CN121844046A

CN121844046A - RNA compositions for delivery of incretins

Info

Publication number: CN121844046A
Application number: CN202480058244.7A
Authority: CN
Inventors: C·米库尔卡; R·菲舍尔; C·L·杜尔贝格; F·叶加内; C·莱因施; A·库恩
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-09-11
Filing date: 2024-09-11
Publication date: 2026-04-10
Also published as: TW202540154A; WO2025057088A1; AU2024343011A1

Abstract

The present disclosure provides compositions (e.g., pharmaceutical compositions) and related techniques (e.g., components thereof and/or methods related thereto) for delivering an incretin agent. The present disclosure provides, inter alia, methods of treatment for various diseases using polyribonucleotides encoding incretins.

Description

RNA compositions for delivery of incretins

RELATED APPLICATIONS

The present application claims priority and benefit from U.S. provisional patent application No. 63/662,890 filed on month 21 of 2024 and PCT application No. PCT/IB2023/059007 filed on month 9 11 of 2023, the entire contents of which are incorporated herein by reference.

Background

Obesity is the most common chronic disease worldwide, affecting now about 6.5 hundred million adults. Obesity is considered a starting and key contributor to pre-diabetes, type 2 diabetes (T2D, and complications thereof), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease and renal disease, and premature death. It is estimated that by 2030 the number of obese (BMI >30kg/m ²) people will be over one billion, of which about 10% will have severe class III obesity (BMI >40kg/m ²). Half of all people with obesity live in only nine countries, U.S., china, india, brazil, mexico, russian, egypt, germany and Turkish. In addition, childhood obesity has risen dramatically throughout the world. T2D, NAFLD, NASH, cardiovascular disease and kidney disease are also common and are not associated with obesity. There is a need to develop further therapies for the treatment and/or prevention of obesity and other related diseases.

Disclosure of Invention

Treatment of obesity and T2D with incretins and incretin mimetics, in particular GLP1 and GIP receptor agonists or combinations thereof, has shown great benefit for people suffering from the disease. The availability of this new class of active agents is limited due to production and cost limitations, thereby disabling the necessary treatment for millions of people. The present disclosure recognizes such drawbacks and provides a new therapeutic approach to delivering incretin mimetics through the use of the polyribonucleotide precursors of incretin, thereby eliminating the need to produce incretin itself. The present disclosure provides, inter alia, the polyribonucleotide precursors of incretins as molecular entities, their production, formulation and administration for the treatment of obesity and its sequelae, including T2D, early T1D, cardiovascular disease, kidney disease, NASH and NAFLD. The present disclosure also provides methods of using these agents to treat diseases, including T2D, early T1D, cardiovascular disease, kidney disease, NASH, and NAFLD, that are not associated with obesity. The present disclosure also provides methods of using these agents to treat the sequelae of NASH, including liver fibrosis and cirrhosis.

The present disclosure also recognizes that such treatment methods, i.e., delivery of polyribonucleotide precursors to incretins, present additional benefits over current therapies, including, but not limited to, greater accessibility to obese people who may not be available with existing products due to limited supply, high price, lack of medical insurance, lower injection volume formulations compared to commercially available peptide-based products, lower patient treatment discontinuation rates due to factors such as gastrointestinal side effects, and improved characteristics such as optimized pharmacokinetic profile.

Has wider accessibility to obese people who are not available with current products due to limited supply, high price, lack of health insurance, the present formulation requires lower injection volumes than commercially available peptide-based products, less frequent discontinuation of patient treatment due to factors such as gastrointestinal side effects, and improved properties such as improved pharmacokinetic profiles. In some embodiments, improved pharmacokinetics has the advantage of reduced frequency of administration by therapeutic agents having longer duration of action.

In one aspect, the present disclosure provides a composition comprising a polyribonucleotide encoding an incretin agent. In some embodiments, the incretin agent is a GLP1 receptor agonist. In some embodiments, the incretin agent is a GIP receptor agonist. In some embodiments, the incretin agent is a GLP1/GIP dual receptor agonist. In some embodiments, the incretin agent is a GLP1/GCG dual receptor agonist. In some embodiments, the incretin agent is a GLP1/GIP/GCG tri-receptor agonist.

In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence as set forth in any one of SEQ ID NOs 5-7, 63-64, 69-70 and 74-75. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence as set forth in any one of SEQ ID NOs 8-9, 62 and 72. In some embodiments, the incretin agent comprises an incretin peptide having the amino acid sequence set forth in SEQ ID NO. 11. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence as set forth in any one of SEQ ID NOs 12-14. In some embodiments, the incretin agent comprises an incretin peptide having the amino acid sequence set forth in SEQ ID NO. 15.

In some embodiments, the incretin peptide is optionally fused to the signal peptide through the N-terminus of the incretin peptide, optionally through a linker peptide. In some embodiments, the signal peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 16-39 and 65-67. In some embodiments, the signal peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 16-21 and 65-67. In some embodiments, the signal peptide has the amino acid sequence shown as SEQ ID NO. 17. In some embodiments, the signal peptide has the amino acid sequence shown as SEQ ID NO. 65. In some embodiments, the signal peptide has the amino acid sequence shown as SEQ ID NO. 66. In some embodiments, the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 41-45, 52-61 and 108-152.

In some embodiments, the incretin agent comprises an incretin peptide optionally fused to one or more additional incretin peptides by one or more linking peptides. In some embodiments, one or more of the connecting peptides comprises an amino acid sequence as set forth in any one of SEQ ID NOs 1-5, 68 or 156. In some embodiments, the incretin agent comprises an incretin peptide fused to two or more incretin peptides. In some embodiments, the incretin agent comprises at least one GLP1 receptor agonist and at least one GIP receptor agonist. In some embodiments, the incretin agent comprises at least two GLP1 receptor agonists. In some embodiments, the incretin agent comprises at least two GIP receptor agonists.

In some embodiments, the incretin agent comprises one or more furin cleavage sites (furin CLEAVAGE SITE). In some embodiments, one or more furin cleavage sites are located between adjacent incretin peptides. In some embodiments, one or more furin cleavage sites comprise the amino acid sequence shown as SEQ ID NO. 153. In some embodiments, the incretin agent comprises one or more units, each comprising, from N-terminus to C-terminus, a GLP1 receptor agonist-connecting peptide-furin cleavage site-GIP receptor agonist, e.g., wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 76, 77, 78, 79, 80, 81), two units (e.g., SEQ ID NO: 82), or four units (e.g., SEQ ID NO: 83). In some embodiments, the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 76-83, 94-97, 102-107.

In some embodiments, the incretin agent comprises a half-life extending moiety. In some embodiments, the half-life extending moiety comprises albumin (e.g., human serum albumin). In some embodiments, the human serum albumin comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO 159. In some embodiments, the human serum albumin comprises the amino acid sequence shown as SEQ ID NO. 159. In some embodiments, the incretin agent comprises albumin (e.g., human serum albumin) fused to one or more units, each comprising, from N-terminus to C-terminus, (i) a GLP1 receptor agonist-connecting peptide (e.g., SEQ ID NO: 98), (ii) a GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 100), (iii) a GLP1 receptor agonist-connecting peptide-furin cleavage site (e.g., SEQ ID NO: 102), or (iv) a GLP1 receptor agonist-connecting peptide-furin cleavage site-GIP receptor agonist, e.g., wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 104), two units (e.g., SEQ ID NO: 106), or four units (e.g., SEQ ID NO: 107). In some embodiments, the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 98, 100, 102, 104, 106, 107, or any combination thereof.

In some embodiments, the half-life extending moiety comprises an Albumin Binding Domain (ABD). In some embodiments, ABD is derived from protein G of Streptococcus (Streptococcus) strain GI48 and/or protein PAB of megagoldens (Finegoldia magna), such as ABD035 and SA21. In some embodiments, the half-life extending moiety comprises an ABD that binds to domain II of human serum albumin and does not overlap or interfere with binding to an FcRn binding site on albumin. In some embodiments, the half-life extending moiety comprises ABDCon. In some embodiments, the half-life extending moiety comprises an Albumin Binding Domain (ABD) derived from bacterial protein Sso7d, such as M11.12 and M18.2.5, from the hyperthermophilic archaea (hyperthermophilic archaeon) sulfolobus (Sulfolobus solfataricus). In some embodiments, the half-life extending moiety comprises a DARPin that binds albumin.

In some embodiments, the ABD comprises an immunoglobulin domain or fragment thereof that binds albumin. In some embodiments, the ABD comprises a fully human domain antibody (dAb), such as an AlbudAb, that binds albumin. In some embodiments, the ABD comprises an albumin binding Fab, such as dsFv CA645. In some embodiments, the ABD comprises a heavy chain only (VHH) antibody, such as a nanobody, that binds albumin. In some embodiments, a VHH antibody comprises a VHH domain having Complementarity Determining Region (CDR) sequences HCDR1, HCDR2 and/or HCDR3 as shown in SEQ ID NO: 191 (GFTLDYYA), SEQ ID NO: 192 (IASSGGST) and/or SEQ ID NO: 193 (AAAVLECRTVVRGYDY), respectively. In some embodiments, the VHH antibody comprises an amino acid sequence that has at least 90%, 95% or 99% identity to SEQ ID NO 154. In some embodiments, the VHH antibody comprises the amino acid sequence set forth in SEQ ID NO. 154. In some embodiments, the incretin agent comprises a VHH antibody that binds to albumin fused to a unit comprising, from N-terminus to C-terminus, (i) a GLP 1-connecting peptide (e.g., SEQ ID NO: 99), (ii) a GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 101), or (iii) a GLP1 receptor agonist-connecting peptide-furin-GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 103 or 105). In some embodiments, the incretin comprises an amino acid sequence as set forth in any one of SEQ ID NOs 99, 101, 103, 105.

In some embodiments, the half-life extending moiety does not comprise an Fc domain, such as from a human IgG, optionally from a human IgG1, igG2, igG3, or IgG4. In some embodiments, the half-life extending moiety comprises an Fc domain, such as from a human IgG, optionally from a human IgG1, igG2, igG3, or IgG4. In some embodiments, the human IgG is human IgG4. In some embodiments, the incretin agent comprises an IgG4 Fc domain fused to a unit comprising, from N-terminus to C-terminus, (i) a GLP1 receptor agonist-connecting peptide (e.g., SEQ ID NO: 10, 89, 90, 91), (ii) a GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 92, 93), or (iii) a GLP1 receptor agonist-connecting peptide-furin-GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 94, 95, 96, 97). In some embodiments, the IgG4 Fc domain comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO: 155. In some embodiments, the IgG4 Fc domain comprises the amino acid sequence shown as SEQ ID NO: 155. In some embodiments, the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 10 and 89-97.

In some embodiments, the Fc domain comprises one or more mutations in one or both Fc constant domains that increase the half-life of the incretin agent and/or induce dimerization. In some embodiments, the one or more mutations comprise one or more mutations in the CH3 domain. In some embodiments, the one or more mutations that induce dimerization comprise (i) Y349C, T366S, L368A and/or Y407V (according to EU numbering), or (ii) S354C and/or T366W (according to EU numbering). In some embodiments, the one or more mutations comprise Y349C, T366S, L A and Y407V ("FcKIH-b", according to EU numbering), or S354C and T366W ("FcKIH-a", according to EU numbering). In some embodiments, the incretin agent comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an incretin peptide fused to a first Fc domain, wherein the first Fc domain comprises mutations Y349C, T366S, L368A and Y407V ("FcKIH-b", according to EU numbering), and wherein the second polypeptide chain comprises an incretin peptide fused to a second Fc domain, wherein the second Fc domain comprises mutations S354C and T366W ("FcKIH-a", according to EU numbering).

In some embodiments, the one or more mutations that increase the half-life of the incretin agent comprise M428L and N434S ("LS", numbering according to EU). In some embodiments, the incretin agent comprises an Fc domain having a FcKIH-a mutation on a first polypeptide chain and an Fc domain having a FcKIH-b mutation on a second polypeptide chain, wherein the Fc domain on each polypeptide chain is independently fused to one or more units comprising, from N-terminus to C-terminus, (i) a GLP1 receptor agonist-connecting peptide (e.g., SEQ ID NO: 84, 85, 86, 87), or (ii) a GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 88). In some embodiments, the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 84-88.

In some embodiments, the Fc domain comprises one or more mutations that eliminate effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q). In some embodiments, the one or more mutations that eliminate effector activity of the Fc domain (e.g., binding to an Fc gamma receptor or C1 q) comprise the mutations L234S, L T and G236R ("STR", numbering according to EU). In some embodiments, the one or more mutations that eliminate effector activity of the Fc domain (e.g., binding to an Fc gamma receptor or C1 q) comprise mutations L234A and L235A ("LALA", numbering according to EU). In some embodiments, the one or more mutations that abrogate the effector activity of the Fc domain (e.g., binding to the Fc gamma receptor or C1 q) comprise the following mutations L234A/L235A/P329G ("LALAPG", numbering according to EU).

In some embodiments, the half-life extending moiety comprises a VNAR that binds albumin. In some embodiments, the half-life extending moiety comprises an XTEN sequence.

In some embodiments, the polyribonucleotide has a ribonucleic acid sequence that is at least 90% identical to any of SEQ ID NOS 177-185 and 224-256. In some embodiments, the polyribonucleotide has a ribonucleic acid sequence as shown in any one of SEQ ID NOs 177-185 and 224-256.

In some embodiments, the polyribonucleotide comprises at least one non-coding sequence component that enhances RNA stability and/or translation efficiency. In some embodiments, at least one non-coding sequence component comprises a 5' cap structure, a 5' UTR, a 3' UTR, and/or a polyA tail.

In some embodiments, the polyribonucleotide comprises in the 5 'to 3' direction an a.5 'UTR, b.signal peptide coding sequence, c.incretin peptide coding sequence, d.3' UTR, and e.polyA tail.

In some embodiments, the polyribonucleotide comprises in the 5 'to 3' direction (1) a 5 'UTR, b a signal peptide coding sequence, c an incretin peptide coding sequence, d a linker peptide coding sequence, e a half-life extending moiety coding sequence, f 3' UTR, and g.polyA tail, or (2) a 5 'UTR, b signal peptide coding sequence, c a half-life extending moiety coding sequence, d a linker peptide coding sequence, e an incretin peptide coding sequence, f 3' UTR, and g.polyA tail.

In some embodiments, the incretin peptide is encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to the wild-type coding sequence, wherein the codon optimized and/or increased G/C content does not alter the sequence of the encoded amino acid sequence.

In some embodiments, the polyribonucleotide comprises at least one modified ribonucleotide. In some embodiments, the polyribonucleotide comprises a modified nucleoside that replaces uridine. In some embodiments, the polyribonucleotide comprises a modified nucleoside that replaces each uridine. In some embodiments, the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

In some embodiments, the polyribonucleotide comprises a 5' cap structure. In some embodiments, the polyribonucleotide comprises a 5' UTR. In some embodiments, the polyribonucleotide comprises a 3' UTR. In some embodiments, the polyribonucleotide comprises a polyA tail. In some embodiments, the polyA tail comprises at least 100 nucleotides. In some embodiments, the polyribonucleotide is mRNA.

In some embodiments, the polyribonucleotide is formulated as a liquid, formulated as a solid, or a combination thereof. In some embodiments, the polyribonucleotides are formulated for injection. In some embodiments, the polyribonucleotides are formulated for intraperitoneal or intravenous administration.

In some embodiments, the polyribonucleotide is formulated or intended to be formulated as a lipid particle. In some embodiments, the polyribonucleotide is formulated or intended to be formulated as a lipid nanoparticle. In some embodiments, the polyribonucleotides are encapsulated within a lipid nanoparticle. In some embodiments, the lipid nanoparticle is a pancreatic and/or intestinal targeting lipid nanoparticle. In some embodiments, the lipid nanoparticle is a cationic lipid nanoparticle.

In some embodiments, the lipid forming the lipid nanoparticle comprises a. A polymer coupled lipid, b. A cationic lipid, and c. A neutral lipid. In some embodiments, the polymer-coupled lipid is a PEG-coupled lipid. In some embodiments, the cationic lipid is an ionizable lipid-like material (lipid).

In some embodiments, the cationic lipid has one of the following structures:

X-1

X-2

X-3

X-4。

In some embodiments, the neutral lipid comprises a helper lipid, such as 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DPSC) and/or cholesterol.

In some embodiments, the cationic lipid is selected from cationic lipids X-2, X-3, or X-4, and the neutral lipid comprises a helper lipid (such as DOTAP, DOPE, or PS) and cholesterol.

In some embodiments, the polymer-coupled lipid is C14-PEG2000.

In some embodiments, the lipid nanoparticle comprises i) about 30 mol% to about 50 mol% cationic lipid, ii) about 1 mol% to 5 mol% PEG conjugated lipid, iii) about 30 mol% to about 50 mol% helper lipid, and iv) about 20 mol% to about 40 mol% cholesterol.

In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid, about 40 mol% helper lipid, about 22.5 mol% cholesterol, and about 2.5 mol% PEG conjugated lipid.

In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-2, X-3, or X-4, about 40 mol% DOTAP, DOPE, or PS, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000.

In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-2, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-3, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-4, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-2, about 40. 40 mol% DOPE, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-3, about 40. 40 mol% DOPE, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-4, about 40. 40 mol% DOPE, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-2, about 40. 40 mol% PS, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-3, about 40. 40 mol% PS, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-4, about 40. 40 mol% PS, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000.

In some embodiments, the lipid nanoparticle is formulated for intraperitoneal (i.p.) delivery. In some embodiments, the lipid nanoparticle has an average size of about 50-150 nm. In some embodiments, the composition comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients. In some embodiments, the composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the composition comprises an aqueous buffer solution. In some embodiments, the aqueous buffer solution includes sodium ions.

In another aspect, the present disclosure provides a method of treating a disease state in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a composition comprising one or more of the polyribonucleotides described herein.

