and/or 3' terminus. In some embodiments, wherein the sense strand and antisense strand comprise 1 nucleotide substitution, the substitution is at the 3 'last nucleotide position of the sense strand and correspondingly at the 5' first nucleotide position of the antisense strand.

In some embodiments, the dsRNA comprises at least one modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of: 2 '-O-methyl modified nucleotides, 2' -F modified nucleotides, nucleotides comprising a 5 '-phosphorothioate group and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, 2' -deoxy-2 '-fluoro modified nucleotides, 2' -deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2 '-amino-modified nucleotides, 2' -alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates and nucleotides containing a non-natural base. In some embodiments, the 2' hydroxyl of the nucleotides of all uracil or cytosine bases in the sense strand and/or antisense strand of the dsRNA is modified with a methoxy group.

In some embodiments, the dsRNA molecule inhibits expression of CKIP-1 by at least 50%, preferably by at least 70%. in some embodiments, the dsRNA molecule inhibits expression of a pro-inflammatory cytokine such as I L-6, TNF- α, and/or I L-17A.

In a second aspect, the invention also provides an expression vector comprising a nucleotide sequence encoding the dsRNA molecule of the invention operably linked to a transcriptional regulatory element.

In a third aspect, the invention also provides a pharmaceutical composition comprising a dsRNA molecule of the invention or an expression vector of the invention, and a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides a method of treating arthritis, in particular rheumatoid arthritis, in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention. In some embodiments, the method further comprises administering to the subject an additional therapeutic agent for the treatment of arthritis, particularly rheumatoid arthritis.

In a fifth aspect, the invention provides the use of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for the treatment of arthritis, in particular rheumatoid arthritis, in a subject in need thereof.

In a sixth aspect, the present invention provides a method of treating an inflammatory disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention. In some embodiments, the method further comprises administering to the subject an additional therapeutic agent for treating an inflammatory disease.

In a seventh aspect, the invention provides the use of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for the treatment of an inflammatory disease in a subject in need thereof.

In an eighth aspect, the present invention provides a method of treating a bone metabolism-related disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention. In some embodiments, the method further comprises administering to the subject an additional therapeutic agent for treating a bone metabolism-related disease.

In a ninth aspect, the invention provides the use of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention for the preparation of a medicament for the treatment of a bone metabolism related disease in a subject in need thereof.

In various aspects of the invention, wherein the subject is a human.

Brief Description of Drawings

FIG. 1 shows a pool of candidate siRNA sequences. TT at the 3' end of each sequence is a dangling end which is not complementary with the target sequence.

FIG. 2, vector map showing the over-expression vector for dual luciferase assay.

FIG. 3 shows the inhibitory effect of si-TD137 on CKIP-1 expression in a dual luciferase assay.

FIG. 4, shows the inhibitory effect of si-TD141 on CKIP-1 expression in a dual luciferase assay.

FIG. 5, shows the inhibitory effect of si-TD176 on CKIP-1 expression in a dual luciferase assay.

FIG. 6, shows the inhibitory effect of si-7 on CKIP-1 expression in a dual luciferase assay.

Figure 7, shows siRNA reduces CIA mouse clinical score.

FIG. 8, shows changes in body weight of CIA mice after siRNA treatment.

FIG. 9, shows that siRNA affects the expression of proinflammatory cytokines in joint tissues of CIA mice.

Detailed Description

A, define

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

Unless otherwise indicated, nucleic acid sequences referenced herein are written in a 5 'to 3' direction. The term "nucleic acid" refers to DNA or RNA or modified forms thereof, which comprise purine or pyrimidine bases present in DNA (adenine "a", cytosine "C", guanine "G", thymine "T") or in RNA (adenine "a", cytosine "C", guanine "G", uracil "U"). The double stranded RNA nucleic acid molecules provided herein can also comprise a "T" base at the 3' end, even if the "T" base is not naturally occurring in RNA. In some cases, these bases may be denoted as "dT" to distinguish the deoxyribonucleotides present in a ribonucleotide chain.

A nucleic acid molecule is "targeted" by a nucleic acid molecule described herein when it selectively reduces or inhibits expression of a gene, or is capable of hybridizing to a gene transcript (i.e., its mRNA) under stringent conditions.

As used herein, "CKIP-1" refers to CKIP-1 gene or protein (also known as P L EKHO 1). examples of sequences of CKIP-1 include, but are not limited to, human: Genbank No. NM-016274.4, mouse: Genbank No. NM-023320.2, rat: Genbank No. NM-001025119.1 and cynomolgus monkey: Genbank No. XM-001098879 and XM-001098774.

As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the CKIP-1 gene, including mRNA which is the product of RNA processing of a primary transcript.

As used herein, unless otherwise indicated, the term "complementary," when used to describe the relationship of a first nucleotide sequence to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under specific conditions to an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by those skilled in the art. For example, such conditions may be stringent conditions, wherein the stringent conditions may include: 400mM NaCl, 40mM PIPES pH6.4, 1mM EDTA, at 50 ℃ or 70 ℃ for 12-16 hours, followed by washing. Other conditions may also be applied, such as physiologically relevant conditions that may be encountered in vivo. The skilled person will be able to determine the set of conditions most suitable for the two sequence complementarity test, depending on the final application of the hybridizing nucleotide.

This includes base pairing of an oligonucleotide or polynucleotide comprising the first nucleotide sequence with an oligonucleotide or polynucleotide comprising the second nucleotide sequence over the full length of the first and second nucleotide sequences. These sequences may be referred to herein as being "fully complementary" to each other. However, when it is stated herein that the first sequence is "substantially complementary" to the second sequence, the two sequences may be fully complementary or form one or more, but typically no more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under conditions most relevant to their end use. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches when referring to the definition of complementarity. For example, in a dsRNA comprising one oligonucleotide of 19 nucleotides in length and another oligonucleotide of 21 nucleotides in length, the longer oligonucleotide comprises a 19 nucleotide sequence that is fully complementary to the shorter oligonucleotide, which may also be referred to as "fully complementary".

As used herein, a "complementary" sequence may also comprise or be formed entirely of non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, so long as the above-mentioned requirements for its hybridization ability are met. These non-Watson-Crick base pairs include, but are not limited to, G: U Wobble or Hoogstein base pairing.

The terms "complementary", "fully complementary" and "substantially complementary" may be used herein with respect to base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, which terms are understood in accordance with the context in which they are used.

As used herein, a polynucleotide that is "substantially complementary to at least a portion" of messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of a target mRNA (e.g., mRNA encoding CKIP-1) that includes a 5 'UTR, Open Reading Frame (ORF), or 3' UTR. For example, a polynucleotide is complementary to at least a portion of CKIP-1 if the sequence of the polynucleotide is substantially complementary to an uninterrupted portion of the mRNA encoding CKIP-1.

WO99/32619(Fire et al) discloses the use of dsRNA of at least 25 nucleotides in length for inhibiting the expression of the C.elegans gene, dsRNA has also been found to degrade target RNA in other organisms including plants (see, e.g., WO99/53050, Waterhouse et al and WO 99/61631, Heifetz et al), Drosophila (see, e.g., Yang, D.et al, curr. biol. (2000) 10: 1191-1200) and mammals (see WO00/44895, L immer and DE10100586.5, Kreutr et al). this natural mechanism has now become a hotspot for the development of novel drugs for the treatment of diseases caused by gene abnormalities or deleterious regulation.

The term "double-stranded RNA" or "dsRNA" as used herein refers to a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands as described above. Typically, the majority of the nucleotides of each strand are ribonucleotides, but as detailed herein, each or both strands may also comprise at least one non-ribonucleotide, such as a deoxyribonucleotide and/or a modified nucleotide. In addition, "dsRNA" as used in the present specification may include chemical modifications to ribonucleotides, including modifications at multiple nucleotides, and including all types of modifications disclosed herein or known in the art.

The two strands forming the duplex structure may be different portions of the same larger RNA molecule, or they may be separate RNA molecules. If the two strands are separate RNA molecules, such dsRNA is often referred to in the literature as siRNA ("short interfering RNA"). If the two strands are part of a larger molecule and are linked by an uninterrupted nucleotide chain between the 3 '-end of one strand and the 5' -end of the other strand forming a duplex structure, the linked RNA strand is referred to as a "hairpin loop", "short hairpin RNA" or "shRNA". If the two strands are covalently linked by means other than an uninterrupted strand between the 3 '-end of one strand and the 5' -end of the other strand forming a duplex structure, the linking structure is referred to as a "linker". The RNA strands may have the same or different number of nucleotides. In addition to duplex structures, dsRNA may comprise one or more nucleotide overhangs. Typically, the majority of the nucleotides of each strand are ribonucleotides, but as detailed herein, each or both strands may also comprise at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide.

As used herein, "overhang" refers to one or more unpaired nucleotides that protrude from the duplex structure of a dsRNA when the 3 'end of one strand of the dsRNA exceeds the 5' end of the other strand, or vice versa. "blunt-ended" or "blunt-ended" means that there are no unpaired nucleotides at the ends of the dsRNA, i.e., there is no nucleotide overhang. By "blunt-ended" dsRNA is meant dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. For clarity, a chemical cap or non-nucleotide chemical moiety coupled to the 3 '-end or 5' -end of a dsRNA is not considered in determining whether the dsRNA has a overhang or a blunt end.

The term "antisense strand" refers to a strand of a dsRNA that comprises a region of substantial complementarity to a target sequence. The term "complementary region" as used herein refers to a region of the antisense strand that is substantially complementary to a sequence (e.g., a target sequence) as defined herein. If the complementary region is not fully complementary to the target sequence, the mismatch may be located in the interior or in a terminal region of the molecule. Typically, the most tolerated mismatches are in the terminal region (excluding the overhangs described herein), e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5 'and/or 3' end, or the endmost 1 nucleotide.

The term "sense strand" as used herein refers to a strand of a dsRNA comprising a region substantially complementary to a region of the antisense strand.

The term "subject" or "individual" as used herein means a mammal, particularly a primate, particularly a human.

As used herein, "treating" an individual having a disease or condition means that the individual's symptoms are partially or fully alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment and/or cure. Prevention refers to prevention of the underlying disease and/or prevention of worsening of symptoms or disease progression. Treatment also includes any dsRNA provided, expression vectors, and any pharmaceutical use of the compositions provided herein.

As used herein, "therapeutic effect" means an effect resulting from treatment of an individual that alters, typically ameliorates or improves a symptom of a disease or disease condition, or cures the disease or disease condition.

As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect upon administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, block, or partially block the symptoms of the disease or disorder. For example, if a given clinical treatment that reduces a measurable parameter associated with a disease or condition by at least 25% is considered an effective treatment, then a therapeutically effective amount of a drug for treating the disease or condition is the amount necessary to reduce the parameter by at least 25%.

The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent (e.g., dsRNA). Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

As used herein, "expression vector" includes a vector capable of expressing a nucleotide sequence of interest operably linked to regulatory sequences capable of effecting the expression of such DNA fragments, such as a promoter region. Such additional fragments may include promoter and terminator sequences, and optionally may include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like.

As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter may be operably linked to a nucleotide sequence encoding a dsRNA such that the promoter regulates or mediates transcription of the nucleotide sequence.

Nucleic acid molecules targeting CKIP-1

The inventor designs, synthesizes and screens dsRNA molecules capable of obviously inhibiting CKIP-1 expression. Surprisingly, the dsRNA molecules selected both reduce inflammation and promote bone repair, and thus can be effectively used for the treatment of Arthritis, such as Rheumatoid Arthritis (RA).

In one aspect, the invention provides a nucleic acid molecule, e.g., dsRNA molecule, targeting CKIP-1 comprising a sense strand selected from table 1 and a corresponding complementary antisense strand.

In some preferred embodiments, the nucleic acid molecule targeting CKIP-1 comprises sense and antisense strands in table 1 corresponding to si-TD060, si-TD062, si-TD066, si-TD068, si-TD070, si-TD074, si-TD080, si-TD082, si-TD089, si-TD096, si-TD137, si-TD140, si-TD141, si-TD143, si-TD176, si-TD178, si-TD181, si-TD362, si-TD364, si-378, si-TD726, si-TD730, si-7, si-10.

In some more preferred embodiments, the nucleic acid molecule targeting CKIP-1 comprises the sense and antisense strands in table 1 corresponding to si-TD137, si-TD141, si-TD176, si-7.

In some embodiments, the sense strand and/or antisense strand of the nucleic acid molecule additionally has a overhang of at least one nucleotide at the 3' end. For example, the sense strand and/or antisense strand additionally has a overhang of 1, 2 or 3 nucleotides at the 3' end. For example, in some embodiments, the overhang is TT (i.e., dTdT). In some embodiments, the sense strand and the antisense strand of the nucleic acid molecule comprise an additional overhang TT at the 3' end.

In some embodiments, the sense strand and/or antisense strand in the nucleic acid molecule comprises at least 1, e.g., 1 or 2, nucleotide substitutions. For example, the substitution is within 6, 5, 4, 3, or 2 nucleotides of the 5 'and/or 3' terminus. In some embodiments, the sense strand and the antisense strand of the nucleic acid molecule comprise 1 nucleotide substitution at the 3 'last nucleotide position of the sense strand and correspondingly the 5' first nucleotide position of the antisense strand. The substitution will result in a mismatch with the target sequence, however the mismatch as defined is tolerated without significantly or without affecting the activity of the dsRNA.

In some embodiments, the dsRNA of the invention comprises at least one modified nucleotide. The modified nucleotides may comprise modifications of the phosphate group, the ribose group and/or the base.

For example, modifications of the phosphate group in nucleotides include modifications to the oxygen in the phosphate group, such as phosphorothioate (phosphorothioate) and Boranophosphate (Boranophosphate) modifications. The oxygen in the phosphate group is replaced with sulfur and borane, respectively, as shown in the following formula. Both modifications stabilize the structure of the nucleic acid, maintaining high specificity and high affinity for base pairing.

The modifications to the 2 '-hydroxyl of the pentose sugar of the nucleotide include, but are not limited to, 2' -fluoro modification (2 '-fluoro modification), 2' -methoxy modification (2 '-OME), 2' -deoxyethyl modification (2 '-MOE), 2' -2, 4-dinitrophenol modification (2 '-DNP modification), locked nucleic acid modification (L NA modification), 2' -Amino modification (2 '-Amino modification), 2' -deoxyxy modification (2 '-deoxyribose modification), 3' -cholesterol modification (3 '-cholesterol modification), and 4' -thymidine modification (exemplified by the following structures:

modification of a base in a nucleotide refers to modification of a base in a nucleotide group, and the modification of a base enhances interaction of the base, thereby enhancing an effect on a target mRNA. For example, 5 ' -bromouracil (5 ' -bromo-uracil) and 5 ' -iodouracil (5 ' -iodo-uracil) modifications in which bromine or iodine is introduced into the 5 ' -position of uracil are commonly used base modification methods, and other examples include N3-methyluracil (N3-methyl-uracil) modification, 2,6-diaminopurine (2, 6-diaminopropurine) modification, and the like.