In some embodiments, the method further comprises administering one or more DPP-4 inhibitors. In some embodiments, one or more DPP-4 inhibitors and compositions are administered simultaneously. In some embodiments, the one or more DPP-4 inhibitors and the composition are administered sequentially. In some embodiments, one or more DPP-4 inhibitors are administered prior to the composition. In some embodiments, one or more DPP-4 inhibitors are administered after the composition. In some embodiments, the one or more DPP-4 inhibitors comprise sitagliptin (sitagliptin), vildagliptin (vildagliptin), saxagliptin (saxagliptin), linagliptin (linagliptin), gemagliptin (gemigliptin), alagliptin (anagliptin), tigliptin (TENELIGLIPTIN), alogliptin (alogliptin), trelagin (TRELAGLIPTIN), aogliptin (omarigliptin), ebagliptin (evogliptin), agogliptin (gosogliptin), duloxetine (dutogliptin), neogliptin (neogliptin), dulgliptin (retagliptin), dulgliptin (denagliptin), colagliptin (cofroglipin), fogliptin (fotagliptin), pragliptin (prusogliptin), huperzine (berberine), or any combination thereof. In some embodiments, the one or more DPP-4 inhibitors are administered orally.

In some embodiments, the disease state is obesity or an obesity-related disorder. In some embodiments, the obesity-related disorder is pre-diabetes, type 2 diabetes (T2D), early stage type 1 diabetes (T1D), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease, kidney disease, or an elevated risk of premature death. In some embodiments, cardiovascular (CV) disease comprises major cardiovascular events (MACEs), including CV death, non-fatal myocardial infarction, non-fatal stroke, and/or heart failure with ejection fraction retention (HFpEF).

In some embodiments, the method improves weight management of the individual. In some embodiments, the method reduces weight gain or induces weight loss in the subject. In some embodiments, the disease state is diabetes. In some embodiments, the method improves glycemic control in the individual. In some embodiments, the method reduces HbA1c of the individual. In some embodiments, the diabetes is pre-diabetes, type 2 diabetes (T2D), or early stage type 1 diabetes (T1D).

In some embodiments, the disease state is a Cardiovascular (CV) disease. In some embodiments, the cardiovascular disease comprises major cardiovascular events (MACEs), including CV death, non-fatal myocardial infarction, non-fatal stroke, and/or heart failure with ejection fraction retention (HfpEF). In some embodiments, the method improves blood pressure and/or blood lipid in the individual.

In some embodiments, the disease state is kidney disease. In some embodiments, the disease state is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the disease state is non-alcoholic steatohepatitis (NASH) and optionally its sequelae liver fibrosis and cirrhosis.

In some embodiments, administering the composition to the subject comprises administering one or more doses of the composition to the subject. In some embodiments, one or more doses of the composition are administered to the individual daily, every other day, or once a week. In some embodiments, one or more doses of the composition are administered to the individual less frequently than once a week. In some embodiments, one or more doses of the composition are administered to the individual every 2, 3, or 4 weeks. In some embodiments, the composition is administered by injection. In some embodiments, the composition is administered subcutaneously, intravenously, intramuscularly, or intraperitoneally. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered non-invasively (e.g., orally or nasally). In some embodiments, administration of the composition results in expression of the incretin agent in the subject. In some embodiments, the composition is administered in a volume of less than 0.5 mL.

In another aspect, the present disclosure provides the use of any of the compositions comprising one or more of the polyribonucleotides described herein for treating a disease state in a subject in need thereof.

In another aspect, the present disclosure provides a method of producing an incretin agent comprising administering to a cell a composition comprising a polyribonucleotide described herein such that the cell expresses and secretes the incretin agent.

In another aspect, the present disclosure provides an incretin agent comprising an incretin peptide fused to a signal peptide. In some embodiments, the incretin peptide is fused to the signal peptide through the N-terminus of the incretin peptide, optionally through a linker peptide. In some embodiments, the signal peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 16-39 and 65-67. In some embodiments, the signal peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 16-21 and 65-67. In some embodiments, the signal peptide has the amino acid sequence shown as SEQ ID NO. 17. In some embodiments, the signal peptide has the amino acid sequence shown as SEQ ID NO. 65. In some embodiments, the signal peptide has the amino acid sequence shown as SEQ ID NO. 66. In some embodiments, the incretin agent comprises an incretin peptide fused to a signal peptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs 41-45, 52-61 and 108-152.

In some embodiments, the incretin agent comprises an incretin peptide optionally fused to one or more additional incretin peptides by one or more linking peptides. In some embodiments, one or more of the connecting peptides comprises an amino acid sequence as set forth in any one of SEQ ID NOs 1-5, 68 or 156. In some embodiments, the incretin agent comprises an incretin peptide fused to two or more incretin peptides.

In some embodiments, the incretin agent comprises at least one GLP1 receptor agonist and at least one GIP receptor agonist. In some embodiments, the incretin agent comprises at least two GLP1 receptor agonists. In some embodiments, the incretin agent comprises at least two GIP receptor agonists. In some embodiments, the incretin agent comprises one or more furin cleavage sites. In some embodiments, one or more furin cleavage sites are located between adjacent incretin peptides. In some embodiments, one or more furin cleavage sites comprise the amino acid sequence shown as SEQ ID NO. 153. In some embodiments, the incretin agent comprises one or more units, each comprising, from N-terminus to C-terminus, a GLP1 receptor agonist-connecting peptide-furin cleavage site-GIP receptor agonist, e.g., wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 76, 77, 78, 79, 80, 81), two units (e.g., SEQ ID NO: 82), or four units (e.g., SEQ ID NO: 83). In some embodiments, the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 76-83, 94-97, 102-107.

In some embodiments, the incretin agent comprises a half-life extending moiety. In some embodiments, the half-life extending moiety comprises albumin (e.g., human serum albumin). In some embodiments, the human serum albumin comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO 159. In some embodiments, the human serum albumin comprises the amino acid sequence shown as SEQ ID NO. 159.

In some embodiments, the incretin agent comprises albumin (e.g., human serum albumin) fused to one or more units, each comprising, from N-terminus to C-terminus, (i) a GLP1 receptor agonist-connecting peptide (e.g., SEQ ID NO: 98), (ii) a GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 100), (iii) a GLP1 receptor agonist-connecting peptide-furin cleavage site (e.g., SEQ ID NO: 102), or (iv) a GLP1 receptor agonist-connecting peptide-furin cleavage site-GIP receptor agonist, e.g., wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 104), two units (e.g., SEQ ID NO: 106), or four units (e.g., SEQ ID NO: 107). In some embodiments, the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 98, 100, 102, 104, 106, 107, or any combination thereof.

In some embodiments, the half-life extending moiety comprises an Albumin Binding Domain (ABD). In some embodiments, ABD is derived from protein G of streptococcus strain GI48 and/or protein PAB of megagoldens, such as ABD035 and SA21. In some embodiments, the half-life extending moiety comprises an ABD that binds to domain II of human serum albumin and does not overlap or interfere with binding to an FcRn binding site on albumin. In some embodiments, the half-life extending moiety comprises ABDCon. In some embodiments, the half-life extending moiety comprises an ABD derived from a bacterial protein Sso7d, such as M11.12 and M18.2.5, from the hyperthermophilic archaea sulfolobus. In some embodiments, the half-life extending moiety comprises a DARPin that binds albumin. In some embodiments, the ABD comprises an immunoglobulin domain or fragment thereof that binds albumin. In some embodiments, the ABD comprises a fully human domain antibody (dAb), such as an AlbudAb, that binds albumin. In some embodiments, the ABD comprises an albumin binding Fab, such as dsFv CA645.

In some embodiments, the ABD comprises a heavy chain only (VHH) antibody, such as a nanobody, that binds albumin. In some embodiments, a VHH antibody comprises a VHH domain having Complementarity Determining Region (CDR) sequences HCDR1, HCDR2 and/or HCDR3 as shown in SEQ ID NO: 191 (GFTLDYYA), SEQ ID NO: 192 (IASSGGST) and/or SEQ ID NO: 193 (AAAVLECRTVVRGYDY), respectively. In some embodiments, the VHH antibody comprises an amino acid sequence that has at least 90%, 95% or 99% identity to SEQ ID NO 154. In some embodiments, the VHH antibody comprises the amino acid sequence set forth in SEQ ID NO. 154. In some embodiments, the incretin agent comprises a VHH antibody that binds to albumin fused to a unit comprising, from N-terminus to C-terminus, (i) a GLP 1-connecting peptide (e.g., SEQ ID NO: 99), (ii) a GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 101), or (iii) a GLP1 receptor agonist-connecting peptide-furin-GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 103 or 105). In some embodiments, the incretin comprises an amino acid sequence as set forth in any one of SEQ ID NOs 99, 101, 103, 105.

In some embodiments, the half-life extending moiety does not comprise an Fc domain, such as from a human IgG, optionally from a human IgG1, igG2, igG3, or IgG4. In some embodiments, the half-life extending moiety comprises an Fc domain, such as from a human IgG, optionally from a human IgG1, igG2, igG3, or IgG4. In some embodiments, the human IgG is human IgG4. In some embodiments, the incretin agent comprises an IgG4 Fc domain fused to a unit comprising, from N-terminus to C-terminus, i) a GLP1 receptor agonist-connecting peptide (e.g., SEQ ID NO: 10, 89, 90, 91), (ii) a GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 92, 93), or (iii) a GLP1 receptor agonist-connecting peptide-furin-GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 94, 95, 96, 97). In some embodiments, the IgG4 Fc domain comprises an amino acid sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO 155. In some embodiments, the IgG4 Fc domain comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 10, 89-97.

In some embodiments, the Fc domain comprises one or more mutations in one or both Fc constant domains that increase the half-life of the incretin agent and/or induce dimerization. In some embodiments, the one or more mutations comprise one or more mutations in the CH3 domain. In some embodiments, the one or more mutations that induce dimerization comprise (i) Y349C, T366S, L368A and/or Y407V (according to EU numbering), or (ii) S354C and/or T366W (according to EU numbering).

In some embodiments, the one or more mutations comprise Y349C, T366S, L A and Y407V ("FcKIH-b", according to EU numbering), or S354C and T366W ("FcKIH-a", according to EU numbering). In some embodiments, the incretin agent comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an incretin peptide fused to a first Fc domain, wherein the first Fc domain comprises mutations Y349C, T366S, L368A and Y407V ("FcKIH-b", according to EU numbering), and wherein the second polypeptide chain comprises an incretin peptide fused to a second Fc domain, wherein the second Fc domain comprises mutations S354C and T366W ("FcKIH-a", according to EU numbering).

In some embodiments, the half-life extending moiety comprises a VNAR that binds albumin.

In some embodiments, the half-life extending moiety comprises an XTEN sequence.

In another aspect, the present disclosure provides an incretin agent comprising husec a signal peptide, an incretin peptide comprising a GLP1 incretin peptide or a fragment or mutant thereof, wherein the GLP1 incretin peptide comprises an amino acid sequence having an A8G substitution mutation compared to the wild-type GLP1 amino acid sequence.

In another aspect, the present disclosure provides a polyribonucleotide encoding an incretin agent comprising husec signal peptide, an incretin peptide comprising a GLP1 incretin peptide or fragment or mutant thereof, wherein the GLP1 incretin peptide comprises an amino acid sequence having an A8G substitution mutation compared to the wild-type GLP1 amino acid sequence.

In another aspect, the present disclosure provides an incretin agent comprising husec signal peptide, an incretin peptide comprising a GIP incretin peptide or a fragment or mutant thereof, wherein the GIP incretin peptide comprises an amino acid sequence having an A2G substitution mutation compared to the wild-type GIP amino acid sequence.

In another aspect, the present disclosure provides a polyribonucleotide encoding an incretin agent comprising husec signal peptide, incretin peptide comprising a GIP incretin peptide or fragment or mutant thereof, wherein the GIP incretin peptide comprises an amino acid sequence having an A2G substitution mutation compared to the wild-type GIP amino acid sequence.

Drawings

FIG. 1 illustrates an exemplary therapeutic strategy for delivering and in vivo expressing an incretin agent using polyribonucleotides as described herein.

FIG. 2 shows a schematic representation of exemplary polyribonucleotides encoding incretins.

Fig. 3 illustrates an exemplary design of an incretin agent as described herein. In particular, FIG. 3 shows a schematic representation of the polyribonucleotides encoding a signal peptide ("SP") and a single incretin peptide (this configuration is referred to herein as "I:1 x") (top) and a schematic representation of the translated incretin protein (bottom).

Fig. 4 illustrates an exemplary design of an incretin agent as described herein. Specifically, FIG. 4 shows a schematic representation of a polyribonucleotide encoding a signal peptide ("SP") and two incretin peptides separated by a linker peptide ("L1") and a furin cleavage site ("F") (this configuration is referred to herein as "I: 2X") and a schematic representation of the translated protein (bottom).

Fig. 5 illustrates an exemplary design of an incretin agent as described herein. Specifically, FIG. 5 shows a schematic representation of the polyribonucleotides (top) and a schematic representation of the translated protein (bottom) encoding a signal peptide ("SP") and four incretin peptides each separated by a linker peptide ("L1") furin cleavage site ("F"), a configuration referred to herein as "I:4 x".

Fig. 6A-6B illustrate exemplary incretins, including incretins having a signal peptide ("SP"), a GLP1 incretin peptide and a (GGGGS) ₂ linker peptide (fig. 6A), and incretins having a signal peptide ("SP"), a GLP1 incretin peptide, (GGGGS) ₂ linker peptide, a furin cleavage site and a GIP incretin peptide (fig. 6B). The signal peptide cleavage sites are indicated in fig. 6A and 6B. In addition, furin cleavage sites are indicated in fig. 6B, such that upon expression, the GIP incretin peptide is cleaved from the GLP1 incretin peptide.

Fig. 7 illustrates an exemplary design of an incretin agent as described herein. In particular, FIG. 7 shows a schematic representation (top) of a polyribonucleotide encoding a signal peptide ("SP") and an incretin agent (e.g., I:1x, I:2x, or I:4x incretin agent as described herein) fused to a half-life extension ("HLE") domain, such as human serum albumin ("HSA") or albumin binding domain ("ABD"), via a linker peptide ("L2"), and a schematic representation (bottom) of a translated protein.

Figures 8A-8B illustrate exemplary incretins that can be encoded by the polyribonucleotides described herein, including more than one incretin peptide and a half-life extending (HLE) domain. FIG. 8A shows an incretin agent having a signal peptide ("SP"), a first GLP1 incretin peptide, a linker peptide, a second GLP1 incretin peptide, a second linker peptide (GGGGS) ₃, and a half-life extending (HLE) domain that is Human Serum Albumin (HSA). FIG. 8B shows an incretin agent having a signal peptide ("SP"), a first GLP1 incretin peptide, a linker peptide, a second GLP1 incretin peptide, a second linker peptide (GGGGS) ₃, and a half-life extension (HLE) domain of a VHH domain that binds to HSA. Furin and SP cleavage sites within the incretin agent are indicated with arrows such that upon expression, the signal peptide is cleaved and the first GLP1 incretin peptide is cleaved from the second GLP1 incretin peptide, and the second GLP1 incretin peptide remains fused to the HLE domain (HSA or anti-HSA VHH). This design produces two incretin peptides with different half-lives and activities.

Fig. 9 illustrates an exemplary incretin agent that may be encoded by one or more of the polyribonucleotides described herein, including more than one incretin peptide and a half-life extending (HLE) domain. Specifically, the incretin in fig. 9 has a signal peptide ("SP"), a first GLP1 incretin peptide, a connecting peptide (GGGGS) ₂, a first GIP incretin peptide, a second connecting peptide (GGGGS) ₂, a second GLP1 incretin peptide, a third connecting peptide (GGGGS) ₂, a second GIP incretin peptide, a fourth connecting peptide (GGGGS) ₃, and a half-life extension (HLE) domain that is Human Serum Albumin (HSA). Furin and SP cleavage sites within the incretins are indicated by arrows. This design resulted in four separate incretin peptides, with the second GIP incretin peptide remaining fused to the HLE domain.

FIG. 10 shows an exemplary design of polyribonucleotides encoding an incretin agent comprising an incretin peptide fused to an Fc domain, wherein the incretin peptide may be one (I: 1 x), two (I: 2 x) or four (I: 4 x) incretin peptides (top). When two of the polyribonucleotides encoding two separate strands are expressed, the two polypeptide strands combine and produce a dimeric (e.g., homodimeric) structure (bottom). Each polypeptide chain also includes a Signal Peptide (SP) and a linking peptide (L2). In some embodiments, the Fc domain includes mutations that abrogate effector function (e.g., mutations such as STR, LALA, LALAPG) and/or mutations that extend half-life (e.g., mutations such as YTE, LS, etc.).

FIG. 11 illustrates an exemplary incretin agent that may be encoded by one or more of the polyribonucleotides described herein, including more than one incretin peptide on more than one polypeptide chain. Specifically, each polypeptide chain of the incretin agent in fig. 11 has a signal peptide ("SP"), GLP1 incretin peptide, a connecting peptide (GGGGS) 3, and an Fc domain. One or both Fc domains contain a "LS" mutation (M428L/N434S according to EU numbering) to extend the half-life of the incretin agent. When two polypeptide chains are expressed, they combine to form a homodimeric structure as shown in fig. 11. The SP cleavage site within the incretins is indicated by the arrow.

FIG. 12 illustrates an exemplary incretin agent that may be encoded by one or more of the polyribonucleotides described herein, including more than one incretin peptide on more than one polypeptide chain. Specifically, each polypeptide chain of the incretin agent has a Signal Peptide (SP), GLP1 incretin peptide, a linker peptide, a GIP peptide, a second linker peptide (GGGGS) ₃, and an Fc domain. One or both Fc domains contain LS mutations. When two polypeptide chains are expressed, they combine to form a homodimeric structure as shown in fig. 12. Furin and SP cleavage sites within the incretins are indicated by arrows.

Fig. 13 shows an exemplary design of two polyribonucleotides, each encoding a polypeptide chain (top) comprising an incretin peptide fused to an Fc domain. In each polypeptide chain (incretin-Fc fusion), there are a Signal Peptide (SP) and one, two or four incretin peptides (I: 1x, I:2x or I:4 x) fused to the Fc domain by a linker peptide (L2), and each Fc domain has a modification (e.g., a knob-in-hole mutation) that induces heterodimerization. When two polypeptide chains are expressed, they bind to each other to form a heterodimeric incretin.

FIG. 14 illustrates an exemplary incretin agent that may be encoded by one or more of the polyribonucleotides described herein, including more than one incretin peptide on more than one polypeptide chain. Specifically, each polypeptide chain of the incretin agent in fig. 14 has a Signal Peptide (SP), GLP1 or GIP incretin peptide, a connecting peptide (GGGGS) ₃, and an Fc domain. One or both Fc domains contain a "LS" mutation (M428L/N434S), "STR" mutation (L234S, L T and G236R mutations according to EU numbering) to silence Fc effector function, and a "knob structure" mutation to induce heterodimerization. When two polypeptide chains are expressed, they combine to form a heterodimeric incretin agent containing two polypeptide chains with different incretin peptides. The SP cleavage site within the incretins is indicated by the arrow.