In some embodiments, the dsRNA of the invention comprises at least one modified nucleotide selected from the group consisting of: 2 ' -O-methyl modified nucleotides, 2 ' -F modified nucleotides, nucleotides comprising a 5 ' -phosphorothioate group and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, and/or, for example, the modified nucleotides are selected from the group consisting of: 2 ' -deoxy-2 ' -fluoro modified nucleotides, 2 ' -deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2 ' -amino-modified nucleotides, 2 ' -alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing non-natural bases. The at least one modified nucleotide may, for example, enhance the stability of the dsRNA and/or reduce the immunogenic effect of the dsRNA. The modified nucleotide may be on the sense strand and/or on the antisense strand.

In some embodiments, the dsRNA comprises at least one 2 '-O-methyl modified ribonucleotide and/or at least one nucleotide comprising a 5' -phosphorothioate group.

In some embodiments, the 2' hydroxyl group of the nucleotides of all uracil or cytosine bases in the sense strand and/or antisense strand of the dsRNA of the invention is modified with a methoxy group.

In some embodiments, the 2' hydroxyl groups of the nucleotides of all uracil or cytosine bases in the sense strand of the dsRNA of the invention are modified with a methoxy group.

In some embodiments, the 2' hydroxyl groups of all nucleotides in the sense strand and/or antisense strand of the dsRNA of the invention are modified with a methoxy group.

In some embodiments, the 2' hydroxyl groups of all nucleotides in the sense strand of the dsRNA of the invention are modified with methoxy groups.

In some embodiments, the 5' end of the sense strand and/or antisense strand of the dsRNA of the invention is phosphorylated.

In some embodiments, the sense strand and/or antisense strand of a dsRNA of the invention comprises a 3' cholesterol modification.

In some embodiments, the 2' hydroxyl groups of the nucleotide groups of all uracil bases or cytosine bases in the sense strand of the dsRNA of the invention are modified with fluorine (F).

In some embodiments, the dsRNA of the invention comprises a locked nucleic acid modification in the sense strand.

In some embodiments, all nucleotides in the sense strand and/or antisense strand of the dsRNA of the invention comprise a phosphorothioate modification.

In some embodiments, the dsRNA molecule is an siRNA.

In other embodiments, the dsRNA molecule is a shRNA (short hairpin RNA). It is within the ability of one skilled in the art to design suitable shrnas based on siRNA sequences.

The dsRNA of the invention can be obtained by techniques conventional in the art, such as solid phase synthesis or solution phase synthesis. Modified nucleotides can be introduced by using modified nucleotide monomers in the synthesis.

In another aspect, the invention provides an expression vector comprising a nucleotide sequence encoding a nucleic acid molecule of the invention, such as a dsRNA, wherein the nucleotide sequence is operably linked to a transcriptional regulatory element, such as a promoter or the like. A recombinant vector capable of expressing the dsRNA molecule can be delivered to and persist in a target cell. Alternatively, vectors can be used which provide for transient expression of the nucleic acid molecule. If desired, the vector may be administered repeatedly. Once expressed, the dsRNA molecule interacts with the target mRNA and produces an RNA interference response. In general, shRNAs are particularly suitable for generation in this way.

The expression vector may be a linear construct, a circular plasmid vector, or a viral vector (including but not limited to adenovirus, adeno-associated virus, lentiviral vectors, and the like). In the case of siRNA, a single strand of siRNA can be transcribed by promoters on two separate expression vectors; alternatively, each single strand of the siRNA may be transcribed from a promoter that are all located on the same expression plasmid. In the case of shRNA, shRNA strands are transcribed from a single expression vector.

The promoter driving expression of the dsRNA in the expression vector of the invention can be a eukaryotic RNA polymerase I (e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV early promoter or actin promoter or U1snRNA promoter), or generally an RNA polymerase III promoter (e.g., U6snRNA or 7SKRNA promoter) or a prokaryotic promoter (e.g., T7 promoter, provided that the expression vector also encodes T7RNA polymerase required for transcription of the T7 promoter).

The dsRNA can remarkably inhibit the expression of CKIP-1 in cells. In some embodiments, expression of CKIP-1 is inhibited by 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 even 100%. Preferably, the dsRNA of the invention is capable of inhibiting the expression of CKIP-1 by at least 50%. More preferably, the dsRNA of the invention is capable of inhibiting the expression of CKIP-1 by at least 70%.

When the terms "inhibit expression", "down-regulate expression", "suppress expression", and the like are directed to the CKIP-1 gene, they refer herein to at least partially inhibiting expression of the CKIP-1 gene, as indicated by a decrease in the level of expression of CKIP-1 in a first cell or population of cells in which the CKIP-1 gene is transcribed and which has been treated such that expression of the CKIP-1 gene is inhibited, as compared to a second cell or population of cells which is substantially identical to the first cell or population of cells but which has not been so treated (control cells). The degree of inhibition is generally expressed in the following manner:

(control cell CKIP-1 expression level-treated cell CKIP-1 expression level)/control cell CKIP-1 expression level x 100%

Wherein the expression level may be an mRNA level or a protein level. It is clear to the person skilled in the art how to determine the mRNA level or the corresponding protein level of a particular gene.

Surprisingly, the dsRNA of the invention can also inhibit the expression of proinflammatory cytokines I L-6, TNF- α and/or I L-17A, particularly, the dsRNA of the invention can obviously inhibit the expression of proinflammatory cytokines I L-6.

In some embodiments, expression of the proinflammatory cytokines I L-6, TNF- α and/or I L-17A is inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, even 100%.

TABLE 1 dsRNA inhibiting CKIP-1 expression

Third, pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising at least one dsRNA of the invention or an expression vector comprising a nucleotide sequence encoding said dsRNA, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is for use in the treatment of inflammatory diseases, such as Arthritis, in particular Rheumatoid Arthritis (RA).

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, intra-articular or epicutaneous administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the dsRNA molecule, can be entrapped in a material, such as a liposome, to protect the compound from acids and other natural conditions that can inactivate the compound. In some embodiments, the dsRNA of the invention can be delivered by a cationic liposome delivery system.

Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like, (2) oil-soluble antioxidants such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), lecithin, propyl gallate, α -tocopherol, and the like, and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

These compositions may also contain, for example, preservatives, wetting agents, emulsifying agents and dispersing agents.

Prevention of the presence of microorganisms can be ensured by sterilization procedures or by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium oxide in the composition. Prolonged absorption of the injectable drug can be achieved by incorporating into the composition a delayed absorption agent, such as monostearate salts and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Conventional media or agents, except insofar as any is incompatible with the active compound, may be present in the pharmaceutical compositions of the invention. Additional active compounds may also be incorporated into the composition.

Therapeutic compositions generally must be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. For sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form is generally that amount of the composition which produces a therapeutic effect. Typically, this amount ranges from about 0.01% to about 99%, for example from about 0.1% to about 70%, for example, preferably from about 1% to about 30%, of the active ingredient, in combination with a pharmaceutically acceptable carrier, based on 100%.

Dosage regimens may be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be scaled down or up as required by the exigencies of the therapeutic condition. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifics of the dosage unit forms of the invention are defined and directly dependent upon (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of formulating such active compounds for use in the treatment of sensitivity in an individual.

For administration of the dsRNA molecules of the invention, the dosage range can be from about 0.0000001 to 100mg/kg of recipient body weight. Exemplary treatment regimens may be weekly, biweekly, every three weeks, every four weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, even every 12 months, or slightly shorter initial dosing intervals (e.g., weekly to every three weeks) followed by longer dosing intervals (e.g., monthly to even every 12 months).

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain amounts of the active ingredients effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without toxicity to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in conjunction with the particular composition employed, the age, sex, weight, condition, general health and medical history of the patient being treated, and like factors well known in the medical arts.

Fourth, disease treatment

The inventor designs more than 200 siRNA molecules aiming at CKIP-1 gene, and screens out the siRNA molecules which can obviously inhibit CKIP-1 expression (example 1-3). Experiments show that the CKIP-1 molecule can obviously inhibit the expression of CKIP-1 protein in human osteoblasts (example 5), and the dsRNA can promote the expression of phenotype genes of the human osteoblasts, thereby promoting the differentiation of the osteoblasts (examples 6 and 7). In mouse and cynomolgus models, administration of the dsRNA of the invention can significantly ameliorate arthritis progression (examples 8 and 9).

More surprisingly, the inventors found that the dsRNA of the invention can inhibit the expression of proinflammatory cytokines I L-6, TNF- α and/or I α 0-17A (examples 4 and 8). The dsRNA of the invention can significantly inhibit the expression of proinflammatory cytokines I α 1-6. TNF- α is mainly expressed by macrophages, synovial lining cells and activated T cells of inflamed joints. in RA inflamed joints, TNF- α is one of the most predominant proinflammatory cytokines and can induce the production of other proinflammatory factors such as I L-1 β, I L-6 and I L-8. in the induction process of CIA, I L-6 receptor neutralizing antibodies can completely eliminate inflammatory responses, indicating that I L-6 plays an important role in the process of initiating arthritis.

Previous pharmaceutical research on CKIP-1 has mainly focused on inhibiting bone destruction or repairing bone injury, and the present inventors have for the first time found that the dsRNA targeting CKIP-1 of the present invention is capable of inhibiting the expression of pro-inflammatory cytokines and thus can be used to treat inflammation. The ability of the dsRNA of the invention to target CKIP-1 to inhibit inflammation is particularly advantageous in the treatment of arthritis, particularly rheumatoid arthritis. Since in RA, the early symptoms are mainly joint inflammation, bone destruction occurs several years later (referred to as "late bone destruction"). The dsRNA targeting CKIP-1 can simultaneously inhibit inflammation and repair bone injury, and can be advantageously used in various stages of RA treatment without limitation to later bone destruction.

Thus, in another aspect, the invention provides a method of treating arthritis, in particular rheumatoid arthritis, in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention.

In another aspect, the invention also provides the use of a dsRNA of the invention or an expression vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of arthritis, in particular rheumatoid arthritis, in a subject in need thereof.

Arthritis that can be treated by the dsRNA molecule of the invention or the expression vector of the invention or the pharmaceutical composition of the invention includes, but is not limited to, rheumatoid arthritis, osteoarthritis, idiopathic arthritis, ankylosing spondylitis, psoriatic arthritis, infectious arthritis, Juvenile arthritis (Juvenile arthritis), reactive arthritis, gouty arthritis, and the like.

The dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention may also be used in combination with additional therapeutic agents for the treatment of arthritis, in particular rheumatoid arthritis. Such additional therapeutic agents include, but are not limited to, non-steroidal anti-inflammatory drugs, hormones, anti-rheumatic drugs, and the like.

In another aspect, the invention provides a method of treating a proinflammatory cytokine (e.g., I L-6, TNF- α and/or I L-17A) -associated inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention.

In another aspect, the invention also provides the use of a dsRNA of the invention or an expression vector of the invention or a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of an inflammatory disease associated with a pro-inflammatory cytokine (e.g., I L-6, TNF- α, and/or I L-17A) in a subject in need thereof.

Inflammatory diseases associated with the proinflammatory cytokines (e.g., I L-6, TNF- α and/or I L-17A) include, but are not limited to, inflammatory bowel disease, inflammation caused by infection, inflammation caused by injury, inflammation of the respiratory system, inflammation associated with cancer, etc. inflammatory diseases associated with the proinflammatory cytokines (e.g., I L-6, TNF- α and/or I L-17A) also include arthritis, such as the arthritis listed above, particularly rheumatoid arthritis.

Inflammatory diseases associated with other proinflammatory cytokines (e.g., I L-6, TNF- α, and/or I L-17A) that can be treated by a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention include, but are not limited to, systemic lupus erythematosus, crohn's disease, psoriasis, colitis, ileitis, glomerulonephritis, asthma, dermatitis (including contact dermatitis and atopic dermatitis), vasculitis, chronic bronchitis, chronic prostatitis, appendicitis, pancreatitis, pelvic inflammatory disease, polymyositis, chronic obstructive pulmonary disease, and the like.

The dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention may also be used in combination with additional therapeutic agents for the treatment of inflammatory diseases, in particular inflammatory diseases associated with pro-inflammatory cytokines (e.g., I L-6, TNF- α and/or I L-17A). The additional therapeutic agents are, for example, inhibitors targeting TNF- α, including but not limited to infliximab, etanercept, adalimumab, golimumab and certolizumab ozolomide, I L-6 blockers, including but not limited to tolizumab (Tocilizumab), I L-17A blockers, including but not limited to secukinumab.

In another aspect, the present invention provides a method of treating a bone metabolism-related disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention.

In another aspect, the invention also provides the use of a dsRNA of the invention or an expression vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for a bone metabolism related disease in a subject in need thereof.

The bone metabolism-related diseases include, but are not limited to, osteomalacia, osteo-deficiency, osteolytic bone disease, renal bone disease, osteogenesis imperfecta, bone destruction caused by cancer bone metastasis, and the like.

The dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention may also be used in combination with additional therapeutic agents for the treatment of diseases associated with bone metabolism.

In another aspect, the invention provides a method of reducing the level of a proinflammatory cytokine (e.g., I L-6, TNF- α, and/or I L-17A) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention.

Preferably, in each of the above aspects of the invention, the subject is a human.

In some embodiments, the dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention is administered intra-articularly. In some embodiments, the dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention is administered systemically.

Examples

A further understanding of the present invention may be obtained by reference to certain specific examples which are set forth herein and are intended to be illustrative of the invention only and are not intended to limit the scope of the invention in any way. Obviously, many modifications and variations of the present invention are possible without departing from the spirit thereof, and these modifications and variations are therefore also within the scope of the invention as claimed.

Example 1 sequence design and Synthesis of siRNA targeting CKIP-1

Designing candidate siRNA according to homologous regions of human CKIP-1mRNA and monkey CKIP-1mRNA sequences to obtain a candidate siRNA sequence pool, comprehensively analyzing off-target effects of the candidate siRNA sequence pool, finally rejecting candidate siRNA sequences with high off-target scores by combining seed region matching scores, and obtaining and synthesizing 208 siRNA candidate sequences aiming at CKIP-1. 8 unrelated NC sequences were additionally designed and synthesized as negative controls in the screening assay. The synthesized gene siRNA sequences of 208 entries, and 8 NC sequences are shown in FIG. 1.