FIG. 15 shows exemplary concentrations (pg/ml) of GLP1 (7-37) in HEK293t17 cell supernatants transfected with polyribonucleotides encoding GLP1 (7-37) at 3, 6, 24, 48 and 72 hours post-transfection (GLP1n=4+/-SD; ns=insignificant; ^* p<0.05,^** p < 0.01).

FIG. 16 shows exemplary concentrations (pg/ml) of GLP1 (7-37) with K34R mutation in supernatant of HEK293t17 cells transfected with polyribonucleotides encoding GLP1 (7-37) - (K34R) 3, 6, 24, 48 and 72 hours post-transfection (GLP1n=4+/-SD; ns=insignificant; ^* p<0.05,^** p < 0.01).

FIG. 17 shows exemplary concentrations (pg/ml) of GIP (1-42) in supernatant of HEK293t17 cells transfected with polyribonucleotides encoding GIP (1-42) 3, 6, 24, 48 and 72 hours post-transfection (GIPn=6+/-SD; ns=insignificant; ^* p<0.05,^** p < 0.01).

FIG. 18 shows the concentrations (pg/ml) of an exemplary GLP1 incretin in HEK29t17 cell supernatants transfected with polyribonucleotides encoding incretins containing a viral signal peptide ("viral SP") or husec signal peptide ("husec") and codon optimized with different strategies ("opt 1" versus "optp"). Specific incretins include viral SP-GLP1 (7-37), viral SP-GLP1 (7-37) - (K34R), husec-GLP-1 (7-37) -A8G (opt 1), husec-GLP-1 (7-37) -A8G-linked peptide (opt 1), husec-GLP-1 (7-37) -A8G (optp) and husec-GLP-1 (7-37) -A8G-linked peptide (optp) incretins.

FIG. 19 shows the concentrations (ng/ml) of an exemplary GIP incretin in HEK29t17 cell supernatants transfected with polyribonucleotides encoding incretins containing a viral signal peptide ("viral SP") or husec signal peptide ("husec") and codon optimized with different strategies ("opt 1" versus "optp"). Specific incretins include the viruses SP-GIP (1-42), husec-GIP (1-42) -A2G (opt 1) and GIP (1-42) -A2G (optp).

FIG. 20 shows a schematic representation of the theoretical cleavage sites of various signal peptides within the amino acid sequence of an incretin agent. FIG. 20 also indicates that the A8G mutation promotes correct N-terminal processing of GLP1 incretins with husec signal peptide.

FIG. 21 shows a schematic of the theoretical cleavage sites of various signal peptides within the amino acid sequence of an incretin agent. Figure 21 also indicates that A2G mutation promotes correct N-terminal processing of GIP incretins with husec signal peptide.

FIGS. 22A-22B show schematic diagrams of HEK293 reporter cell lines overexpressing GLP1R (A) and GIPR (B) to be utilized in assays to determine the bioactivity of the exemplary GLP1 and GIP incretins of example 7.

FIG. 23 shows the results from a biological activity assay for an exemplary GLP1 incretin agent. Specifically, the results are expressed as fold induction relative to control samples.

Fig. 24 shows the results from the bioactivity assay of exemplary GIP incretins. Specifically, the results are expressed as fold induction relative to control samples.

Figure 25 shows the in vitro activity (GIP expression) of certain exemplary incretins tested.

Figure 26 shows GIP biological activity of certain exemplary GIP-containing incretins tested.

Figure 27 shows the in vitro activity (GLP 1 expression) of certain exemplary incretins tested.

FIG. 28 shows GLP1 bioactivity of certain exemplary GLP 1-containing incretins tested.

FIG. 29 shows a comparison of GIP expression (A) and GIP bioactivity (B) in exemplary candidates with different signal peptides (husec vs gD 1).

FIG. 30 shows a comparison of GLP1 expression (A) and GLP1 bioactivity (B) in exemplary candidates with different signal peptides (husec vs gD 1).

Figure 31 shows a comparison of GIP expression (a) with GIP bioactivity (B) with and without various half-life extending (HLE) moieties.

FIG. 32 shows a comparison of GLP1 expression (A) with and without various half-life extending (HLE) moieties to GLP1 bioactivity (32B).

FIG. 33 shows a comparison of GIP expression (A) and GIP bioactivity (B) in an exemplary incretin containing both GIP and GLP1, wherein the sequence of the GIP and GLP1 peptides encoded by a single polyribonucleotide is altered.

FIG. 34 shows a comparison of GLP1 expression (A) and GLP1 bioactivity (B) in an exemplary incretin containing both GIP and GLP1, wherein the sequence of the GIP and GLP1 peptides encoded by a single polyribonucleotide is altered.

Definition of the definition

About the term "about" as used herein to refer to a value, refers to a value in a context similar to the value mentioned. In general, one of ordinary skill in the art will recognize, after familiarity with the context, the relative degree of variation covered by the "about" in this context. For example, in some embodiments, the term "about" may encompass a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the mentioned values.

Reagent As used herein, the term "reagent" may refer to a physical entity. In some embodiments, the agent may be characterized by specific features and/or effects. For example, as used herein, the term "therapeutic agent" refers to a physical entity that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, the agent may be any chemical class of compound, molecule, or entity, including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or combination or complex thereof.

Aliphatic the term "aliphatic" refers to a straight (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a mono-or bicyclic hydrocarbon (also referred to herein as "cycloaliphatic") that is fully saturated or contains one or more unsaturated units but is not aromatic, having a single point of attachment or more than one point of attachment to the remainder of the molecule. Unless otherwise indicated, aliphatic groups contain 1 to 12 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms (e.g., C _1-6). In some embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms (e.g., C _1-5). In other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms (e.g., C _1-4). In other embodiments, the aliphatic group contains 1-3 aliphatic carbon atoms (e.g., C _1-3), and in other embodiments, the aliphatic group contains 1-2 aliphatic carbon atoms (e.g., C _1-2). Suitable aliphatic groups include, but are not limited to, straight or branched chain, substituted or unsubstituted alkyl, alkenyl or alkynyl groups, and hybrids thereof. Preferred aliphatic groups are C _1-6 alkyl groups.

Alkyl the term "alkyl", used alone or as part of a larger moiety, refers to a saturated, optionally substituted, straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C _1-12、C_1-10、C_1-8、C_1-6、C_1-4、C_1-3 or C _1-2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

Alkylene the term "alkylene" refers to a divalent alkyl group. In some embodiments, "alkylene" is a divalent straight or branched chain alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., - (CH ₂)_n -, where n is a positive integer, e.g., 1 to 6,1 to 4, 1 to 3, 1 to 2, or 2 to 3, an optionally substituted alkylene chain is a polymethylene group that is optionally substituted with one or more methylene hydrogen atoms, suitable substituents include those described below for substituted aliphatic groups, and also include those described herein, it will be appreciated that the two substituents of an alkylene group may together form a ring system, in certain embodiments, the two substituents may together form a 3 to 7 membered ring, the substituents may be on the same or different atoms, suffix "-subunit" or "-subunit" when appended to certain groups herein is intended to mean a difunctional moiety of the group, e.g., "-subunit" or "-subunit" when appended to "cyclopropyl" is intended to become "cyclopropyl" as "cyclopropyl" (cyclopropylene or "cyclopropyl" (cyclopropylenyl) and means, e.g., difunctional cyclopropyl),。

The term "alkenyl", used alone or as part of a larger moiety, refers to an optionally substituted straight or branched or cyclic hydrocarbon group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C _2-12、C_2-10、C_2-8、C_2-6、C_2-4 or C _2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having from about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl and cycloheptenyl.

The term "alkynyl", used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C _2-12、C_2-10、C_2-8、C_2-6、C_2-4 or C _2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

Amino acids in its broadest sense, the term "amino acid" as used herein refers to a compound and/or substance that can be incorporated, or already incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, the amino acid has the general structure H ₂ N-C (H) (R) -COOH. In some embodiments, the amino acid is a naturally occurring amino acid. In some embodiments, the amino acid is an unnatural amino acid, in some embodiments, the amino acid is a D-amino acid, and in some embodiments, the amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "non-standard amino acid" refers to any amino acid other than a standard amino acid, whether synthetically prepared or obtained from natural sources. In some embodiments, amino acids (including carboxy-terminal amino acids and/or amino-terminal amino acids in polypeptides) may contain structural modifications as compared to the general structures described above. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of amino groups, carboxylic acid groups, one or more protons, and/or hydroxyl groups) as compared to the general structure. In some embodiments, such modifications may, for example, alter the circulatory half-life of a polypeptide containing a modified amino acid as compared to a polypeptide containing an otherwise identical unmodified amino acid. In some embodiments, such modifications do not significantly alter the activity associated with polypeptides containing modified amino acids as compared to polypeptides containing otherwise identical unmodified amino acids. It will be clear from the context that in some embodiments the term "amino acid" may be used to refer to a free amino acid, and in some embodiments the term "amino acid" may be used to refer to an amino acid residue of a polypeptide.

Aryl the term "aryl" refers to mono-and bi-cyclic systems having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an "aryl" group contains a total of six to twelve ring members (e.g., C6-C12). The term "aryl" may be used interchangeably with the term "aryl ring". In certain embodiments, "aryl" refers to an aromatic ring system that may bear one or more substituents, including but not limited to phenyl, biphenyl, naphthyl, anthracenyl, and the like. Unless otherwise indicated, "aryl" is a hydrocarbon. In some embodiments, an "aryl" ring system is an aromatic ring (e.g., phenyl) fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aromatic rings include fused aromatic rings, including、And。

Relatives as this term is used herein, two events or entities are "related" to one another if the presence, level, degree, type, and/or form of one event or entity is related to the presence, level, degree, type, and/or form of another event or entity. For example, a particular entity (e.g., polypeptide, genetic stamp, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder or condition if its presence, level, and/or form is associated with the occurrence, susceptibility, severity, stage, etc., of that disease, disorder or condition (e.g., in a related population). In some embodiments, two or more entities are physically "bound" to each other if they interact directly or indirectly such that the entities are physically close to each other and/or remain in close proximity. In some embodiments, two or more entities physically bound to each other are covalently linked to each other, and in some embodiments, two or more entities physically bound to each other are not covalently linked to each other, but are non-covalently bound, for example, by hydrogen bonding, van der Waals interactions (VAN DER WAALS interactions), hydrophobic interactions, magnetic interactions, and combinations thereof.

Co-administration the term "co-administration" as used herein refers to the use of a composition herein (e.g., a pharmaceutical composition) and one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents comprise at least one polyribonucleotide encoding another therapeutic agent (e.g., an incretin agent). The combined use of the compositions (e.g., pharmaceutical compositions) and additional therapeutic agents described herein can be performed simultaneously or separately (e.g., sequentially in any order). In some embodiments, the compositions described herein (e.g., pharmaceutical compositions) and additional therapeutic agents can be combined in one pharmaceutically acceptable excipient, or they can be placed in separate excipients and delivered to the target cells or administered to the individual at different times. Each of such cases is contemplated to fall within the meaning of "co-administration" or "combination" provided that the compositions (e.g., pharmaceutical compositions) and additional therapeutic agents described herein are delivered or administered sufficiently close in time that there is at least some temporal overlap in the biological effects produced by each on the target cells or treated individual.

Combination therapy the term "combination therapy" as used herein refers to those instances in which an individual is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents (e.g., two or more incretins agents)). In some embodiments, two or more regimens may be administered simultaneously, in some embodiments such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered prior to any dose of the second regimen), in some embodiments such agents are administered in an overlapping dosing regimen. In some embodiments, administration of a combination therapy may involve administration of one or more agents or modalities to an individual receiving other agents or modalities in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even at the same time), although in some embodiments, two or more agents or active portions thereof may be administered together in a combined composition. In some embodiments, the combination therapy comprises polyribonucleotides encoding two or more incretins.

Comparably, as used herein, the term "comparably" refers to two or more agents, entities, conditions, sets of conditions, etc., that may not be consistent with each other, but that are sufficiently similar to allow comparison therebetween so that one of ordinary skill in the art will appreciate that conclusions can be drawn reasonably based on the observed differences or similarities. In some embodiments, a set of comparable conditions, an environment, an individual, or a population is characterized by a plurality of substantially identical features and one or a small number of varying features. In this context, those of skill in the art will understand what degree of consistency is required in any given environment to render two or more such agents, entities, situations, sets of conditions, etc., as comparable. For example, one of skill in the art will understand that when characterized by a sufficient number and type of substantially identical features to ensure that differences in results or observed phenomena obtained under or in different groups of environments, individuals or populations are caused by or indicative of changes in the different those features, the environments, individuals or populations of the groups are comparable to one another.

Corresponding to the term "corresponding to" as used herein refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to designate the position/identity of a structural component in a compound or composition relative to another compound or composition (e.g., relative to an appropriate reference compound or composition). For example, in some embodiments, a monomer residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, one of skill in the art will appreciate that for simplicity, residues in a polypeptide are typically specified using a canonical numbering system based on the reference to the relevant polypeptide, such that an amino acid "corresponding to" a residue at position 190, for example, does not actually need to be the 190 th amino acid in a particular amino acid chain, but corresponds to a residue found in the reference polypeptide, and that the manner of identifying the "corresponding" amino acid will be readily apparent to those of skill in the art. For example, one of skill in the art will recognize a variety of sequence alignment strategies, including software programs such as ,BLAST、CS-BLAST、CUSASW++、DIAMOND、FASTA、GGSEARCH/GLSEARCH、Genoogle、HMMER、HHpred/HHsearch、IDF、Infernal、KLAST、USEARCH、parasail、PSI-BLAST、PSI-Search、ScalaBLAST、Sequilab、SAM、SSEARCH、SWAPHI、SWAPHI-LS、SWIMM or SWIPE, that can be used, for example, to identify "corresponding" residues in polypeptides and/or nucleic acids as described in the present disclosure. Those skilled in the art will also appreciate that in some cases the term "corresponding to" may be used to describe an event or entity that shares a related similarity with another event or entity (e.g., an appropriate reference event or entity). To mention just one example, a gene or protein in one organism may be described as "corresponding to" a gene or protein from another organism, in order to indicate that it performs a similar function or performs a similar function and/or that it exhibits a particular degree of sequence identity or homology, or shares a particular characteristic sequence component in some embodiments.

Cycloaliphatic As used herein, the term "cycloaliphatic" refers to a monocyclic C3-8 hydrocarbon or a bicyclic C6-10 hydrocarbon that is fully saturated or contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment or more than one point of attachment to the remainder of the molecule.

Cycloalkyl As used herein, the term "cycloalkyl" refers to an optionally substituted saturated cyclic mono-or multicyclic ring system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

Derivatization in the context of an amino acid sequence (peptide or polypeptide) that is "derived from" the specified amino acid sequence (peptide or polypeptide) refers to a structural analog of the specified amino acid sequence. In some embodiments, the amino acid sequences derived from a particular amino acid sequence have an amino acid sequence that is identical, substantially identical, or homologous to that of the particular sequence or fragment thereof. The amino acid sequences derived from a particular amino acid sequence may be mutants of that particular sequence or fragment thereof. For example, an incretin agent as used in the present disclosure may include an amino acid sequence derived from two or more incretins agents (e.g., two or more naturally occurring incretins).

Detection the term "detection" is used broadly herein to include any suitable way of determining the presence or absence of a target entity in a sample, or any form of measuring a target entity. Thus, "detecting" may include determining, measuring, assessing or detecting the presence or absence, level, quantity and/or location of a target entity. Including quantitative and qualitative determinations, measurements or evaluations, including semi-quantitative. Such determination, measurement or evaluation may be relative, for example when detecting the target entity relative to a control reference, or absolute. Thus, the term "quantization" when used in the context of a quantization target entity may refer to absolute quantization or relative quantization. Absolute quantification may be accomplished by correlating the detected level of the target entity with a known control standard (e.g., by generating a standard curve). Or relative quantization may be accomplished by comparing detected levels or amounts between two or more different target entities to provide relative quantization of each of the two or more different target entities (i.e., relative to each other).

Dosing regimen one skilled in the art will appreciate that the term "dosing regimen" (or "treatment regimen") may be used to refer to a set of unit doses (typically more than one unit dose) that are individually administered to an individual, typically at intervals of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.

Coding the term "coding" or "encoding" as used herein refers to sequence information that directs the production of a first molecule having a defined nucleotide sequence (e.g., a polyribonucleotide) or a second molecule having a defined amino acid sequence. For example, a DNA molecule may encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase). The RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, cDNA or RNA molecule encodes a polypeptide if transcription and translation of the RNA corresponding to the gene produces the polypeptide in a cell or other biological system. In some embodiments, the coding region of a polyribonucleotide encoding a target antigen refers to a coding strand whose nucleotide sequence is identical to the polyribonucleotide sequence of such target antigen. In some embodiments, the coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such target antigen that can be used as a template for transcription of a gene or cDNA.

Engineering in general, the term "engineering" refers to aspects of a human manual manipulation. For example, a polyribonucleotide is considered "engineered" when two or more sequences that are not linked together in that order in nature are manually manipulated to be directly linked to each other in an engineered polynucleotide and/or when particular residues in the polynucleotide are non-naturally occurring and/or result in a linkage to an entity or moiety that is not linked to it in nature by manual action.

Expression As used herein, the term "expression" of a nucleic acid sequence refers to the production of a gene product from the nucleic acid sequence. In some embodiments, the gene product may be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of the nucleic acid sequence involves one or more of (1) generating an RNA template from the DNA sequence (e.g., by transcription), (2) processing of the RNA transcript (e.g., by splicing, editing, etc.), (3) translating the RNA into a polypeptide or protein, and/or (4) post-translational modification of the polypeptide or protein.