Example 2 screening of siRNA inhibiting CIKP-1 expression Using real-time quantitative PCR

The expression method comprises the following steps of inoculating an hFOB cell (human osteoblast strain purchased from a Chinese academy) into a 96-well cell culture plate, and carrying out siRNA transfection when the cell density reaches about 70%, diluting 0.5 mu l of L ipofanamine 2000 into 25 mu l of opti-MEM containing no serum and antibiotics, fully mixing, diluting 15pmol of RNA into 25 mu l of opti-MEM containing no serum and antibiotics, gently mixing, adding L ipofanamine 2000 diluent into RNA diluent, fully mixing, standing at room temperature for 20min, adding 50 mu l of L ipofanamine 2000 and RNA mixing into the 96-well cell plate inoculated with cells, gently shaking the siRNA to mix uniformly, changing the solution after 5h, extracting RNA after 48 h (a day root microRNA extraction kit), carrying out reverse transcription (TaqCIra kit), carrying out PCR detection (full-format gold PCR) by using a GADPCIH gene kit as an internal reference, and obtaining relative normalized expression values of each relative expression sequence of each negative primer set from KP-1 NC-NC as follows:

CIKP1-F：GGAACCAACCTCTTGTGCTG

CIKP1-R：GTCAACTTCTTGGGTGCCTG

GADPH-F：CATGAGAAGTATGACAACAGCCT

GADPH-R：AGTCCTTCCACGATACCAAAGT

the results showed that 82 sequences with interference efficiencies of 50% and more (see table 2) and 22 sequences with interference efficiencies of 70% and more (shown in bold italics in table 2) were selected from 208 siRNA sequences. These sequences were candidates for further screening.

TABLE 2 siRNA with interference efficiency of 50% or more

siRNA sequences	Normalizing target gene relative expression values
si-TD037	0.472339
si-TD040	0.457801
si-TD042	0.422001
si-TD044	0.398672
si-TD050	0.307432
si-TD057	0.417976
si-TD058	0.412397
si-TD060	0.250549
si-TD061	0.314191
si-TD062	0.198302
si-TD064	0.46957
si-TD065	0.4389
si-TD066	0.30317
si-TD067	0.411764
si-TD068	0.252114
si-TD070	0.281898
si-TD072	0.401834
si-TD074	0.220171
si-TD076	0.334746
si-TD078	0.318811
si-TD080	0.23612
si-TD082	0.297076
si-TD084	0.356374
si-TD087	0.32098
si-TD089	0.238577
si-TD093	0.367916
si-TD094	0.410962
si-TD096	0.17883
si-TD097	0.409968
si-TD098	0.431926
si-TD136	0.356366
si-TD137	0.118522
si-TD138	0.387089
si-TD139	0.335127
si-TD140	0.235433
si-TD141	0.287318
si-TD143	0.169164
si-TD145	0.346415
si-TD176	0.223003
si-TD177	0.410735
si-TD178	0.172067
si-TD179	0.469953
si-TD181	0.224999
si-TD217	0.414913

si-TD221	0.462056
si-TD224	0.490381
si-TD358	0.387057
si-TD362	0.288363
si-TD364	0.275357
si-TD370	0.445778
si-TD372	0.459658
si-TD376	0.387624
si-TD378	0.295441
si-TD380	0.400417
si-TD443	0.396858
si-TD451	0.311861
si-TD480	0.460598
si-TD483	0.377209
si-TD508	0.476182
si-TD509	0.468754
si-TD577	0.424962
si-TD585	0.448536
si-TD587	0.410307
si-TD588	0.441516
si-TD596	0.497351
si-TD598	0.422082
si-TD600	0.487359
si-TD604	0.401307
si-TD607	0.375209
si-TD609	0.476541
si-TD611	0.457187
si-TD717	0.467227
si-TD718	0.450869
si-TD720	0.335688
si-TD723	0.411798
si-TD726	0.270674
si-TD730	0.252773
si-TD734	0.48745
si-TD736	0.416718
si-TD743	0.471836
si-1	0.506191
si-7	0.373945

Example 3 identification of candidate siRNA by Dual luciferase assay

In this example, candidate siRNA sequences selected in example 2 were further identified by dual luciferase assay.

1. Construction of target Gene CKIP-1 overexpression vector

The sequence fragment of CKIP-1 CDS 1-652 is obtained by PCR amplification with upstream and downstream primers respectively carrying SacI and XhoI cleavage sites and protective bases, the amplification product is digested by SacI and XhoI and then inserted into pGP-mirG L O overexpression vector (see figure 2) digested by SacI and XhoI, and pmirGlo-CDS1 vector is obtained, which overexpresses the sequence of the first segment 1-652 of the CDS region of CKIP-1 gene.

The sequence fragment of CKIP-1 CDS 653-1230 is obtained by PCR amplification by using upstream and downstream primers with SacI and XhoI restriction sites and protective bases respectively, the amplification product is digested by SacI and XhoI and then inserted into a pGP-mirG L O overexpression vector (shown in figure 2) digested by SacI and XhoI to obtain pmirGlo-CDS2 vector which overexpresses the sequence of the second segment 653-1230 of the CDS region of the CKIP-1 gene.

2. Cell culture

293T cells were cultured routinely in DMEM medium (Gibco) containing 10% FBS (Gibco) (1.5 mM L-glutamine, 100U/ml penicillin, 100. mu.g/ml streptomycin) in a 5% CO2 saturated humidity incubator at 37 ℃.

3. Cell transfection

293T cells are cultured in a 10cm culture dish until 80-90% of cells are fused, the culture solution is poured off, the cells are washed twice by 3ml of PBS, 1ml of Trypsin-EDTA solution is added, after uniform mixing, the pancreatin solution is carefully sucked off, the cells are placed for 2-3 minutes at 37 ℃, 2ml of complete culture medium is added, the cells are blown to form single cell suspension, the count is carried out by a blood counting plate, and the count is carried out according to the proportion that each hole is about 1 × 10⁵The cell amount of (a) was seeded in a 24-well plate.

Mu.l of Opti-MEM I (50. mu.l/well 3) was added to a 1.5ml EP tube, 30ng of the corresponding plasmid vector (10 ng per well), and the corresponding amount of siRNA (each siRNA was set to a gradient of final concentration: 6.25, 12.5, 25, 50, 100, 300nM, where negative control NC-7 was 25nM), mixed, 150. mu.l of Opti-MEM I (50. mu.l/well 3), and 6. mu.l of transfection reagent L ipo2000 were added to another 1.5ml EP tube, mixed after 5min of standing, the two were mixed, a total of 300. mu.l volume, and left at room temperature for 20min, medium was removed from the 24 well plates spread the day before, medium was added at 400. mu.l/well, after 20min of standing time, the transfection mixtures were added to the above 24 well plates, 100. mu.l/well, 3 replicates each, and the Blank wells were set, luciferase was added to the wells in a Blank chamber, shaken, washed, and the cells were rinsed after 6 hours.

4. Dual luciferase assay

The experimental materials and reagents include Dual-L mutual Reporter Assay System (Promega, E1960), PBS, 96-well white board (corning cat. #3912), and multi-label microplate detector (Perkinelmer EnSpire).

Removing the culture medium in a cell pore plate to be detected, washing and culturing cells by PBS, removing the PBS, adding 1 × P L B into the cell pore plate according to 100 mul/pore, gently shaking the cell pore plate at room temperature, cracking the cell pore plate for 15min, transferring cell lysate into a small centrifuge tube, centrifuging the cell lysate at 3000rpm for 3min, removing cell debris, taking 30 mul of supernatant, adding the supernatant into a 96-pore white plate, and adding a substrate according to the operation steps recommended by the instruction for detection.