Heteroaliphatic As used herein, the term "heteroaliphatic" or "heteroaliphatic group" refers to an optionally substituted hydrocarbon moiety having one to five heteroatoms in addition to a carbon atom, which may be linear (i.e., unbranched), branched, or cyclic ("heterocyclic") and may be fully saturated or may contain one or more unsaturated units, but which is not aromatic. The term "heteroatom" refers to nitrogen, oxygen or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternary ammonium form of basic nitrogen. The term "nitrogen" also includes substituted nitrogen. Unless otherwise indicated, a heteroaliphatic group contains 1 to 10 carbon atoms, wherein 1 to 3 carbon atoms are optionally and independently replaced by heteroatoms selected from oxygen, nitrogen and sulfur. In some embodiments, the heteroaliphatic group contains 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroaliphatic group contains 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, straight or branched heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, the heteroaliphatic groups of 1 to 10 atoms include the following exemplary groups ：-O-CH₃、-CH₂-O-CH₂、-O-CH₂-CH₂-O-CH₂-CH₂-O-CH₂ and the like.

The terms "heteroaryl" and "heteroaromatic-" used alone or as part of a larger moiety (e.g., "heteroaralkyl" or "heteroarylalkoxy") refer to a monocyclic or bicyclic group having 5 to 10 ring atoms (e.g., 5 to 6 membered monocyclic heteroaryl or 9 to 10 membered bicyclic heteroaryl), having 6, 10, or 14 pi electrons shared in a cyclic array, and having one to five heteroatoms in addition to carbon atoms. Heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo [1,2-a ] pyrimidinyl, imidazo [1,2-a ] pyridinyl, imidazo [4,5-b ] pyridinyl, imidazo [4,5-c ] pyridinyl, pyrrolopyridinyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridinyl, and benzisoxazolyl. As used herein, the terms "heteroaryl" and "heteroaromatic-" also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, wherein the linking group or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaromatic ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, xin -linyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido [2,3-b ] -1, 4-oxazin-3 (4H) -one, 4H-thieno [3,2-b ] pyrrole, and benzisoxazolyl. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring", "heteroaryl" or "heteroaromatic", any of which include an optionally substituted ring.

Heteroatom As used herein, the term "heteroatom" refers to nitrogen, oxygen or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternary ammonium form of basic nitrogen.

Heterocycle As used herein, the terms "heterocycle (heterocycle)", "heterocyclyl", "heterocyclic group" and "heterocycle (heterocyclic ring)" are used interchangeably and refer to a stable 3-to 8-membered monocyclic, 6-to 10-membered bicyclic or 10-to 16-membered polycyclic heterocyclic moiety which is saturated or partially unsaturated and has one or more, such as one to four heteroatoms, as defined above, in addition to carbon atoms. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. By way of example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3, 4-dihydro-2H-pyrrolyl), NH (as in pyrrolidyl) or NR ⁺ (as in N-substituted pyrrolidyl). The heterocycle may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom may be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, pyrazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepine, thiozepine, morpholinyl, and thiomorpholinyl. The heterocyclic group may be monocyclic, bicyclic, tricyclic or polycyclic, preferably monocyclic, bicyclic or tricyclic, more preferably monocyclic or bicyclic. Bicyclic heterocycles also include groups in which the heterocycle is fused to one or more aromatic rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1, 3-dihydroisobenzofuranyl, 2, 3-dihydrobenzofuranyl, and tetrahydroquinolinyl. Bicyclic heterocycles may also be 7-to 11-membered spiro-fused heterocycles of spiro ring systems (e.g., having one or more heteroatoms (e.g., one, two, three, or four heteroatoms) as defined above in addition to carbon atoms). Bicyclic heterocycles may also be bridged ring systems (e.g., 7 to 11 membered bridged heterocycles having one, two or three bridging atoms).

Homology As used herein, the term "homology" or "homolog" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered "homologous" to each other if the sequences of the polynucleotide molecules and/or polypeptide molecules are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered "homologous" to each other if the sequences of the polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., contain residues with related chemical properties at corresponding positions). For example, as is well known to those skilled in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids that are similar to one another, and/or as having "polar" or "nonpolar" side chains. One amino acid substitution may be generally considered a "homologous" substitution for another amino acid of the same type.

Identity the term "identity" as used herein refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered to be "substantially identical" to each other if the sequences between the polynucleotide molecules and/or polypeptide molecules are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. The calculation of the percent identity of two nucleic acid or polypeptide sequences may be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. When a position in a first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison performed with the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two nucleotide sequences may be determined using the GAP program in the GCG software package using the nwsgapdna.

Increasing, inducing, or decreasing as used herein, such terms or grammatically comparable comparison terms indicate measured values relative to a comparable reference. For example, in some embodiments, the evaluation value achieved with a provided composition (e.g., a pharmaceutical composition) may be "increased" relative to the evaluation value obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, the evaluation value achieved in an individual may be "increased" relative to an evaluation value obtained in the same individual under different conditions (e.g., before or after an event; or in the presence or absence of an event, such as administration of a composition (e.g., a pharmaceutical composition) as described herein), or in a different comparable individual (e.g., a comparable individual than a target individual previously exposed to a disorder (e.g., in the absence of administration of a composition (e.g., a pharmaceutical composition) as described herein). In some embodiments, the comparison term refers to a statistically relevant difference (e.g., having a prevalence and/or magnitude sufficient to achieve a statistical correlation). Those skilled in the art will recognize or will be able to readily determine the degree of difference and/or prevalence needed or sufficient to achieve such statistical significance in a given context. In some embodiments, the term "reduce" or equivalent term refers to a reduction in the level of an evaluation value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or more, as compared to a comparable reference. In some embodiments, the term "reduce" or equivalent terms refer to complete or substantially complete inhibition, i.e., to zero or substantially to zero. In some embodiments, the term "increase" or "induction" refers to an increase in the level of an evaluation value of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500% or more, as compared to a comparable reference.

Sequencing as used herein, with respect to a polynucleotide or polyribonucleotide, "sequencing" refers to the sequence along the 5 'to 3' character of the polynucleotide or polyribonucleotide. As used herein, with respect to a polypeptide, "in order" refers to the order of features that move along the polypeptide from N-terminal-most features to C-terminal-most features. "in order" does not mean that there may be no additional features in the listed features. For example, if features A, B and C of a polynucleotide are described herein as "feature a, feature B, and feature C in order," this description does not exclude feature D, e.g., located between features a and B.

Ionizable the term "ionizable" refers to a compound or group or atom that is charged at a certain pH. In the context of ionizable amino lipids, such lipids or functional groups or atoms thereof carry a positive charge at a certain pH. In some embodiments, the ionizable amino lipid is positively charged at an acidic pH. In some embodiments, the ionizable amino lipid is predominantly neutral at physiological pH values (e.g., about 7.0-7.4 in some embodiments), but becomes positively charged at lower pH values. In some embodiments, the ionizable amino lipid may have a pKa in the range of about 5 to about 7.

The term "isolated" means altered or removed from a natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide, partially or completely isolated from coexisting materials in its natural state, is "isolated. The isolated nucleic acid or protein may be present in a substantially purified form, or may be present in a non-natural environment (such as, for example, a host cell).

Lipid as used herein, the terms "lipid" and "lipid-like material" are broadly defined as molecules comprising one or more hydrophobic moieties or groups and optionally also comprising one or more hydrophilic moieties or groups. Molecules comprising a hydrophobic moiety and a hydrophilic moiety are also commonly denoted amphiphiles.

RNA lipid nanoparticle as used herein, the term "RNA lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and an RNA molecule, such as one or more polyribonucleotides as provided herein. In some embodiments, the RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, the RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-coupled lipid (e.g., a PEG-coupled lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., zaverage) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, the RNA lipid nanoparticle may have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, the average size of the lipid nanoparticle is determined by measuring the average particle size. In some embodiments, RNA lipid nanoparticles can be prepared by mixing a lipid with an RNA molecule described herein.

Neutralization as used herein, the term "neutralization" refers to an event in which a binding agent (e.g., an antibody) binds to a biologically active site (e.g., a receptor binding protein) of a virus, thereby inhibiting parasitic infection of a cell. In some embodiments, the term "neutralization" refers to an event in which the binding agent eliminates or significantly reduces the ability to infect cells.

Nucleic acid/polynucleotide as used herein, the term "nucleic acid" refers to a polymer of at least 10 nucleotides or more. In some embodiments, the nucleic acid is or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises a Peptide Nucleic Acid (PNA). In some embodiments, the nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, the nucleic acid is or comprises a double stranded nucleic acid. In some embodiments, the nucleic acid comprises both a single-stranded portion and a double-stranded portion. In some embodiments, the nucleic acid comprises a backbone comprising one or more phosphodiester linkages. In some embodiments, the nucleic acid comprises a backbone comprising both phosphodiester linkages and non-phosphodiester linkages. For example, in some embodiments, the nucleic acid may comprise a backbone comprising one or more phosphorothioate linkages or 5' -N-phosphoramidite linkages and/or one or more peptide linkages, e.g., in a "peptide nucleic acid". In some embodiments, the nucleic acid comprises one or more or all of the natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more or all non-natural residues. In some embodiments, the unnatural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxo-adenosine, 8-oxo-guanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the non-natural residues comprise one or more modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose) as compared to the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (e.g., RNA or polypeptide). In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by complementary template-based polymerization, e.g., in vivo or in vitro), replication in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、20、225、250、275、300、325、350、375、400、425、450、475、500、600、700、800、900、1000、1500、2000、2500、3000、3500、4000、4500、5000、5500、6000、6500、7000、7500、8000、8500、9000、9500、10,000、10,500、11,000、11,500、12,000、12,500、13,000、13,500、14,000、14,500、15,000、15,500、16,000、16,500、17,000、17,500、18,000、18,500、19,000、19,500 or 20,000 or more residues or nucleotides in length.

Pharmaceutically effective amount the term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that achieves a desired response or desired effect, alone or in combination with other doses. In the case of treating a particular disease (e.g., obesity), in some embodiments, the desired response involves inhibition of the course of the disease (e.g., obesity). In some embodiments, such inhibition may comprise slowing the progression of a disease (e.g., obesity) and/or interrupting or reversing the progression of a disease (e.g., obesity). In some embodiments, the desired response in the treatment of a disease (e.g., obesity) may be or include delaying or preventing the onset of the disease (e.g., obesity) or disorder (e.g., a disorder associated with obesity). The effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on the disease (e.g., obesity) or disorder to be treated (e.g., a disorder associated with obesity), the severity of such disease (e.g., obesity) or disorder (e.g., a disorder associated with obesity), the individual parameters of the patient (including, for example, age, physiological condition, body shape, and weight), the duration of treatment, the type of concomitant therapy (if present), the particular route of administration, and the like. Thus, the dosage of a composition described herein (e.g., a pharmaceutical composition) may depend on various such parameters. In cases where the response in the patient is insufficient at the initial dose, a higher dose (or effectively higher doses achieved by different, more topical routes of administration) may be used.

Polypeptide As used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, the polypeptide has an amino acid sequence that occurs in nature. In some embodiments, the polypeptide has an amino acid sequence that is not found in nature. In some embodiments, the polypeptide has an engineered amino acid sequence in that it is designed and/or produced by human manual action. In some embodiments, the polypeptide may comprise, or consist of, a natural amino acid, an unnatural amino acid, or both. In some embodiments, the polypeptide may comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, the polypeptide may comprise a D-amino acid, an L-amino acid, or both. In some embodiments, the polypeptide may comprise only D-amino acids. In some embodiments, the polypeptide may comprise only L-amino acids. In some embodiments, the polypeptide may include one or more pendant groups or other modifications, such as modification of one or more amino acid side chains or attachment thereto at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, and the like, including combinations thereof. In some embodiments, the polypeptide may be circular, and/or may comprise a circular portion. In some embodiments, the polypeptide is not cyclic and/or does not comprise any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity or structure, in which case it is used herein to refer to polypeptides that share a related activity or structure and thus may be considered members of the same class or family of polypeptides. For each such class, the present description provides and/or those skilled in the art will recognize exemplary polypeptides within that class for which the amino acid sequence and/or function is known, and in some embodiments such exemplary polypeptides are reference polypeptides of the class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit significant sequence homology or identity to a reference polypeptide of that class (in some embodiments to all polypeptides within that class), share a common sequence motif (e.g., a characteristic sequence component), and/or share common activity (in some embodiments, at a comparable level or within a specified range). For example, in certain embodiments, a member polypeptide has an overall sequence homology or identity of at least about 30-40%, and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or comprises at least one region (e.g., a conserved region, which in certain embodiments may be or comprise a characteristic sequence element) that exhibits very high sequence identity, typically greater than 90%, even greater than 95%, 96%, 97%, 98% or 99%. such conserved regions typically comprise at least 3-4 and typically at most 35 or more amino acids, and in some embodiments, the conserved regions comprise at least one segment of at least 2, 3, 4, 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 or more contiguous amino acids. In some embodiments, the related polypeptide may comprise or consist of a fragment of the parent polypeptide.

Prevention as used herein, the term "prevention" or "prevention" when used in connection with the occurrence of a disease, disorder, and/or condition refers to reducing the risk of developing the disease, disorder, and/or condition, and/or delaying the onset of one or more features or symptoms of the disease, disorder, or condition. Prevention may be considered complete when the onset of a disease, disorder or condition has been delayed for a predetermined period of time.

Reference as used herein, the term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of a target is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the testing and/or determining of the reference or control is performed substantially simultaneously with the testing and/or determining of the target. In some embodiments, the reference or control is a historical reference or control optionally embodied in a tangible medium. Typically, as will be appreciated by those skilled in the art, the reference or control is determined or characterized under conditions or circumstances comparable to those assessed. Those skilled in the art will appreciate that when sufficient similarity exists, it may prove reasonable to rely on and/or compare to a particular possible reference or control.

Ribonucleic acid (RNA) or polyribonucleotide As used herein, the terms "ribonucleic acid", "RNA" or "polyribonucleotide" refer to a polymer of ribonucleotides. In some embodiments, the RNA is single stranded. In some embodiments, the RNA is double stranded. In some embodiments, the RNA comprises both a single-stranded portion and a double-stranded portion. In some embodiments, the RNA can comprise a backbone structure as described in the definition of "nucleic acid/polynucleotide" above. The RNA can be a regulatory RNA (e.g., siRNA, microrna, etc.) or a messenger RNA (mRNA). In some embodiments, the RNA is mRNA. In some embodiments, where the RNA is mRNA, the RNA typically comprises a poly (a) region at its 3' end. In some embodiments, where the RNA is mRNA, the RNA typically comprises a cap structure at its 5' end that is recognized by the art, e.g., for recognizing the mRNA and ligating it to the ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNAs include RNAs synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).

Ribonucleotides As used herein, the term "ribonucleotide" encompasses both unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (a) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides can include one or more modifications including, but not limited to, for example, (a) terminal modifications, such as 5 'terminal modifications (e.g., phosphorylation, dephosphorylation, coupling, reverse bonding, etc.), 3' terminal modifications (e.g., coupling, reverse bonding, etc.), base modifications (b) such as substitutions with modified bases, stabilized bases, destabilized bases, or bases or coupled bases that base pair with an amplification partner profile, (c) sugar modifications (e.g., at the 2 'position or the 4' position) or substitutions of sugar, and (d) internucleoside bond modifications, including modifications or substitutions of phosphodiester bonds. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

Risk as will be understood from the context, "risk" of a disease, disorder, and/or condition refers to the likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, the risk is expressed as a risk relative to a risk associated with a reference sample or a reference sample set. In some embodiments, the reference sample or group of reference samples has a known risk of a disease, disorder, condition, and/or event. In some embodiments, the reference sample or set of reference samples is from an individual that is comparable to a particular individual. In some embodiments, the risk may reflect one or more genetic attributes, e.g., which may predispose an individual to (or not) developing a particular disease, disorder, and/or condition. In some embodiments, the risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.

Specificity the term "specificity" when used herein in reference to an agent having activity is understood by those skilled in the art to mean that the agent distinguishes between potential target entities, states or cells. For example, in some embodiments, an agent is said to "specifically" bind to its target if the agent preferentially binds to its target in the presence of one or more competing surrogate targets. In many embodiments, the specific interaction depends on the presence of specific structural features (e.g., epitopes, gaps, binding sites) of the target entity. It should be understood that the specificity need not be absolute. In some embodiments, specificity may be assessed relative to the specificity of the target binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, the specificity is assessed relative to the specificity of a reference specific binding member. In some embodiments, the specificity is assessed relative to the specificity of a reference non-specific binding member.

Substituted or optionally substituted the compounds of the invention may contain an "optionally substituted" moiety, as described herein. In general, the term "substituted" (whether preceded by the term "optionally") means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. "substituted" applies to self-structures (e.g.,Means at leastAnd (3)Means at least、、Or (b)) In explicit or implicit one or more hydrogens. Unless otherwise indicated, an "optionally substituted" group may have suitable substituents at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from the specified group, the substituents may be the same or different at each position. Combinations of substituents contemplated by the present invention are preferably those resulting in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that does not substantially change when subjected to a process that allows it to be produced, detected, and in certain embodiments, recovered, purified, and used for one or more of the purposes provided herein. The group described as "substituted" preferably has 1 to 4 substituents, more preferably 1 or 2 substituents. The group described as "optionally substituted" may be unsubstituted or "substituted" as described above.