As a result:

the 4 siRNAs have good inhibition effect on CKIP-1 expression in dual-luciferase assay: si-TD137, si-TD141, si-TD176 and si-7. The measurement results are shown in tables 3 to 6 and FIGS. 3 to 6, respectively.

TABLE 3 Dual luciferase assay results for si-TD137

TABLE 4 results of dual luciferase assay of si-TD141

TABLE 5 Dual luciferase assay results of si-TD176

TABLE 6 results of dual luciferase assay of si-7

Example 4 inhibition of proinflammatory cytokine expression by siRNA targeting CKIP-1

RAW264.7 mouse peritoneal macrophage cell line (purchased from cell bank of Chinese academy of sciences, Shanghai) was cultured in complete DMEM medium containing 10% fetal bovine serum, 100U penicillin and streptomycin, and cultured overnight in a constant temperature carbon dioxide (5%) incubator at 37 ℃ until the cell fusion degree reached 70-80% for experiments.

In vitro, the small interfering RNA aiming at CKIP-1 prepared in the above or a sense strand methoxy modified sequence thereof is used for transfecting mouse macrophages to serve as a drug treatment group (RNAi group), the cells are used as a transfection reagent group (MOCK group) by singly using a transfection reagent X-TremenE siRNA transfection reagent (purchased from Roche under the code of 4476093001), 3 experiments are repeated for at least 3 times in parallel in each group, when the mouse macrophages are transfected, L PS (purchased from Sigma under the code of L2630-10 MG) is added for stimulation for 6 hours after the small interfering RNA with the final concentration of 30 nM. is transfected for 24 hours, cell supernatants of each group are collected, proinflammatory cytokine secretion is detected, and the cells of each group are collected to detect the expression level of the proinflammatory cytokine mRNA.

Determination of the Effect of siRNA on the inhibition of TNF- α and I L-6 protein secretion

Detecting the inhibition efficiency of the secretion level of TNF- α and I L-6 in the cell supernatant by using an E L ISA method, specifically, using Mouse TNF alpha E L ISA(eBioscience, cat. No. 88-7324-88) and Mouse I L-6E L ISA(eBioscience, cat # 88-7064-88) kit, which was tested according to its instructions, and the concentrations of TNF- α and I L-6 were calculated by plotting a standard curve.

The cytokine inhibitory efficiency was calculated as follows:

the cytokine inhibitory efficiency was equal to [ (L PS group-treated group)/(L PS group-blank control group) ] × 100%.

The results of the measurements are shown in tables 7 and 8 below:

TABLE 7

Remarking: p <0.05 was statistically significantly different from the MOCK group.

TABLE 8

Remarking: p <0.05 was statistically significantly different from the MOCK group.

As can be seen from the table, each siRNA candidate and its methylation modification can inhibit L PS-induced I L-6 and TNF- α secretion of RAW264.7 mouse macrophage, and inhibit I L-6 secretion to a significant level.

Determination of the Effect of siRNA on the inhibition of TNF- α and I L-6 mRNA expression

TNF- α and I L-6 mRNA levels in the harvested RAW264.7 cells are detected by a real-time fluorescent quantitative PCR (real-time PCR) method, specifically, TRIzol reagent (Invitrogen, Cat. No. 15596018) is used for extracting total RNA of the cells, TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (All-formula gold, Cat. No. AT341-02) kit is used for reverse transcription Synthesis of cDNA, and the inhibition efficiency of siRNA to L PS-induced mouse peritoneal macrophage I L-6 and TNF- α expression is detected by a fluorescent quantitative PCR method.

GAPDH gene was used as an internal reference gene in Real-time PCR method, and the sequences of the primers used are shown in Table 9

TABLE 9

	Forward (5 '-3')	Reverse direction (5 '-3')
Mouse TNF- α	TCAGCGAGGACAGCAAGG	AGTGAGTGAAAGGGACAGAACC
Mouse I L-6	CCTTCTTGGGACTGATGCTG	TTGGGAGTGGTATCCTCTGTGA
Mouse GAPDH	CCTTCATTGACCTCAACTACATGG	CTCGCTCCTGGAAGATGGTG

In the fluorescent quantitative PCR method, the nucleic acid inhibition efficiency is calculated as follows:

the siRNA inhibition efficiency ═ [ (L PS group cytokine gene copy number/L PS group GAPDH gene copy number-treatment group cytokine gene copy number/treatment group GAPDH gene copy number)/(L PS group cytokine gene copy number/L PS group GAPDH gene copy number-blank control group cytokine gene copy number/blank control group GAPDH gene copy number) ] × 100%

The results are shown in Table 10:

watch 10

Remarking: p <0.05 was statistically significantly different from the MOCK group.

As can be seen from Table 10, each siRNA candidate significantly inhibited L PS-induced I L-6 mRNA expression of mouse macrophages, but did not significantly inhibit TNF- α mRNA expression.

It can be seen that the siRNA targeting CKIP-1 of the present invention can inhibit the levels of the proinflammatory cytokines I L-6 and TNF- α, particularly I L-6, thereby inhibiting inflammation, particularly I L-6 and/or TNF- α associated inflammation, such as in RA.

Example 5 inhibitory Effect of siRNA targeting CKIP-1 on CKIP-1 protein expression

Human osteoblast strain hFOB1.19, purchased from the cell bank of the Chinese academy of sciences, and the culture medium was DMEM-F12 medium (purchased from Gibco Co.) containing 10% fetal bovine serum. The human osteoblast line hfob1.19 was transferred to a 24-well plate for overnight culture to allow adherence. Human osteoblast-like cell strain hFOB1.19 was transfected with siRNA targeting CKIP-1 as a treatment group, and the above cells were transfected with nonspecific nucleic acid as a negative control group (NC group). Each group was replicated at least 3 times in 2 replicates. When human osteoblasts were transfected, the final concentration of nucleic acid was 20. mu.M. After 72 hours of transfection, cells were collected and the amount of CKIP-1 protein expression was measured.

The content of CKIP-1 protein in osteoblast-like cells was detected by immunoblotting according to the method described in the literature (molecular cloning, A laboratory Manual, scientific Press, 2005). The CKIP-1 antibody used for the immunoblot detection was purchased from Santa Cruz Biotechnology (cat # sc-376355), and the internal reference antibody was GADPH (Santa Cruz Biotechnology, cat # sc-166574).

In the immunoblotting method, the nucleic acid inhibitory activity was calculated as follows, according to the following equation, × 100% for the nucleic acid inhibitory activity [1- (light intensity value of treatment group CKIP-1 western blot band/light intensity value of treatment group GAPDH western blot band)/(light intensity value of control group CKIP-1 western blot band/light intensity value of control group GAPDH western blot band) ].

As a result: si-7 can obviously inhibit the expression of CKIP-1 protein in the human osteoblast strain hFOB1.19. There were statistical differences (P <0.05) compared to control NC. The measurement results are shown in Table 11.

TABLE 11

	Inhibition ratio (%) for CKIP-1 protein expression
NC	0.0
si-7	74.5*

Example 6 Effect of siRNA targeting CKIP-1 on osteoblast differentiation

In analogy to example 5, CKIP-1siRNA was assayed for the expression levels of human osteoblast strain hFOB1.19 phenotypic genes alkaline phosphatase (A L P), type I collagen (CO L1), Osteopontin (OPN), Bone Sialoprotein (BSP) and Osteocalcin (OC) mRNA as a function of time using the primers shown in Table 12. the assay results are shown in Table 13.