Suitable monovalent substituents on the substitutable carbon atom of an "optionally substituted" group are independently halogen ;-(CH₂)_0-4R⁰;-(CH₂)_0-4OR⁰;-O(CH₂)_0-4R⁰、-O-(CH₂)_0-4C(O)OR⁰;-(CH₂)_0-4CH(OR⁰)₂;-(CH₂)_0- ₄SR⁰;-(CH₂)_0-4Ph, which may be substituted with R ⁰, - (CH ₂)_0-4O(CH₂)_0-1 Ph which may be substituted with R ⁰, -ch=chph which may be substituted with R ⁰, - (CH ₂)_0-4O(CH₂)_0-1 -pyridinyl which may be substituted with R ⁰ for ;-NO₂;-CN;-N₃;-(CH₂)_0-4N(R⁰)₂;-(CH₂)_0-4N(R⁰)C(O)R⁰;-N(R⁰)C(S)R⁰;-(CH₂)_0-4N(R⁰)C(O)NR⁰ ₂;-N(R⁰)C(S)NR⁰ ₂;-(CH₂)_0-4N(R⁰)C(O)OR⁰;-N(R⁰)N(R⁰)C(O)R⁰;-N(R⁰)N(R⁰)C(O)NR⁰ ₂;-N(R⁰)N(R⁰)C(O)OR⁰;-(CH₂)_0-4C(O)R⁰;C(S)R⁰;-(CH₂)_0-4C(O)OR⁰;-(CH₂)_0-4C(O)SR⁰;-(CH₂)_0-4C(O)OSiR⁰ ₃;-(CH₂)_0-4OC(O)R⁰;-OC(O)(CH₂)_0-4SR⁰;-(CH₂)_0-4SC(O)R⁰;-(CH₂)_0-4C(O)NR⁰ ₂;-C(S)NR⁰ ₂;-C(S)SR⁰;-SC(S)SR⁰、-(CH₂)_0- ₄OC(O)NR⁰ ₂;-C(O)N(OR⁰)R⁰;-C(O)C(O)R⁰;-C(O)CH₂C(O)R⁰;-C(NOR⁰)R⁰;-(CH₂)_0-4SSR⁰;-(CH₂)_0-4S(O)₂R⁰;-(CH₂)_0-4S(O)₂OR⁰;-(CH₂)_0-4OS(O)₂R⁰;-S(O)₂NR⁰2;-(CH₂)_0-4S(O)R⁰;-N(R⁰)S(O)₂NR⁰ ₂;-N(R⁰)S(O)₂R⁰;-N(OR⁰)R⁰;-C(NH)NR⁰ ₂;-P(O)₂R⁰;-P(O)R⁰ ₂;-OP(O)R⁰ ₂;-OP(O)(OR⁰)₂;SiR⁰ ₃;-(C_1-4 linear or branched alkylene) O-N (R ⁰)₂; or- (C _1-4 linear or branched alkylene) C (O) O-N (R ⁰)₂, wherein each R ⁰ may be substituted as defined below and is independently hydrogen, C _1-6 aliphatic, -CH ₂Ph、-O(CH₂)_0-1Ph、-CH₂ - (5 to 6 membered heteroaromatic ring), or 3 to 6 membered saturated, partially unsaturated or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, or, in spite of having the definition above, two independently occurring R ⁰ together with intervening atoms form a 3 to 12 membered unsaturated partially unsaturated or monocyclic aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen or sulfur, which may be defined below.

Suitable monovalent substituents on R ⁰ (OR the ring formed by two independently occurring R ⁰ together with the intervening atoms) are independently halogen, - (CH ₂)_0-2R^l, - (halo R^l)、-(CH₂)_0-2OH、-(CH₂)_0-2OR^l、-(CH₂)_0-2CH(OR^l)₂、-O( halo R^l)、-CN、-N₃、-(CH₂)_0-2C(O)R^l、-(CH₂)_0-2C(O)OH、-(CH₂)_0-2C(O)OR^l、-(CH₂)_0-2SR^l、-(CH₂)_0-2SH、-(CH₂)_0-2NH₂、-(CH₂)_0-2NHR^l、-(CH₂)_0-2NR^l ₂、-NO₂、-SiR^l ₃、-OSiR^l ₃、-C(O)SR^l、-(C_1-4 straight OR branched alkylene) C (O) OR ^l OR-SSR ^l, wherein each R ^l is unsubstituted OR substituted with only one OR more halogen groups in the case of the preceding "halo" and is independently selected from C _1-4 aliphatic, -CH ₂Ph、-O(CH₂)_0-1 Ph OR 3 to 6 membered saturated, partially unsaturated OR aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen OR sulfur suitable divalent substituents on the saturated carbon atoms of R ⁰ include =o and =s.

Suitable divalent substituents on the saturated carbon atoms of an "optionally substituted" group include the following: =o ("pendant oxy ")、=S、=NNR^* ₂、=NNHC(O)R^*、=NNHC(O)OR^*、=NNHS(O)₂R^*、=NR^*、=NOR^*、-O(C(R^* ₂))_2-3O- or-S (C (R ^* ₂))_2-3 S-, wherein each independently occurring R ^* is selected from hydrogen, a substituted C _1-6 aliphatic, as may be defined below, or an unsubstituted 5-to 6-membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur suitable divalent substituents bonded to the ortho-substitutable carbon of the" optionally substituted "group include-O (CR ^* ₂)_2-3 O-, wherein each independently occurring R ^* is selected from hydrogen, a substituted C _1-6 aliphatic, as may be defined below, or an unsubstituted 5-to 6-membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur).

Suitable substituents on the aliphatic radical of R ^* include halogen, -R ^l, - (halo R ^l)、-OH、-OR^l, -O (halo R ^l)、-CN、-C(O)OH、-C(O)OR^l、-NH₂、-NHR^l、-NR^l ₂ or-NO ₂, wherein each R ^l is unsubstituted or substituted with only one or more halogen groups if preceded by a "halo" group, and are independently C _1-4 aliphatic, -CH ₂Ph、-O(CH₂)_0-1 Ph, or a 3-to 6-membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the substitutable nitrogen of an "optionally substituted" group include -R^†、-NR^† ₂、-C(O)R^†、-C(O)OR^†、-C(O)C(O)R^†、-C(O)CH₂C(O)R^†、-S(O)₂R^†、-S(O)₂NR^† ₂、-C(S)NR^† ₂、-C(NH)NR^† ₂, or-N (R ^†)S(O)₂R^†; wherein each R ^† is independently hydrogen, a substituted C _1-6 aliphatic, unsubstituted-OPh, as may be defined below, or an unsubstituted 3-to 6-membered saturated, partially unsaturated, or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or two independently occurring R ^†, in spite of the definition above, together with intervening atoms form an unsubstituted 3-to 12-membered saturated, partially unsaturated, or aromatic mono-or bi-ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic radical of R ^† are independently halogen, -R ^l, - (halo R ^l)、-OH、-OR^l, -O (halo R ^l)、-CN、-C(O)OH、-C(O)OR^l、-NH₂、-NHR^l、-NR^l ₂ or-NO ₂, wherein each R ^l is unsubstituted or substituted with only one or more halogen groups if preceded by a "halo" group, and are independently C _1-4 aliphatic, -CH ₂Ph、-O(CH₂)_0-1 Ph or a 3-to 6-membered saturated, partially unsaturated or aromatic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

Individual as used herein, the term "individual" refers to an organism to which the compositions described herein are to be administered, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical individuals include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the individual has a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, the individual is susceptible to a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, the individual exhibits one or more symptoms or features of a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, the individual exhibits one or more non-specific symptoms of a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, the individual does not exhibit any symptoms or features of a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, the individual is a human having one or more characteristics that characterize a susceptibility or risk of a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, the individual is a patient. In some embodiments, the individual is an individual who is administered and/or has been administered a diagnosis and/or therapy.

An individual having a "disease, disorder, and/or condition (e.g., obesity, a condition associated with obesity, etc.) has been diagnosed with and/or exhibits one or more symptoms of the disease, disorder, and/or condition.

An individual susceptible to a "predisposed" disease, disorder and/or condition (e.g., obesity, an obesity-related disorder, etc.) is a person at a higher risk of developing the disease, disorder and/or condition (e.g., obesity, an obesity-related disorder, etc.) than the general public. In some embodiments, an individual susceptible to a disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.) may not be diagnosed with the disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.). In some embodiments, an individual susceptible to a disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.) may express symptoms of the disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.). In some embodiments, an individual susceptible to a disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.) may not express symptoms of the disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.). In some embodiments, an individual susceptible to a disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.) will develop the disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.). In some embodiments, an individual susceptible to a disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.) will not develop the disease, disorder, and/or condition (e.g., obesity, an obesity-related disorder, etc.).

Therapy the term "therapy" refers to the administration or delivery of an agent or intervention that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect (e.g., that has proven statistically likely to have such an effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that is useful for alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, inhibit, present one or more symptoms or features of a disease, disorder, and/or condition, delay the onset thereof, reduce the severity thereof, and/or reduce the incidence thereof.

Treatment as used herein, the term "treatment" or "treatment" refers to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., obesity-related disorders, etc.). Treatment may be administered to individuals that do not express signs of a disease, disorder, and/or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, treatment may be administered to an individual that expresses only early signs of a disease, disorder, and/or condition (e.g., obesity, a condition associated with obesity, etc.), e.g., for the purpose of reducing the risk of developing a pathology associated with the disease, disorder, and/or condition. In some embodiments, the treatment may be administered to an individual at a later stage of the disease, disorder, and/or condition (e.g., obesity, a condition associated with obesity, etc.).

Compounds of the present disclosure include those generally described above, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless indicated otherwise. For purposes of this disclosure, chemical elements are identified according to the periodic Table of the elements, CAS version, handbook of CHEMISTRY AND PHYSICS, 75 th edition. In addition, general principles of organic chemistry are described in "Organic Chemistry", thomas Sorrell, university Science Books, sausalato 1999 and "March' S ADVANCED Organic Chemistry", 5 th edition, editions: smith, m.b. and March, j., john Wiley and Sons, new york:2001, the entire contents of which are hereby incorporated by reference.

Unless otherwise indicated, structures depicted herein are intended to include all stereoisomeric (e.g., mirror-image or non-mirror-image) forms of the structures, as well as all geometric or topographical isomeric forms of the structures. For example, the R and S configurations of each stereocenter are contemplated as part of the present disclosure. Thus, single stereochemical isomers as well as enantiomeric, non-enantiomeric, and geometric (or conformational) mixtures of the compounds provided are within the scope of the present disclosure. For example, in some cases, the compounds provided exhibit one or more stereoisomers of the compounds, and each stereoisomer is represented, individually and/or as a mixture, unless otherwise indicated. Unless otherwise indicated, all tautomeric forms of the provided compounds are within the scope of the disclosure.

Unless otherwise indicated, structures depicted herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structure (including substitution of deuterium or tritium for hydrogen, or 13C or 14C enriched carbon for carbon) are within the scope of the present disclosure.

Detailed Description

Incretins and their use in the treatment of diseases

Incretins are peptide hormones released in the Gastrointestinal (GI) tract in response to glucose consumption, which stimulate the pancreas to secrete insulin and reduce glucagon production, thereby lowering blood glucose levels. Incretins exert their effects by binding to their corresponding receptors on pancreatic beta cells, resulting in insulin release. Glucagon-like peptide-1 (GLP 1) and glucose-dependent insulinotropic polypeptide (GIP) are two incretins that were found to play a role in postprandial insulin secretion. GIP is primarily responsible for the release of insulin in response to glucose uptake. GLP1 stimulates satiety, slows gastric emptying, reduces glucagon secretion, and reduces food intake, resulting in weight loss. It has been shown that activation of the GIP and GLP1 receptors produces a cumulative effect when insulin is secreted. See Chim, US pharm.2022; 47 (10): 18-22, which is incorporated herein by reference in its entirety.

Due to their role in controlling blood glucose, satiety, etc., incretins and incretin mimetics have the potential to treat a variety of diseases including obesity, pre-diabetes, type 2 diabetes (T2D, and complications thereof), early stage type 1 diabetes (e.g., within 3 months after diagnosis of T1D), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease (e.g., characterized by major cardiovascular events (MACEs), including CV death, non-fatal myocardial infarction, non-fatal stroke, or heart failure with ejection fraction retention (HFpEF)), kidney disease, and an elevated risk of premature death. Such chronic diseases are prevalent throughout the world and often exist as co-diseases.

Obesity and obesity control method

Obesity is the most common chronic disease worldwide affecting about 6.5 hundred million adults. Obesity is considered as a starting point and key contributor to pre-diabetes, type 2 diabetes (T2D, and its complications), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease and renal disease, and premature death. Obesity can place considerable economic burden, including additional direct medical costs, productivity costs (absences, attendance, disability support, premature death), transportation costs (including increased CO ₂ footprint), human capital accumulation costs (school absences, highest education achieved). It is estimated that by 2030 the number of obese (BMI >30kg/m ²) people will be over one billion, of which about 10% will have severe class III obesity (BMI >40kg/m ²). Half of all men with obesity live in only nine countries, U.S., china, india, brazil, mexico, russian, egypt, germany and Turkish. Childhood obesity also rises dramatically throughout the world.

Obesity was declared as a disease only in 2011 by the american society of clinical endocrinologists (American Association of Clinical Endocrinologists) (AACE) and is managed based on severity, beginning with lifestyle/behavioral interventions, and increasing physical activity, followed by pharmacotherapy, and finally bariatric surgery.

Based on short-term studies, overweight or obese patients with T2D were suggested to lose weight. Look AHEAD studies investigating the long-term effects of weight loss on cardiovascular disease in more than 5100 people with T2D stopped in vain after almost 10 years 2012, as it was shown that intensive lifestyle interventions focused on weight loss did not reduce the rate of cardiovascular events in overweight or obese adults with T2D.

In summary, to date, drug therapies have had limited success for obesity with only modest placebo-corrected weight loss, e.g., 3% for Xenical (orlistat), 4-5% for Belviq (lorcaserin) and Contrave (naltrexone SR (naltrexone SR)/bupropion SR (bupropion SR)), while also having social limiting side effects of Xenical and adverse central nervous effects of the central acting agents. Acomplia (rimonabant) was refused by the FDA because there is a concern that use of it might increase suicidal ideation and depression.

The most effective intervention for obesity remains bariatric surgery, however, only 1% of eligible patients undergo the surgery due to perceived serious complications (including mortality). Bariatric surgery has been shown to significantly alter endocrine hormone release, which has stimulated research interest in the endogenous trophic stimulation hormone pathway, aimed at essentially mimicking the effects of weight loss surgery through a chemical agent called an "incretin mimetic".

Existing treatments using incretin mimetics

Current therapies using incretin mimetics include glucagon-like peptide-1 (GLP 1) receptor agonists such as Trulicity (dulcin) (dulaglutide)), byetta (exenatide), ozempic/Rybelsus (injectable/oral semaglutinin (semaglutide)), victoza (liraglutide (liraglutide)) and Suliqua (liraglutide (lixisenatide), in combination with insulin glargine alone), which are approved for lowering blood glucose in humans with T2D without the need for continuous examination of blood glucose levels. Additional benefits are weight loss (2-4%) and positive effects on cardiovascular and renal parameters. Recent studies combine GLP1 receptor agonists with GIP receptor agonists and/or glucagon (GCG) receptor agonists (dual/triple agonists) activity with the aim of better controlling blood glucose and greater degrees of weight loss. Glycemic control and weight loss were demonstrated with the GLP1/GCG receptor dual agonist SAR425899, however, the procedure was discontinued due to unacceptable gastrointestinal side effects in 2019. Recently, the GLP1/GIP receptor dual agonist telpofungin (tirzepatide) (now marketed as Mounjaro) was approved as an injectable drug for adults with T2D, which is used to improve blood glucose along with diet and exercise. GIP functions to regulate energy balance through cell surface receptor signaling in brain and adipose tissue. SURPASS-2 studies demonstrated the non-inferior efficacy and superiority of telipopeptide relative to semaglutin in lowering blood glucose. However, weight loss is only a secondary endpoint.

Phase 3 trials have been conducted in non-diabetic, overweight/obese patient populations with weight loss as the primary endpoint-see the SCALE (liraglutide), STEP-1 (semraglutide) and SURMOUNT-1 (telpopeptide) trials. The SCALE trial demonstrated 5.4% placebo-corrected weight loss and less progression to prediabetes at a dose of 3 mg liraglutide once daily plus lifestyle intervention. The STEP-1 test produced 12.4% weight loss in overweight or obese participants who were 2.4 mg semaglutin per week plus lifestyle intervention. The SURMOUNT-1 trial showed 17.8% placebo-corrected weight loss at the highest weekly dose of 15 mg telipopeptide. Cardiac metabolism measurements were improved in all studies. Gastrointestinal side effects are as expected, especially at the beginning of treatment, and are manageable. Approved obese products now include Saxenda (liraglutide injected 3 mg a day) and Wegovy (semraglutide injected once a week). The FDA granted rapid channel qualification for telpofungin for treating adults with obesity or overweight with weight-related co-morbidity 10 at 2022, which may result in approval of the indication at the beginning of 2024 based on rolling submissions of further data of the SURMOUNT study series. Topline results recently published from the SELECT test Wegovy ℃ was tested in patients with established cardiovascular disease and overweight/obese but without T2D, and 2.4. 2.4 mg semaglutin was found to reduce the risk of major adverse cardiovascular events in overweight or obese adults by 20%. Another study using an incretin mimetic is OASIS-1, which is a study of oral administration of 25 or 50 mg semaglutin in non-diabetic overweight/obese patients. 14 Oral semaglutin at a mg maintenance dose has been approved as Rybelsus for T2D. Phase 2 results for the triple receptor agonist (GIP/GLP 1/GCG receptor) ritalunin (retratutide) LY3437943 have recently been disclosed, showing 22.1% placebo-corrected weight loss in people with obesity 48 weeks after treatment. It has also recently been shown that semaglutin shows benefits in early T1D (within 3 months after T1D diagnosis). In a small study, all participants no longer needed prandial insulin, and most participants no longer needed basal insulin.

Exemplary treatments for obesity/T2D using incretin mimetics are shown in table 1 below.

TABLE 1 exemplary incretin mimetics for treating obesity/T2D

The present disclosure recognizes, among other things, current problems in the incretin mimetic market for the treatment of obesity, pre-diabetes, T2D, early T1D, NAFLD, NASH, cardiovascular disease, kidney disease, and elevated risk of premature death, including, but not limited to, limited supply, high price, lack of health insurance covering such treatments, frequent injections (e.g., once a week), high injection volumes, and gastrointestinal side effects.

The present disclosure provides, inter alia, more effective and cost-effective ways of using incretins and incretin mimetics for the treatment of obesity, pre-diabetes, T2D, early T1D, NAFLD, NASH, cardiovascular disease, kidney disease, and increased risk of premature death by delivering incretins and incretin mimetics encoded by one or more polyribonucleotides (collectively encompassed by the term "incretin agent"). In some embodiments, the polyribonucleotides encoding an incretin agent are used in the therapeutic treatment of obesity and/or pre-diabetes, T2D, early T1D, NAFLD, NASH, cardiovascular disease, kidney disease, and elevated risk of premature death (e.g., obesity-related disease) with improved properties compared to known incretin mimetic therapies, including requiring fewer injections (no more than once a week), lower injection volumes (e.g., no more than 0.5 ml), and fewer or less severe side effects. In addition, the polyribonucleotides used to deliver the incretins provide expression of the incretins in cells at therapeutically relevant levels, comparable to the dosages of current peptide-based therapies.