TABLE 12

Table 13:

p <0.05 was statistically different from the NC group.

As a result, A L P, CO L1A 1 and OPN begin to express in the early stage of osteoblast differentiation, and BSP and OC begin to express only in the stage of osteoblast maturation function, action 72h, the expression levels of A L P, CO L1, OPN, BSP and OC in the si-7 group are remarkably increased compared with those in the NC group.

Experimental results show that the siRNA targeting CKIP-1 can promote the expression of phenotype genes of a human osteoblast strain hFOB1.19, so that osteoblast differentiation can be promoted.

Example 7 Effect of siRNA targeting CKIP-1 on bone matrix mineralization deposition Rate

Calcium deposition is a key functional mineralization marker for mature osteoblasts during osteoblast formation in vitro. As described above, the human osteoblast strain hFOB1.19 was transfected with siRNA targeting CKIP-1 as a treatment group, and the above cells were transfected with nonspecific nucleic acid as a negative control group (NC group). The final concentration of nucleic acid at transfection was 20. mu.M. The frequency of gap transfection was once a week, with 4 replicates in each group. The amount of calcium deposited in the human osteoblast-like cell line hFOB1.19 was determined by calcium staining 7, 14 and 21 days after the first transfection.

The measurement results are shown in Table 14. After 21 days of first transfection of human osteoblasts, the calcium deposition amount of the treated group is obviously higher than that of the NC group, which verifies that the siRNA of the invention can promote differentiation of human preosteoblasts into mature osteoblasts on a functional level.

Table 14:

p < 0.05: there were statistical differences compared to the NC group.

Example 8 evaluation of siRNA in vivo Activity by mouse CIA model

A collagen-induced arthritis (CIA) model is established by subcutaneous injection of the tail root of a II type collagen in a male DBA mouse of 8-10 weeks old, and the specific method comprises the following steps of taking a proper amount of 2mg/m L bovine II type collagen, mixing the mixture with an equivalent amount of incomplete Freund's adjuvant, fully emulsifying, taking the emulsified mixture, and injecting 100 mu g of II type collagen per mouse at the tail root part of the tail.

The 5-grade semi-quantitative score standard is used as the judgment standard of the clinical severity of arthritis: 0: no red swelling; 1: erythema is associated with mild swelling and is confined to the midfoot or ankle joints; 2: mild swelling extends from the ankle joint to the midfoot; 3: moderate swelling extends from the ankle joint to the metatarsal joint; 4: the ankle joint, foot and toes are severely swollen.

When the severity of the double hind limbs meets the condition that the average value of the animal score of each group is about 1 point by visual evaluation, animals are randomly grouped: carrier group, injecting blank liposome into ankle joint cavity; NC (negative control) group, injecting liposome carrying negative control sequence into ankle joint cavity; a treatment group, injecting liposomes respectively loaded with Si-7, Si-137, Si-141 or Si-176 into the ankle joint cavity; positive control group, positive drug yisaipu (Etanercept, 12.5mg active ingredient per bag, purchased from shanghai china healthcare pharmaceutical limited) was administered.

Animals in each group were administered 6 consecutive hind limb bilateral ankle joint cavity injections from day 0, day 7, day 14, day 21, day 28 and day 35 of the study group at a dose of 4 μ g siRNA/5 μ l liposome/ankle joint. Wherein the positive drug Yisaipu is administered by subcutaneous injection at a dose of 7.5mg/kg body weight.

Effect of siRNA treatment on clinical scores and body weight of CIA mice

Mice were scored for double hind limb ankle swelling starting from the day of enrollment, 2 times per week, and statistically analyzed by the addition of double hind limb scores. The clinical scoring results are shown in figure 7 and table 15 below. At the same time, the body weight of the mice was recorded 1 time per week, and the results are shown in fig. 8 and table 16.

The results showed that the mice in each group gained weight and no weight loss occurred. Si-7-Ome, Si-137-Ome, Si-141-Ome and Si-176-Ome remarkably reduce the clinical score of arthritis of a mouse CIA model, the inhibition rates are respectively 50%, 60%, 70% and 60%, and the effect is better than that of a positive drug Yisaipu (40% inhibition).

TABLE 15 inhibition of mouse CIA clinical scores

Remarking: represents P <0.05 compared to the vehicle group, represents P < 0.01 compared to the vehicle group, and represents P < 0.001 compared to the vehicle group.

TABLE 16 Effect on weight changes in mice

Effect of siRNA treatment on proinflammatory factor expression in articular tissue of CIA mice

After the CIA model mouse dies, leg fur is cut off by scissors, ankle joint parts are exposed, parts below the knee joint are cut off by using bone tongs, the parts are ground by cooling with liquid nitrogen and transferred into an enzyme-free tube, cell total RNA is extracted by using a TRIzol reagent (Invitrogen, product number 15596018), cDNA is synthesized by reverse transcription by using a TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (All-formula gold, product number AT341-02) kit, and the inhibition efficiency of the CKK-7, Si-137, Si-141 and Si-176 on the expression of CKCKK-1, I L-6, TNF- α and I L-17A mRNA of the CIA model mouse joint tissues is detected by a fluorescence quantitative PCR method.

The primers used for I L-6, TNF- α, and the reference gene GAPDH were as above CKIP-1, I L-17A primers are shown in Table 17:

TABLE 17

	Forward (5 '-3')	Reverse direction (5 '-3')
Mouse I L-17A	CTCCACCGCAATGAAGACC	CCCTCTTCAGGACCAGGATC
Mouse CKIP-1	TTTCTCGGCCTTGGGAAAAAC	GAGGCACATCGGCTCTTCT

In the fluorescent quantitative PCR method, the expression inhibition efficiency was calculated as follows:

inhibition efficiency ([ (vector cytokine gene copy number/vector GAPDH gene copy number-treatment cytokine gene copy number/treatment GAPDH gene copy number)/(vector cytokine gene copy number/vector GAPDH gene copy number-normal control cytokine gene copy number/normal control GAPDH gene copy number) ] × 100%

The measurement results are shown in table 18 and fig. 9:

watch 18

Remarking: p <0.05, statistically significant difference compared to vehicle group; p < 0.01, with statistically significant differences compared to vehicle group; p < 0.001, with statistically significant differences compared to vehicle group; p < 0.0001, with statistically significant differences compared to vehicle group; the # P < 0.0001 has a statistically significant difference compared with the normal control group.

As can be seen, the si7-OMe, the si137-OMe, the si141-OMe and the si176-OMe all remarkably inhibit the expression of mRNA of joint tissues CKIP-1, I L-6, TNF- α and I L-17A of CIA mice, the inhibition rate is more than 50 percent, and the inhibition effect on the mRNA of CKIP-1 is stronger than that of the Yiseipu (41.37 percent) of positive drugs.

MicroCT detection

The detection of MicroCT is carried out by Scancoviva 40. And (3) placing the mouse hind paw into a Micro CT sample bulb tube for three-dimensional CT scanning reconstruction. After the scanning is finished, the trabecular bone three-dimensional microstructure is analyzed by adopting matched software, and the space structure parameters of the trabecular bone are collected.

4. Pathology detection

The hind limbs of the mice are fixed in 4% formaldehyde solution, and are embedded in paraffin after EDTA decalcification. And (4) continuously sectioning, and checking pathological changes of joints and bone erosion conditions by adopting HE staining.

5. Bone morphometric analysis

Mice were intraperitoneally injected with xylenol orange (90mg/kg) 12 days prior to sacrifice and calcein (10mg/kg) 2 days prior to sacrifice. After the mice were sacrificed, the hind paws were removed and discontinuous 10 μm sections were made using a non-decalcifying microtome, and the sections were stained with 1% methylene blue and observed with an optical microscope, while the unstained sections were observed with a fluorescence microscope. The metatarsals in the paw were used for bone morphometry analysis.

Compared with a model control group, each siRNA administration group plays a positive role in improving inflammation and bone injury of a rheumatoid arthritis model and delaying disease progression, and shows a good treatment effect.

Example 9 validation of siRNA Effect in monkey rheumatoid arthritis model

1. Animal molding and administration

The experiment selects 3-6 years old female normal cynomolgus monkey, the collagen induces arthritis modeling method is referred to the relevant literature, and the cynomolgus monkey receives bovine II type collagen immunity on the 0 th day and the 21 st day respectively. After the onset of disease, the drug is administered in the form of topical administration to the joint. Small nucleic acids employ liposome delivery systems.

The grouping is as follows: carrier group, injecting blank liposome into joint cavity; NC (negative control) group, injecting liposome carrying negative control sequence into ankle joint cavity; treatment group, injecting liposome respectively loaded with Si-7, Si-137, Si-141 or Si-176 into articular cavity; positive control group, positive drug yisaipu (Etanercept, purchased from china pharmaceuticals, ltd., shanghai) was administered. Each group of 3 animals was administered by intra-articular injection once a week for 6 consecutive weeks.

2. Index detection

Detection by MicroCT, pathology, bone morphology, etc. is similar to mouse experiments.

Compared with animals in a control group, the siRNA administration group shows good treatment effect on the aspects of improving the disease condition, particularly reducing bone injury, maintaining bone function and the like on a rheumatoid joint model.

Claims (26)

1. A double-stranded rna (dsrna) molecule comprising a sense strand and an antisense strand selected from the group consisting of:

   1) the sense strand shown in SEQ ID NO. 63 and the antisense strand shown in SEQ ID NO. 64;

   2) the sense strand shown in SEQ ID NO. 71 and the antisense strand shown in SEQ ID NO. 72;

   3) the sense strand shown as SEQ ID NO. 83 and the antisense strand shown as SEQ ID NO. 84; and

   4) the sense strand shown in SEQ ID NO. 161 and the antisense strand shown in SEQ ID NO. 162.
2. The dsRNA molecule of claim 1, the sense strand and/or antisense strand additionally has a overhang of at least one nucleotide at the 3' end.
3. The dsRNA molecule of claim 2, said sense strand and/or antisense strand additionally having a overhang of 2 nucleotides at the 3' end, preferably said overhang is TT.
4. The dsRNA molecule of claim 1, wherein the sense strand and the antisense strand comprise 1 or 2 nucleotide substitutions within 6, 5, 4, 3, or 2 nucleotides of the 5 'and/or 3' terminus.
5. The dsRNA molecule of claim 4, wherein the sense strand and the antisense strand comprise 1 nucleotide substitution at the 3 'last nucleotide position of the sense strand and correspondingly the 5' first nucleotide position of the antisense strand.
6. The dsRNA molecule of any one of claims 1-5, comprising at least one modified nucleotide.
7. The dsRNA molecule of claim 6, said modified nucleotide being selected from the group consisting of: 2 '-O-methyl modified nucleotides, 2' -F modified nucleotides, nucleotides comprising a 5 '-phosphorothioate group and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, 2' -deoxy-2 '-fluoro modified nucleotides, 2' -deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2 '-amino-modified nucleotides, 2' -alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates and nucleotides containing a non-natural base.
8. The dsRNA molecule of claim 6, wherein the 2' hydroxyl groups of all nucleotides of uracil bases or cytosine bases in the sense strand and/or antisense strand are modified with a methoxy group.
9. The dsRNA molecule of any one of claims 1 to 8, which is an siRNA or shRNA.
10. The dsRNA molecule of any one of claims 1-9, which inhibits expression of CKIP-1 by at least 50%, preferably by at least 70%.
11. The dsRNA molecule of any one of claims 1-10, which inhibits expression of pro-inflammatory cytokines such as TNF- α, I L-6 and/or I L-17A.
12. An expression vector comprising a nucleotide sequence encoding the dsRNA molecule of any one of claims 1-11 operably linked to a transcriptional regulatory element.
13. A pharmaceutical composition comprising the dsRNA molecule of any one of claims 1-11 or the expression vector of claim 12, and a pharmaceutically acceptable carrier.
14. A method of treating arthritis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the dsRNA molecule of any one of claims 1-11 or the expression vector of claim 12 or the pharmaceutical composition of claim 13.
15. The method of claim 14, further comprising administering to the subject an additional therapeutic agent for treating arthritis.
16. Use of the dsRNA molecule of any one of claims 1-11 or the expression vector of claim 12 or the pharmaceutical composition of claim 13 in the manufacture of a medicament for treating arthritis in a subject in need thereof.
17. The method of claim 14 or 15, or the use of claim 16, wherein the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, idiopathic arthritis, ankylosing spondylitis, psoriatic arthritis, infectious arthritis, juvenile arthritis, reactive arthritis, gouty arthritis, preferably rheumatoid arthritis.
18. A method of treating an inflammatory disease, in particular a proinflammatory cytokine such as TNF- α, I L-6 and/or I L-17A-related inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the dsRNA molecule of any one of claims 1-11 or the expression vector of claim 12 or the pharmaceutical composition of claim 13.
19. The method of claim 18, further comprising administering to the subject an additional therapeutic agent for treating an inflammatory disease, in particular a proinflammatory cytokine such as TNF- α, I L-6 and/or I L-17A-related inflammatory disease.
20. Use of the dsRNA molecule of any one of claims 1-11 or the expression vector of claim 12 or the pharmaceutical composition of claim 13 for the preparation of a medicament for the treatment of an inflammatory disease, in particular a proinflammatory cytokine such as TNF- α, I L-6 and/or I L-17A associated inflammatory disease in a subject in need thereof.
21. The method of claim 18 or 19, or the use of claim 20, wherein the inflammatory disease is selected from the group consisting of inflammatory bowel disease, inflammation caused by infection, inflammation caused by injury, inflammation of the respiratory system, and inflammation associated with cancer.
22. The method of claim 18 or 19, or the use of claim 20, wherein the inflammatory disease is selected from systemic lupus erythematosus, crohn's disease, psoriasis, colitis, ileitis, glomerulonephritis, asthma, dermatitis (including contact dermatitis and atopic dermatitis), vasculitis, chronic bronchitis, chronic prostatitis, appendicitis, pancreatitis, pelvic inflammation, polymyositis, and chronic obstructive pulmonary disease.
23. A method of treating a bone metabolism-related disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the dsRNA molecule of any one of claims 1-11 or the expression vector of claim 12 or the pharmaceutical composition of claim 13.
24. The method of claim 23, further comprising administering to the subject an additional therapeutic agent for treating a bone metabolism-related disease.
25. Use of the dsRNA molecule of any one of claims 1-11 or the expression vector of claim 12 or the pharmaceutical composition of claim 13 for the manufacture of a medicament for treating a bone metabolism related disease in a subject in need thereof.
26. The method of claim 23 or 24, or the use of claim 25, wherein the bone metabolism-related disease is selected from the group consisting of osteomalacia, osteohalisteresis, osteolytic bone disease, renal bone disease, osteogenesis imperfecta and bone destruction caused by cancer bone metastases.