For delivery of incretins polyribonucleotides of agents

The present disclosure utilizes, inter alia, RNA technology as a modality for direct expression in an individual of an incretin agent that activates GLP1, GIP and/or GCG receptors as a novel class of therapeutic agents effective in treating disease states such as obesity, pre-diabetes, T2D, early T1D, NAFLD, NASH, cardiovascular disease, kidney disease and/or elevated risk of premature death. In some embodiments, a polyribonucleotide as described herein encodes an incretin agent or fragment or mutant thereof found in nature. In some embodiments, a polyribonucleotide as described herein encodes an incretin agent or fragment or mutant thereof that has been modified from its native form.

As used herein, the term "incretin agent" refers to an agent comprising incretin or an incretin mimetic (wherein incretin and incretin mimetic are collectively referred to herein as "incretin peptides"). Exemplary incretins include GLP1, GIP and GCG. Exemplary incretin mimetics are shown, for example, in table 1. In some embodiments, the incretin agent is a biologically active portion or fragment of incretin or incretin mimetic. In some embodiments, the incretin agent comprises an incretin peptide as part of a fusion. For example, in some embodiments, the incretin agent is an incretin peptide fused to another peptide moiety (e.g., a half-life extending (HLE) domain).

In some embodiments, the incretin agent comprises a GLP1 receptor agonist, such as GLP1. In some embodiments, the incretin agent comprises a GIP receptor agonist, such as GIP. In some embodiments, the incretin agent comprises dual GIP and GLP1 receptor agonists. In some embodiments, the incretin agent comprises a triple GIP, GLP1, and GCG receptor agonist.

Exemplary incretin peptides

In some embodiments, the incretin agent comprises a wild-type (i.e., unmutated) incretin peptide sequence or fragment thereof. For example, in some embodiments, the incretin agent comprises any of the incretin peptides as set forth in SEQ ID NOs 5-15 and 62-64.

In some embodiments, the incretin agent comprises an incretin peptide or fragment thereof having at least one mutated amino acid residue compared to a wild-type reference sequence. In some embodiments, the mutated amino acid residue comprises a substitution of a natural amino acid residue with another natural amino acid residue. In some embodiments, the incretin agent comprises any of the incretin peptides as set forth in SEQ ID NOs 5-10. In some embodiments, the mutated amino acid residue may confer dual or triple activation properties to the incretins. For example, in some embodiments, one or more amino acid substitutions are introduced into the GLP1, GIP or GCG peptide sequences (e.g., as shown in SEQ ID NOS: 12-15 and 62-64) to impart binding properties to two or more of the GLP1, GIP and/or GCG receptors.

In some embodiments, the incretin agent has an amino acid sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to any of the incretin peptides detailed in table 2. In some embodiments, the incretin agent comprises any one of the incretin peptides detailed in table 2 below, or a combination or mutant thereof.

TABLE 2 exemplary incretin peptides (mutations shown in bold)

Connecting peptide

In some embodiments, the incretins described herein include a single incretin peptide (this configuration is referred to herein as "I:1 x") (see, e.g., FIG. 3). In some embodiments, the incretin peptide is fused to another peptide (e.g., half-life extension (HLE) domain) via a linker peptide (see, e.g., fig. 4-14). In some embodiments, the linker peptide contains at least one Gly (G) amino acid residue. Suitable linking peptides can be readily selected and can have a variety of lengths, such as 1 amino acid (e.g., gly) to 25 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids (e.g., at least 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids). Exemplary linking peptides include glycine polymer (G) _n, glycine-serine polymers (including, for example, (GS) _n、(GGGGS：SEQ ID NO: 1)_n and (GGGS: SEQ ID NO: 2) _n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linking peptides known in the art. Glycine and glycine-serine polymers are relatively unstructured and are therefore capable of acting as neutral tethers between components. Glycine is even closer to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, rev. Computational chem. 11:173-142 (1992)). In some embodiments, the linker peptide comprises the amino acid sequence of SEQ ID NO 3 (GGGGSGGGGSGGGGGGGS or "(G ₄S)₄" linker peptide) or SEQ ID NO 4 (GGGGSGGGGSGGGGSGGGGSGGGGS or "(G ₄S)₅" linker peptide). In some embodiments, the linker peptide comprises the amino acid sequence of SEQ ID NO. 68 (GGGGSGGGGS or "(G ₄S)₂" linker peptide), SEQ ID NO. 156 (GGGSGGGS or "(G ₃S)₂" linker peptide), SEQ ID NO. 157 (GGGGSGGGGSGGGGGGS or "(G ₄S)₃" linker peptide), or GGGGSGGGS (SEQ ID NO. 186).

In some embodiments, the incretin agent comprises an incretin peptide using (G ₄S)₃ linker peptide linked to another peptide (e.g., HLE domain as described herein), the incretins described herein include incretins peptides using (G ₄S)₂ linker peptide linked to another incretins peptide).

Cleavage site

In some embodiments, the incretin peptide is fused to another peptide (e.g., another incretin peptide and/or half-life extension (HLE) domain) via a protease cleavage site, such as a furin cleavage site (e.g., a peptide comprising the motif R-X-K/R-R SEQ ID NO: 158, e.g., SEQ ID NO: 160 RRKR or SEQ ID NO: 153 NVRRKR) and optionally any of the above-described linking peptides. In some embodiments, the protease cleavage site (e.g., furin cleavage site) comprises any of SEQ ID NO: 160 RRKR, SEQ ID NO: 153 NVRRKR, SEQ ID NO: 189 RKKR, SEQ ID NO:190 RMQR, or SEQ ID NO: 191 VFRR. The terms "furin cleavage site" and "furin recognition site" are used interchangeably herein and refer to a sequence that facilitates furin cleavage.

In some embodiments, the furin cleavage site is operably linked to a linker peptide (e.g., a glycine linker peptide, e.g., (G ₄S)₂ linker peptide) (e.g., on its C-terminal side). In some embodiments, the incretin agent comprises a plurality (e.g., 2,3,4, or more) incretin peptides, each separated by a furin cleavage site and optionally a linker peptide (e.g., (G ₄S)₂ linker peptide) on the N-terminal side of the furin cleavage site).

Included within the incretin agents described herein are furin cleavage sites, particularly where the incretin peptide is fused to another peptide (e.g., another incretin peptide and/or a half-life extension (HLE) domain), facilitating proper cleavage of the incretin peptide from the other peptide and allowing the incretin peptide to be fully processed and functional upon its expression.

Without wishing to be bound by any theory, in the context of the polyribonucleotides encoding the incretin agent, the cleavage site and the type of cleavage site or recognition site are important to ensure that the N-terminus of the incretin peptide is correctly processed. Certain furin cleavage and recognition sites, as well as placement of those sites relative to the incretin peptide within the incretin agent, can create alternative processing or cleavage sites, ultimately altering the final amino acid sequence of the mature incretin peptide. In such relatively small peptides, such as GLP1 or GIP (or mutants thereof, and other peptides of similar size/nature), any change in amino acid residues can affect the biological activity of the peptide. In some embodiments, the furin recognition/cleavage site is selected and positioned within the incretin agent so as to facilitate proper cleavage of the N-terminus of the incretin peptide, or in other words, to produce a "scarless" N-terminus of the incretin peptide so as to maintain the bioactivity of the incretin peptide. FIGS. 20 and 21 show schematic representations of the locations of theoretical cleavage sites for certain exemplary signal peptides. FIG. 20 indicates that the A8G mutation promotes correct N-terminal processing of GLP1 incretin peptides with husec signal peptide. Figure 21 indicates that the A2G mutation promotes correct N-terminal processing of GIP incretin peptide with husec signal peptide.

This concept and utilization of furin cleavage site at the N-terminus of an incretin peptide linked to another peptide can also be applied to other intestinal peptides (e.g., glucagon) and/or other peptides of comparable size/nature to GLP1 and GIP described herein. This is particularly important in the context of delivering an incretin agent (or other similar peptide) as one or more polyribonucleotides encoding an incretin agent. In addition to post-translational processing (including proper cleavage of one incretin peptide from another), such delivery requires proper translation of the intracellular protein. In some embodiments, an incretin agent comprising one or more incretin peptides fused to another peptide as described herein has been designed and generated to include a protease cleavage site disposed within the incretin agent such that cleavage of the incretin peptide from the other peptide is accurate and does not affect the amino acid sequence of the mature peptide (i.e., yields a scar-free N-terminus). The "scarless" N-terminus referred to herein includes peptides that have been cleaved from another peptide by a cleavage site, and wherein cleavage occurs in such a way that there are no remaining amino acids that are not part of the mature peptide and all amino acids of the mature peptide remain at the N-terminus of the peptide. The scarless N-terminus of incretin peptides (and other similar peptides, e.g., other intestinal peptides, e.g., glucagon) allows the peptide to function properly after processing into a mature peptide.

In some embodiments, the furin cleavage site is placed immediately 5' of the second incretin peptide in the incretin agent to ensure cleavage produces a scar-free N-terminus on the second incretin peptide. Such cleavage may be important for maintaining the function of the mature incretin peptide.

In some embodiments, the furin cleavage site is selected to be compatible with the N-terminal sequence of an incretin peptide (e.g., a wild-type or mutant incretin peptide, e.g., GIP with an A2G mutation or GLP1 with an H1Y mutation and/or an A8G mutation). Mutations as described herein may be introduced into the incretin peptide in order to promote efficient cleavage and maintain the scar-free N-terminus of the incretin peptide.

As disclosed herein, various furin cleavage sequences may be utilized to facilitate proper cleavage. For example, in some embodiments, the furin cleavage site is, for example, NVRRKR (SEQ ID NO: 153), which is derived from human MT-MMP 1 protein. Such furin cleavage sites are derived from human proteins and are compatible with the N-terminal sequences of GIP and GLP1 incretin peptides, including wild-type and mutant GIP and GLP1 incretin peptides. Other human furin cleavage sequences may be utilized, as different human furin cleavage sequences may exhibit different cleavage efficiencies depending on adjacent amino acid sequences (see Izidoro et al, archives of biochemistry and biophysics 2009, 487.2, 105-114, which are incorporated herein by reference in their entirety).

In some embodiments, mutations are introduced into the incretin peptides described herein to promote cleavage of the signal peptide and to produce mature incretin peptides with a scar-free N-terminus. In some embodiments, such mutations include A2G mutations in a GIP incretin peptide (e.g., GIP (1-42) incretin peptide). In some embodiments, such mutations include an A8G mutation in the GLP1 (7-37) incretin peptide. In some embodiments, such mutations may also extend the half-life of the incretin agent, for example, by preventing proteolysis of amino acids in the second position of the incretin peptide (thereby avoiding the generation of a truncated mature incretin peptide that is incorrectly cleaved at the N-terminus and lacks 1 or 2 amino acids). In some embodiments, the mutation is selected such that it increases the probability of correct cleavage (i.e., cleavage at the N-terminus such that the mature incretin peptide is not truncated). In some embodiments, the compatibility of the signal peptide and cleavage site sequences used in the incretins agents described herein depends on the particular adjacent incretin peptide amino acid sequence, particularly the amino acid residue at the N-terminus.

Incretin agents with multiple incretin peptides

In some embodiments, the incretin agent comprises a single incretin peptide. In some embodiments, the incretin agent comprises or more than one incretin peptide. In some embodiments, two or more of the incretin peptides included in the incretin agents described herein are the same or are derived from the same incretin peptide (e.g., are GLP1 receptor agonists). In some embodiments, two or more incretin peptides included in an incretin agent described herein are different or are derived from different incretin peptides.

In some embodiments, the incretin agent comprises a combination of incretin peptides, e.g., fused to a single polypeptide chain. For example, in some embodiments, the incretin agent comprises a GLP1 receptor agonist (e.g., a GLP1 peptide or fragment or mutant thereof) and a GIP receptor agonist (e.g., a GIP peptide or fragment or mutant thereof). In some embodiments, the incretin agent comprises one or more incretin peptides selected from the group consisting of SEQ ID NOs 5-15 and 62-64 and one or more incretin peptides selected from the group consisting of SEQ ID NOs 5-15 and 62-64. In some embodiments, the incretin agent comprises a GLP1 receptor agonist (e.g., a GLP1 peptide or fragment or mutant thereof), a GIP receptor agonist (e.g., a GIP peptide or fragment or mutant thereof), and a GCG receptor agonist (e.g., a fragment of a GCG peptide or mutant thereof). In some embodiments, the incretin agent comprises more than one GLP1 receptor agonist (e.g., GLP1 peptide or fragment or mutant thereof) that are the same or different. In some embodiments, the incretin agent comprises more than one GIP receptor agonist (e.g., a GIP peptide or fragment or mutant thereof) that are the same or different. In some embodiments, the incretin agent comprises more than one copy of the same incretin peptide and/or a combination of different incretin peptides.

In some embodiments, an incretin peptide is fused to another incretin peptide by a linker peptide. In some embodiments, the linker peptide contains at least one Gly (G) amino acid residue. Suitable linking peptides can be readily selected and can have a variety of lengths, such as1 amino acid (e.g., gly) to 25 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids). Exemplary linking peptides include glycine polymer (G) _n, glycine-serine polymers (including, for example, (GS) _n、(GGGGS：SEQ ID NO: 1)_n and (GGGS: SEQ ID NO: 2) _n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linking peptides known in the art. Glycine and glycine-serine polymers are relatively unstructured and are therefore capable of acting as neutral tethers between components. Glycine is even closer to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, rev. Computational chem. 11:173-142 (1992)). In some embodiments, the linker peptide comprises the amino acid sequence of SEQ ID NO 3 (GGGGSGGGGSGGGGGGGS or "(G ₄S)₄" linker peptide) or SEQ ID NO 4 (GGGGSGGGGSGGGGSGGGGSGGGGS or "(G ₄S)₅" linker peptide). In some embodiments, the linker peptide comprises the amino acid sequence of SEQ ID NO. 68 (GGGGSGGGGS or "(G ₄S)₂" linker peptide), SEQ ID NO. 156 (GGGSGGGS or "(G ₃S)₂" linker peptide), SEQ ID NO. 157 (GGGGSGGGGSGGGGGGS or "(G ₄S)₃" linker peptide), or GGGGSGGGS (SEQ ID NO. 186).

In some embodiments, the incretin peptide is fused to another incretin peptide via a protease cleavage site, such as a furin cleavage site (e.g., a peptide comprising the motif R-X-K/R-R SEQ ID NO: 158, e.g., RRKR SEQ ID NO: 160 or SEQ ID NO: 153 NVRRKR) and optionally any of the above-described linking peptides. In some embodiments, the protease cleavage site (e.g., furin cleavage site) comprises any of SEQ ID NO: 160 RRKR, SEQ ID NO: 153 NVRRKR, SEQ ID NO: 189 RKKR, SEQ ID NO:190 RMQR, or SEQ ID NO: 191 VFRR. In some embodiments, the furin cleavage site is operably linked to a linking peptide (e.g., a glycine linking peptide, e.g., (G ₄S)₂ linking peptide) (e.g., 3 'thereof), hi some embodiments, the incretin agent comprises a plurality (e.g., 2, 3, 4, or more) of incretin peptides, each separated by a furin cleavage site and optionally a linking peptide (e.g., a (G ₄S)₂ linking peptide) located 5' of the furin cleavage site).

In some embodiments, the polyribonucleotide encodes an incretin agent comprising a signal peptide and a single incretin peptide (this configuration is referred to herein as "I:1 x") (see, e.g., FIG. 3). In some embodiments, the polyribonucleotide encodes an incretin agent comprising a signal peptide and two incretin agents (this configuration is referred to herein as "I:2 x") separated by a linker peptide and a cleavage site (e.g., a furin cleavage site) (see, e.g., fig. 4). This design allows cleavage of the first incretin peptide from the second incretin peptide after translation of the peptide from the polyribonucleotide. In some embodiments, the polyribonucleotide encodes an incretin agent comprising a signal peptide and four incretin agents each separated by a linking peptide and a cleavage site (e.g., a furin cleavage site) (this configuration is referred to herein as "I:4 x") (see, e.g., fig. 5). This design allows cleavage of four incretin peptides after translation of the incretin peptide from the polyribonucleotide. It will be appreciated by those skilled in the art that while incretins comprising 1,2 and 4 incretins are specifically described herein, incretins having different numbers of incretins separated by cleavage sites and linking peptides may be used. In any of the designs shown in fig. 3-5, the incretin peptide can be any incretin peptide described herein (e.g., GLP1 or GIP incretin peptide, e.g., any of the incretin peptides shown in table 2).

In some embodiments, the polyribonucleotides described herein encode a GIP incretin peptide (e.g., any of the GIP incretin peptides described in table 2) that is upstream or 5' of a GLP1 incretin peptide (e.g., any of the GLP1 incretin peptides described in table 2). In some embodiments, the polyribonucleotides described herein encode a GLP1 incretin peptide (e.g., any of the GLP1 incretin peptides described in table 2) that is upstream or 5' of a GIP incretin peptide (e.g., any of the GIP incretin peptides described in table 2). In some embodiments, the order of the incretin peptides (N-terminal to C-terminal direction) is determined by the manner in which cleavage of the incretin peptide is expected such that the incretin peptide maintains its amino acid sequence and scar-free N-terminal.

In some embodiments, the polyribonucleotides described herein encode an incretin agent having an amino acid sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to any of the incretins agents detailed in table 3. In some embodiments, the polyribonucleotides described herein encode an incretin agent having an amino acid sequence set forth in any one of the incretin agents detailed in table 3.

TABLE 3 exemplary incretin agents comprising more than one incretin peptide (mutations shown in bold, linker peptides shown underlined, furin cleavage sites shown in italics), wherein examples x2 and x4 include linker peptides between each repeat unit and furin cleavage sites

In some embodiments, the present disclosure provides one or more polyribonucleotides encoding an incretin agent comprising a combination of incretin peptides. In such embodiments, one or more polyribonucleotides may encode an incretin agent. In some embodiments, the first polyribonucleotide may encode a first incretin peptide of an incretin agent and the second polyribonucleotide may encode a second incretin peptide of an incretin agent.

Half-life extension (HLE) domains

In some embodiments, the polyribonucleotides described herein encode an incretin agent comprising one or more incretin peptides fused to a half-life extension (HLE) domain (see, e.g., incretin agents shown in fig. 7-14). In some embodiments, where the incretin agent comprises more than one incretin peptide, the HLE domain may be included in the incretin agent described herein to increase the half-life of one or each incretin peptide.

Human Serum Albumin (HSA)

In some embodiments, the incretin agent comprises one or more incretin peptides fused to an HLE domain comprising albumin (e.g., human Serum Albumin (HSA)). In some embodiments, the half-life extending moiety comprises albumin, e.g., human serum albumin. In some embodiments, the Human Serum Albumin (HSA) sequence has at least 90%, 95% or 99% identity to the amino acid sequence shown as SEQ ID NO: 159 (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL), or a fragment or mutant thereof. In some embodiments, the HSA sequence comprises or consists of the amino acid sequence set forth in SEQ ID NO 159 or a fragment or mutant thereof. In some embodiments, the HSA sequence comprises or consists of an amino acid sequence that is a mutant of wild type HSA (i.e., SEQ ID NO: 159) comprising one or more amino acid mutations. In some embodiments, the one or more mutations comprise a mutation at position 573 of SEQ ID NO. 159. In some embodiments, the K residue at position 573 of SEQ ID NO. 159 is substituted with any one of the following amino acid residues A, C, D, F, G, H, I, L, M, N, P, Q, R, S, V, W and Y (SEQ ID NO: 187). In some embodiments, the K residue at position 573 of SEQ ID NO. 159 is substituted with a P residue (SEQ ID NO. 188). In some embodiments, the HSA mutant comprises any of the HSA mutants disclosed in U.S. patent No. 8,748,380, which is hereby incorporated by reference in its entirety.

In some embodiments, the polyribonucleotides described herein encode an incretin agent as shown in fig. 7 fused to an HLE domain (e.g., HSA or HSA mutant as described herein), including the I:1x, I:2x, or I:4x configuration (i.e., 1, 2, or 4 incretin peptides). In some embodiments, the incretin peptide can be a GLP1 or GIP incretin peptide described herein or a mutant thereof. In some embodiments, where the incretin agent comprises more than one incretin peptide, the incretin peptide adjacent to the HLE domain will remain fused to the HLE domain after post-translational processing, and the incretin peptide not adjacent to the HLE domain will be cleaved from the adjacent incretin peptide and HLE domain. Such designs may be used when it is desirable to administer multiple incretin peptides having various half-lives. Such a design may also be desirable where one of the incretin peptides is intended to cross the blood brain barrier (i.e., where the HLE domain is undesirable) and one of the incretin peptides is intended to remain in circulation for a longer period of time (i.e., where the HLEs remain connected).

In some embodiments, the incretin agent comprises more than one incretin peptide separated by a connecting peptide and a protease cleavage site (e.g., a furin cleavage site), and one of the incretin peptides is a GLP1 peptide described herein adjacent to an HLE domain (e.g., HSA or HSA mutant), and one of the incretin peptides is a GIP peptide not adjacent to an HLE (e.g., at the N-terminus of a polypeptide chain). In such embodiments, when the incretin is expressed from the polyribonucleotides described herein, the GIP peptide will be cleaved from the GLP1 peptide linked to the HLE domain such that the GLP1 incretin peptide will have a longer half-life than the GIP incretin peptide. In some embodiments, the incretin agent comprises more than one incretin peptide separated by a connecting peptide and a protease cleavage site (e.g., a furin cleavage site), and one of the incretin peptides is a GIP incretin peptide described herein adjacent to an HLE domain (e.g., HSA or HSA mutant), and one of the incretin peptides is a GLP1 peptide not adjacent to an HLE (e.g., at the N-terminus of a polypeptide chain). In such an embodiment, when the incretin is expressed from the polyribonucleotides described herein, the GLP1 incretin peptide will be cleaved from the GIP peptide linked to the HLE domain such that the GIP incretin peptide will have a longer half-life than the GLP1 incretin peptide (see, e.g., fig. 9).

In some embodiments, the polyribonucleotides described herein encode an incretin agent as shown in fig. 8A, wherein the incretin agent has a signal peptide ("SP"), a GLP1 incretin peptide, a linker peptide, a second GLP1 incretin peptide, a second linker peptide (GGGS) ₃, and a half-life extension (HLE) domain that is Human Serum Albumin (HSA) or a mutant of HSA. In addition, the incretin agent may include a protease cleavage site (e.g., a furin cleavage site) between GLP1 incretin peptides. In such embodiments, upon expression of the incretin agent, the first (N-terminal) GLP1 incretin peptide will be cleaved from the second incretin GLP1 incretin peptide adjacent to the HLE domain. The resulting GLP1 incretin peptide will have two different half-lives (i.e., the GLP1 incretin peptide that remains attached to the HLE domain will have a longer half-life than the cleaved GLP1 incretin peptide).

In some embodiments, the polyribonucleotides described herein encode an incretin agent as shown in fig. 9, wherein the incretin agent comprises a Signal Peptide (SP), a first GLP1 incretin peptide, a linking peptide (GGGS) ₂, a first GIP incretin peptide, a second linking peptide (GGGS) ₂, a second GLP1 incretin peptide, a third linking peptide (GGGS) ₂, a second GIP incretin agent, a fourth linking peptide (GGGS) ₃, and a half-life extension (HLE) domain that is Human Serum Albumin (HSA) or an HSA mutant. Furin and SP cleavage sites within the incretins are indicated by arrows. In such embodiments, when expressing the incretin agent, the first GLP1 incretin peptide, the first GIP incretin peptide, and the second GLP1 incretin peptide will cleave, and the second GIP incretin peptide will remain fused to the HLE domain. The resulting second GIP-HLE fusion will have a longer half-life than the other incretin peptides.

In some embodiments, a polyribonucleotide described herein encodes an incretin agent comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the incretin agent sequences in table 4. In some embodiments, the incretins comprise any one of the incretins detailed in table 4 below, or a combination or mutant thereof.

TABLE 4 exemplary incretins comprising hAlbumin (mutations shown in bold, linker peptides shown underlined, furin cleavage sites shown in italics), wherein examples x2 and x4 include linker peptides between each repeat unit and furin cleavage sites

Albumin binding domains

In some embodiments, the incretin peptide is fused to a half-life extending moiety that binds albumin. Various albumin binding moieties (i.e., albumin binding protein domains) may be used as half-life extending moieties for the incretins described herein (see, e.g., zorzi et al, medChemComm, 2019, 10.7, 1068-1081, which is incorporated herein by reference in its entirety). In some embodiments, the albumin binding protein domain comprises an Albumin Binding Domain (ABD) derived from protein G of streptococcus strain GI48 and/or derived from protein PAB of goldenseal, as described in ABD035 and SA21 (e.g., levy et al, PLoS One, 2014, 9 (2), e87704, which are incorporated herein by reference in their entirety) and ABD094 (NCT 02690142) (e.g., frejd and Kim, exp. Mol. Med., 2017, 49 (3), e306, which are incorporated herein by reference in their entirety). In some embodiments, ABD binds to domain II of human serum albumin and does not overlap or interfere with binding to the FcRn binding site on albumin.

In some embodiments, the ABD comprises an ABDCon (triple helix bundle albumin binding domain), as described in Jacobs et al, protein eng., des. In some embodiments, the ABD is derived from a bacterial protein Sso7d from the hyperthermophilic archaea sulfolobus, such as M11.12 and M18.2.5 (as described in Gao et al, nat. Struct. Biol., 1998, 5 (9), 782-786 and Traxlmayr et al, j. Biol. Chem., 2016, 291 (43), 22496-22508, which is incorporated herein by reference in its entirety). In some embodiments, the ABD comprises a DARPin, as described in plurkthun, annu, rev, pharmacol, toxicol, 2015, 55, 489-511, which is incorporated by reference herein in its entirety.

In some embodiments, the ABD comprises an immunoglobulin domain or fragment thereof. In some embodiments, the ABD comprises a fully human domain antibody (dAb). For example, in some embodiments, the ABD comprises an AlbudAb, as described in Holt et al, protein eng, des. In some embodiments, the ABD comprises a Fab, such as dsFv CA645, as described in Adams et al, mAbs, 2016, 8 (7), 1336-1346, which is incorporated herein by reference in its entirety.

In some embodiments, the ABD comprises a heavy chain only (VHH) antibody, i.e., a nanobody, as described in Steeland et al, drug Discovery Today, 2016, 21 (7), 1076-1113, which is incorporated herein by reference in its entirety. In some embodiments, the ABD comprises a VHH domain comprising one or more of the Complementarity Determining Region (CDR) sequences HCDR1, HCDR2 and/or HCDR3 as shown in SEQ ID NO: 191 (GFTLDYYA), SEQ ID NO: 192 (IASSGGST) and/or SEQ ID NO: 193 (AAAVLECRTVVRGYDY), respectively. In some embodiments, the ABD comprises a VHH domain comprising the CDR sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 191 (GFTLDYYA), SEQ ID NO: 192 (IASSGGST) and SEQ ID NO: 193 (AAAVLECRTVVRGYDY), respectively. In some embodiments, the ABD comprises a VHH domain having at least 90%, 95% or 99% identity to an amino acid sequence as shown in SEQ ID NO: 154 EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS or "aHSA-VHH". In some embodiments, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO. 154. In some embodiments, the ABD comprises a VNAR, as described in Muller et al, mAbs, 2012, 4 (6), 673-685, which is incorporated herein by reference in its entirety.

In some embodiments, the polyribonucleotides described herein encode an incretin agent as shown in fig. 8B, which shows an incretin agent having a Signal Peptide (SP), a first GLP1 incretin peptide, a linker peptide, a second GLP1 incretin peptide, a second linker peptide (GGGS) ₃, and a half-life extension (HLE) domain that is a VHH domain that binds to HSA. Furin and SP cleavage sites within the incretins are indicated by arrows. In such embodiments, when the incretin agent is expressed, the first GLP1 incretin peptide is cleaved from the second GLP1 incretin peptide, and the second GLP1 incretin peptide remains fused to the HLE domain (anti-HSA VHH domain). Thus, the second GLP1 incretin peptide will have a longer half-life than the first GLP1 incretin peptide. In some embodiments, the incretins are those shown in fig. 8B, where one or both of the GLP1 incretins may instead be a different incretins (e.g., the GIP incretins described herein). In some embodiments, a single incretin peptide is fused to a VHH domain that binds to HSA.

Other albumin binding domains are known in the art, see, e.g., zorzi et al, medChemComm, 2019, 10.7, 1068-1081, which is incorporated herein by reference in its entirety.

In some embodiments, a polyribonucleotide described herein encodes an incretin agent comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the incretin agent sequences in table 5. In some embodiments, the incretins comprise any one of the incretins detailed in table 5 below, or a combination or mutant thereof.

TABLE 5 exemplary incretins comprising aHSA-VHH domains (mutations shown in bold, connecting peptides underlined)

XTEN

In some embodiments, the half-life extending moiety is an XTEN sequence as described in U.S. patent nos. 8,673,860 and Podust et al, journal of Controlled Release, 2016, 240, 52-66, which are incorporated herein by reference in their entirety.

In some embodiments, the XTEN sequence comprises from about 100 to about 3000 amino acid residues, preferably from 400 to about 3000 residues, wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of multiple units of two or more non-overlapping sequence motifs selected from the amino acid sequences of table 6 or table 7. In some cases, XTEN comprises a non-overlapping sequence motif, wherein about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of two or more non-overlapping sequences selected from the single motif family of table 6 or table 7, thereby producing a "family" sequence in which the overall sequence remains substantially non-repetitive. Thus, in such embodiments, the XTEN sequence can comprise multiple units of the AD motif family, or AE motif family, or AF motif family, or AG motif family, or AM motif family, or AQ motif family, or BC family, or BD family of non-overlapping sequence motifs of the sequences of table 6. In other cases, XTEN comprises motif sequences from two or more motif families of table 6. In other cases, XTEN comprises motif sequences from one or more motif families of table 7.

TABLE 6 XTEN sequence motif and motif family of 12 amino acids

TABLE 7 XTEN sequence motif and motif family of 12 amino acids

Fc domain with multiple polypeptide chains and incretin agents

In some embodiments, the half-life extending moiety is or comprises an Fc domain of, for example, a human IgG (e.g., human IgG1, igG2, igG3, or IgG 4). In some embodiments, the half-life extending moiety does not comprise an Fc domain of, for example, a human IgG (e.g., human IgG1, igG2, igG3, or IgG 4). In some embodiments, the half-life extending moiety comprises an Fc domain of human IgG4 or a mutant thereof (e.g., as included in a dolapride). In some embodiments, the Fc domain of an IgG4 sequence has at least 90, 95, 96, 97, or 99% identity to SEQ ID NO: 155 (AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG). In some embodiments, the Fc domain of the IgG4 sequence is or comprises the amino acid sequence shown as SEQ ID NO: 155.

Homodimeric incretins

In some embodiments where the incretin agent comprises more than one incretin peptide, the incretin agent comprises two or more incretin peptides on a single polypeptide chain. In some embodiments where the incretin agent comprises more than one incretin peptide, the incretin agent comprises one or more incretin peptides on separate polypeptide chains.

In some embodiments, the individual polypeptide chains multimerize (e.g., dimerize). In some embodiments in which the incretin agent comprises one or more incretin peptides on a single polypeptide chain, the single polypeptide chain comprises two polypeptide chains each comprising an immunoglobulin constant domain, and the two polypeptide chains dimerize via two constant domains that combine to make the Fc domain.

In some embodiments, the Fc domain comprises an IgG4 Fc domain (e.g., as included in a duloxetine). In some embodiments, the Fc domain comprises an IgG1 Fc domain. An exemplary design of an incretin comprising a plurality of polypeptide chains including an Fc domain such that the polypeptide chains dimerize is shown, for example, in fig. 10. The design in FIG. 10 may include an incretin peptide (or other numbers of incretin peptides may be available) in the I:1x, I:2x, or I:4x configuration, and each may be GLP1 or GIP incretin peptide (or any of the mutants described herein). In fig. 10, the two Fc domains are identical. The incretins on each polypeptide chain may be the same or different (or in the case of multiple incretins on a single chain, different combinations of incretins peptides may be contained). An exemplary incretin agent comprising two polypeptide chains that form a homodimer by dimerization of the Fc domains on each polypeptide chain is shown in fig. 11. In FIG. 11, the incretin peptide contains two polypeptide chains, each comprising a Signal Peptide (SP), GLP1 incretin peptide, a connecting peptide (GGGGS) ₃, and an Fc domain. In some embodiments, each polypeptide chain may comprise two or more incretin peptides (see, e.g., fig. 12). In some embodiments, two or more incretin peptides may be the same incretin peptide. In some embodiments, two or more incretin peptides may be different incretin peptides (see, e.g., fig. 12). Where two or more incretin peptides are included on each polypeptide chain, a cleavage site may be introduced to cleave the incretin peptide, leaving one incretin peptide linked to the Fc domain. Those skilled in the art will appreciate that an incretin peptide that remains linked to an Fc domain will have a longer half-life and different activity than other cleaved incretin peptides.

The Fc domains included in the incretins described herein not only allow dimerization of the two polypeptide chains, but may also increase the half-life of the incretins. Other mutations may also be introduced into the Fc domain to increase the half-life of the incretin agent.

Fc mutation of HLE

In some embodiments, the Fc domain within the incretin agent comprises one or more mutations to increase the half-life of the incretin agent. For example, in some embodiments, the Fc domain may include LS mutations within the CH3 region (for enhanced FcRn binding) (see Zalevsky et al, nature biotechnology, 2010, 28.2:157-159, which is incorporated herein by reference). Such mutations are numbered according to EU numbering as M428L and N434S (i.e., M88L and N94S within the CH3 domain), and are referred to herein as "LS". Exemplary incretins having such mutations are shown, for example, in fig. 11, 12 and 14.

In some embodiments, the Fc domain of an IgG4 (LS) sequence is identical to SEQ ID NO: 299 (AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG).

In some embodiments, the Fc domain in an incretin agent comprises one or more mutated amino acid residues that increase half-life. In some embodiments, the Fc domain comprises one of the following mutated amino acid residues, M252Y, S T and T256E ("YTE") according to the EU numbering scheme, to increase half-life. In some embodiments, the Fc domain comprises a combination of mutated amino acid residues, M252Y, S254T and T256E, according to the EU numbering scheme, to increase half-life.

In some embodiments, the Fc domain comprises one of the following mutated amino acid residues, T250Q and M428L ("QL") according to the EU numbering scheme, to increase half-life. In some embodiments, the Fc domain comprises one of the following mutated amino acid residues, H433K and N434F ("KF") according to the EU numbering scheme, to increase half-life. In some embodiments, the second Fc domain comprises one of the following mutated amino acid residues, T307A, E A and N434A ("AAA") according to the EU numbering scheme, to increase half-life. In some embodiments, the Fc domain comprises mutated amino acid residues V308P according to the EU numbering scheme to increase half-life. In some embodiments, the Fc domain comprises one of the following mutated amino acid residues, M252Y, V P and N434Y ("YPY") according to the EU numbering scheme, to increase half-life. In some embodiments, the Fc domain comprises one of the following mutated amino acid residues, H285D, T307Q and A378V ("DQV") according to the EU numbering scheme, to increase half-life. In some embodiments, the Fc domain comprises one of the following mutated amino acid residues, L309D, Q311H, N S ("DHS") according to the EU numbering scheme, to increase half-life. Exemplary Fc mutations are described, for example, in Liu et al, antibodies 9.4:64 (2020), which is hereby incorporated by reference in its entirety.

In some embodiments, a polyribonucleotide described herein encodes an incretin agent comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the incretin agent sequences in table 8. In some embodiments, the incretins comprise any one of the incretins detailed in table 8 below, or a combination or mutant thereof.

TABLE 8 exemplary incretins comprising IgG4 Fc domains (mutations shown in bold, linker peptides shown underlined, furin cleavage sites shown in italics), wherein x4 examples include linker peptides between each repeat unit and furin cleavage sites

Mutations that abrogate Fc effector function

In some embodiments, the Fc domain comprises one or more mutations that abrogate the effector function of the Fc domain included in the incretin agent. By eliminating the Fc effector function of the incretins, delivery of the incretins will be less likely to cause undesirable immune responses due to immune cells triggering cytotoxicity and other effector activities. In some embodiments, the Fc domain of the molecule comprises one or more mutations that silence effector function.

In some embodiments, the one or more mutations in the Fc domain comprise a "STR" modification, or a combination of mutations comprising L234S, L T and G236R according to the EU numbering scheme. When such mutations are introduced into the Fc domain, the Fc domain will exhibit little or no detectable binding to fcγ receptor or C1q and will not promote inflammatory cytokine responses (see, e.g., wilkinson et al, (2021) PLoS One 16.12:e 0260954, which is incorporated herein by reference in its entirety). Exemplary incretins comprising STR modifications are shown, for example, in figure 14.

In some embodiments, the modification that reduces or silences the effector function of an Fc domain included in an incretin agent described herein comprises one or more of the following mutations L234A, L235,235, 235A, P329, 329G, P329,329 329A, N297,297A or N297D according to the EU numbering scheme. In some embodiments, the modification that silences the effector function of the Fc domain comprises mutated amino acid residues L234A and L235A ("LALA") according to the EU numbering scheme. In some embodiments, mutations used to eliminate effector functions of the Fc domain include L234A/L235A/P329G ("LALAPG") according to EU numbering. In some embodiments, the modification that silences the effector function of the Fc domain comprises a mutated amino acid residue according to the EU numbering scheme, L234A, L A and P329A ("LALAPA"). In some embodiments, the modification to silence the effector function of the Fc domain further comprises N297A or N297D according to EU numbering. In some embodiments, modifications that silence the effector function of the Fc domain include Fc mutations of L234A/L235A/P329G and N297A according to EU numbering. In some embodiments, modifications that silence the effector function of the Fc domain include Fc mutations of L234A/L235A/P329G and N297D according to EU numbering. In some embodiments, modifications that silence the effector function of the Fc domain include Fc mutations L234A, L A and N297A, according to EU numbering. In some embodiments, modifications that silence the effector function of the Fc domain include Fc mutations L234A, L A and N297D according to EU numbering. In some embodiments, modifications that silence the effector function of the Fc domain include Fc mutations L234A, L, 235A, P329A and N297A according to EU numbering. In some embodiments, modifications that silence the effector function of the Fc domain include Fc mutations L234A, L, 235, A, P A and N297D according to EU numbering.

In some embodiments, the modification that reduces or silences the effector function of an Fc domain included in an incretin agent described herein comprises a mutation of L234F/L235E/P331S ("FES") according to the EU numbering scheme. In some embodiments, the modification that reduces or silences the effector function of an Fc domain included in an incretin agent described herein comprises a mutation of L234F/L235Q/K322Q ("FQQ") according to the EU numbering scheme. In some embodiments, the modification that reduces or silences the effector function of an Fc domain included in an incretin agent described herein comprises a mutation, A330S/P331S, according to the EU numbering scheme.

Those skilled in the art will appreciate that other modifications known in the art may be used to eliminate effector functions.

Heterodimeric incretins

In some embodiments, one or more of the polyribonucleotides described herein encode an incretin agent comprising more than one incretin peptide on separate polypeptide chains. In some embodiments, the individual polypeptide chains multimerize (e.g., dimerize). In some embodiments in which the incretin agent comprises one or more incretin peptides on a single polypeptide chain, the single polypeptide chain comprises two polypeptide chains each comprising an immunoglobulin constant domain, and the two polypeptide chains dimerize via two constant domains that combine to make the Fc domain.

In some embodiments, the Fc domain within the incretin agent comprises one or more mutations that induce dimerization. For example, in some embodiments, the incretin agent comprises a first polypeptide chain comprising an incretin peptide fused to a constant domain of an immunoglobulin, wherein the constant domain comprises one or more mutations that induce dimerization with a second polypeptide chain comprising an incretin peptide fused to a constant domain of an immunoglobulin. In some embodiments, the constant domains of both the first polypeptide and the second polypeptide contain one or more mutations that induce dimerization. In some embodiments, one or more of the incretin peptides in the first polypeptide and the second polypeptide are different.

One method of inducing dimerization is known as the "knob-to-hole technique" (KIH) which aims at forcing two different constant domains to pair by introducing mutations into the CH3 domain to modify the contact interface. On one CH3 domain, bulky amino acids are replaced with amino acids having short side chains to create a "mortar" and amino acids having large side chains are introduced into the other CH3 domain to create a "pestle". Co-expression of these two constant domains induces dimerization. In some embodiments, the Fc domains described herein utilize KIH techniques, such as those described in WO1998/050431, which is incorporated herein by reference in its entirety. As described herein, the Fc domain of an incretin agent may comprise certain mutations utilizing KIH technology, including, but not limited to, CH3 modifications. In some embodiments, the Fc domain of an incretin comprises a CH3 domain comprising one or more of Y349C, T366S, L A and Y407V (numbering according to EU). In some embodiments, the Fc domain of an incretin comprises a CH3 domain, wherein the CH3 domain comprises each of the following mutations Y349C, T366S, L A and Y407V (numbering according to EU). Such a combination of mutations is referred to herein as "FcKIH-b". In some embodiments, the Fc domain of an incretin comprises a CH3 domain comprising one or more mutations selected from the group consisting of S354C and T366W (numbering according to EU). In some embodiments, the Fc domain of an incretin comprises a CH3 domain, which CH3 domain comprises each of the following mutations S354C and T366W (numbering according to EU). Such a combination of CH3 mutations is referred to herein as "FcKIH-a". In some embodiments, the incretin agent comprises an Fc domain comprising a FcKIH-a sequence and a FcKIH-b sequence.

In some embodiments, the Fc domain within the incretin agent comprises one or more "KiH" mutations and LS mutations.

Thus, in some embodiments, an incretin agent encoded by one or more polyribonucleotides as described herein comprises one or more incretin peptides fused to an Fc domain, wherein the CH3 domain of the Fc domain comprises one or more of Y349C, T366S, L A and Y407V (numbering according to EU). In some embodiments, an incretin agent encoded by one or more polyribonucleotides as described herein comprises one or more incretin peptides fused to an Fc domain, wherein the Fc domain comprises a CH3 domain comprising one or more mutations selected from the group consisting of S354C and T366W (numbering according to EU).

In some embodiments, the incretin agent comprises a heterodimer, e.g., as shown in fig. 13 or fig. 14. Fig. 13 shows an exemplary design of two polypeptide chains comprising an incretin peptide fused to an Fc domain. In each polypeptide chain (incretin-Fc fusion), there is a Signal Peptide (SP) and one, two or four incretin peptides fused to an Fc domain (I: 1x, I:2x or I:4 x), and each incretin agent includes an Fc mutation (e.g., a knob structure mutation) that induces heterodimerization. The Fc domain may also include modifications that eliminate effector function and/or increase half-life as described herein. When one or more polyribonucleotides encoding the two polypeptide chains in fig. 13 (top) are expressed, the two polypeptide chains bind and form a heterodimeric incretin agent (bottom). An exemplary incretin according to the design shown in fig. 13 is shown in fig. 14. Specifically, each polypeptide chain of the incretin agent in fig. 14 has a Signal Peptide (SP), GLP1 or GIP incretin peptide, a connecting peptide (GGGS) ₃, and an Fc domain. One or both of the Fc domains contain a "LS" mutation (M428L/N434S) that extends the half-life of the incretin agent, a "STR" mutation that silences Fc effector function, and a "knob and hole" mutation that promotes heterodimerization. In some embodiments, any mutation described herein can be included in place of or in addition to LS and/or STR mutations. When two peptide chains are expressed, they combine to form a heterodimeric structure containing two peptide chains with different incretin peptides. The SP cleavage site within the incretins is indicated by the arrow.

In some embodiments, the incretin agent comprises two polypeptide chains that bind and form a heterodimeric incretin agent, wherein one polypeptide chain comprises SEQ ID NO: 300 (DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK) ("FcKIH-a (LS and STR) ") and the other polypeptide chain comprises SEQ ID NO: 301(DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK) ("FcKIH-b (LS and STR)").

In some embodiments, a polyribonucleotide described herein encodes an incretin agent comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the incretin agent sequences in table 9. In some embodiments, the incretins comprise any one of the incretins detailed in table 9 below, or a combination or mutant thereof.

TABLE 9 exemplary heterodimeric formed incretins comprising FcKIH-a or FcKIH-b domains (mutations shown in bold, connecting peptides shown underlined)

In some embodiments, any of the exemplary incretins comprising the FcKIH-a domain may be combined with the exemplary incretins comprising the FcKIH-b domain, e.g., in some embodiments, the incretins of SEQ ID NO: 84, 85, 86, 87, 173, or 174 may be combined with the incretins of SEQ ID NO: 88.

Signal peptides

According to certain embodiments, the signal peptide is fused to the encoded incretin peptide described herein, either directly or through a linker peptide. In some embodiments, the open reading frames of the polyribonucleotides described herein encode an incretin agent having a signal peptide that functions, for example, in mammalian cells.

In some embodiments, the signal peptide is a sequence generally characterized by a length of about 15 to 30 amino acids. In many embodiments, the signal peptide is located at the N-terminus of the incretin, but is not limited thereto. In some embodiments, the signal peptide preferably allows for transport of an incretin agent encoded by a polyribonucleotide of the present disclosure bound thereto into a defined cell compartment, preferably a cell surface, endoplasmic Reticulum (ER) or endosomal-lysosomal compartment.

In some embodiments, the ribonucleic acid sequence encoding the signal peptide allows the incretins encoded by the polyribonucleotides to be secreted post-translationally by, for example, cells present in the individual, thus producing a plasma concentration of the bioactive incretins.

In some embodiments, the ribonucleic acid sequence encoding the signal peptide included in the polyribonucleotide consists of or comprises a nucleotide sequence encoding a human signal peptide. In some embodiments, the ribonucleic acid sequence encoding a secretion signal included in the polyribonucleotide consists of or comprises a nucleotide sequence encoding a non-human secretion signal. In some embodiments, the signal peptide may be or comprise a viral signal peptide. In some embodiments, such signal peptide may be or comprise the amino acid sequence of MRVLVLLACLAAASNA (SP 1-2; SEQ ID NO: 17). In some embodiments, the signal peptide may be or comprise the amino acid sequence of MRVMAPRTLILLLSGALALTETWA (husec signal peptide δGS; SEQ ID NO: 65).

In some embodiments, the signal peptide sequence is selected from those included in table 10 below, or a fragment or mutant thereof:

TABLE 10 exemplary Signal peptides

The present disclosure recognizes, among other things, that the selection of signal peptides is important for predicting cleavage sites between signal peptides and incretin peptides. In order for the polyribonucleotides to deliver and express the incretin peptide, wherein the expressed incretin peptide maintains proper function and biological activity, in some embodiments, a signal peptide is selected and included in the incretin agent to effect proper cleavage of incretin to the mature form.

Without wishing to be bound by any theory, in the context of the polyribonucleotides described herein that encode an incretin agent, the cleavage site of the signal peptide and the type or sequence of the signal peptide are important to ensure that the N-terminus of the incretin peptide is properly processed. The signal peptide may contain a specific sequence or structure that results in a surrogate processing or cleavage site, thereby ultimately altering the final amino acid sequence of the mature incretin peptide. In such relatively small peptides, such as GLP1 or GIP (or mutants thereof, and other peptides of similar size/nature), variations in amino acid residues can affect the biological activity of the peptide. In some embodiments, the signal peptide is selected for inclusion in an incretin agent as described herein so as to promote proper cleavage of the N-terminus of the incretin peptide, or in other words, to produce a "scarless" N-terminus of the incretin peptide so as to maintain the biological activity of the incretin peptide. FIGS. 20 and 21 show schematic representations of the locations of theoretical cleavage sites of certain exemplary signal peptides and incretins. FIG. 20 indicates that the A8G mutation promotes correct N-terminal processing of GLP1 incretins with husec signal peptide. Figure 21 indicates that the A2G mutation promotes correct N-terminal processing of GIP incretins with husec signal peptide.

This concept and utilization of specific signal peptides to facilitate cleavage to produce mature peptides with a scar-free N-terminus can also be applied to other intestinal peptides (e.g., glucagon) and/or other peptides of comparable size/nature to GLP1 and GIP described herein. This is important in the context of delivering an incretin agent (or other similar peptide) as one or more polyribonucleotides encoding an incretin agent. In addition to post-translational processing (including proper cleavage of post-translational peptides), such delivery requires proper translation of intracellular proteins. An incretin agent comprising one or more incretin peptides fused to another peptide described herein can be designed and produced such that signal peptide cleavage is accurate and does not affect the amino acid sequence of the mature peptide (i.e., produces a "scar-free" N-terminus). The scarless N-terminus of incretin peptides (and other similar peptides, e.g., other intestinal peptides, e.g., glucagon) allows the peptide to function properly after processing into a mature peptide.

In some embodiments, the polyribonucleotide comprises a signal peptide coding sequence and one or more incretin peptide coding sequences in the 5 'to 3' direction. In some embodiments, the signal peptide coding sequence and the one or more incretin peptide coding sequences encode any of the sequences shown in table 11 below, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to any of the sequences shown in table 11 below.

TABLE 11 exemplary incretins wherein examples x2 and x4 include the connecting peptide between the repeat units and the furin cleavage site

In some embodiments, the polyribonucleotide comprises a signal peptide coding sequence, an incretin peptide coding sequence, a linker peptide coding sequence, and a half-life extending moiety coding sequence in the 5 'to 3' direction. In some embodiments, the polyribonucleotide comprises a signal peptide coding sequence in the 5 'to 3' direction, a half-life extending moiety coding sequence, a linker peptide coding sequence, and an incretin peptide coding sequence.

In some embodiments, the polyribonucleotide comprises a signal peptide coding sequence and one or more incretin peptide coding sequences in the 5 'to 3' direction, each independently separated by a connecting peptide coding sequence and a protease cleavage site coding sequence, e.g., a furin cleavage site coding sequence. In some such embodiments, one or more of the incretin peptide coding sequences is preceded or followed by a linker peptide coding sequence and a half-life extending moiety coding sequence.

Encoding incretins exemplary polyribonucleotides

In some embodiments, the polyribonucleotide comprises a ribonucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the sequences shown in table 12 below.

TABLE 12 exemplary polyribonucleotides encoding incretins wherein examples x2 and x4 include the linking peptide between the various repeat units and the furin cleavage site

Exemplary polyribonucleotide features

The polyribonucleotides described herein encode an incretin agent as described herein. In addition, in some embodiments, the polyribonucleotides described herein include encoding other components, such as signal peptides. In some embodiments, the polyribonucleotides described herein can comprise nucleotide sequences encoding 5 'UTRs and/or 3' UTRs. In some embodiments, a polynucleotide described herein may comprise a nucleotide sequence encoding a polyA tail. In some embodiments, the polyribonucleotides described herein can comprise a 5' cap that can be incorporated during transcription, or that can be joined post-transcriptionally to polyribonucleotides.

5' Cap

One structural feature of mRNA is the cap structure at the 5' end. The natural eukaryotic mRNA contains a 7-methylguanosine cap linked to the mRNA by a 5 'to 5' -triphosphate bridge, resulting in a cap 0 structure (m 7 GpppN). In most eukaryotic and some viral mrnas, further modification may occur at the 2 '-hydroxy-group (2' -OH) of the first and subsequent nucleotides (e.g., the 2 '-hydroxy group may be methylated to form 2' -O-Me), yielding "cap 1" and "cap 2" five-primer, respectively. Diamond et al, (2014) Cytokine & growth Factor Reviews, 25:543-550 reported that cap 0-mRNA was not translated as efficiently as cap 1-mRNA, with the role of 2'-O-Me at the penultimate position of the 5' end of mRNA being decisive. The lack of 2' -O-Me has been shown to trigger innate immunity and activate IFN responses. Daffis et al, (2010) Nature, 468:452-456, and Tust et al (2011) Nature Immunology, 12:137-143.

RNA capping is well studied and described, for example, in Decroly et al, (2012) Nature Reviews 10:51-65, and Ramanthan et al, (2016) Nucleic Acids Res; 44 (16): 7511-7526, the entire contents of which are hereby incorporated by reference. For example, in some embodiments, the 5 'cap structure that may be suitable in the context of the present invention is cap 0 (methylation of the first nucleobase, e.g., m7 GpppN), cap 1 (additional methylation of ribose of the adjacent nucleotide of m7 GpppN), cap 2 (additional methylation of ribose of the 2 nd nucleotide downstream of m7 GpppN), cap 3 (additional methylation of ribose of the 3 rd nucleotide downstream of m7 GpppN), cap 4 (additional methylation of ribose of the 4 th nucleotide downstream of m7 GpppN), ARCA ("anti-reverse cap analogue"), modified ARCA (e.g., phosphorothioate modified ARCA), inosine, N1-methyl-guanosine, 2' -fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

As used herein, the term "5 '-cap" refers to a structure found on the 5' end of an RNA (e.g., mRNA) and generally includes guanosine nucleotides attached to the RNA (e.g., mRNA) through a 5 '-to 5' -triphosphate linkage (also known as Gppp or G (5 ') ppp (5'). In some embodiments, guanosine included in the 5' cap can be modified, for example, by methylation at one or more positions (e.g., at position 7) on the base (guanine) and/or by methylation at one or more positions of ribose. In some embodiments, the guanosine nucleoside included in the 5' cap comprises a 3' o methylation at ribose (3 ' ome). In some embodiments, the guanosine included in the 5' cap comprises a methylation at the guanine 7 position (m 7G). In some embodiments, the guanosine included in the 5' cap comprises a methylation at the guanine 7 position and a 3' O methylation at the ribose (m 7 (3 ' ome)). It will be appreciated that the symbols used in the previous paragraph, such as "(m ₂ ^7,3'-O) G" or "m7 (3' ome)" apply to the other structures described herein.

In some embodiments, providing RNA with a5 '-cap as disclosed herein can be accomplished by in vitro transcription, wherein the 5' -cap is co-transcribed into the RNA strand, or can be linked to the RNA post-transcriptionally using a capping enzyme. In some embodiments, co-transcription capping using the caps disclosed herein increases the capping efficiency of RNA compared to co-transcription capping using an appropriate reference comparator. In some embodiments, increasing capping efficiency may increase the translation efficiency and/or translation rate of the RNA, and/or increase expression of the encoded polypeptide. In some embodiments, the modification to the polynucleotide results in a non-hydrolyzable cap structure that can, for example, prevent uncapping and increase RNA half-life.

In some embodiments, the 5' cap utilized is a cap 0, cap 1, or cap 2 structure. See, for example, fig. 1 of ramamathan et al and fig. 1 of Decroly et al, which are incorporated herein by reference in their entirety. In some embodiments, the RNAs described herein comprise a cap 1 structure. In some embodiments, the RNAs described herein comprise a cap 2 structure.

In some embodiments, the RNAs described herein comprise a cap 0 structure. In some embodiments, the cap 0 structure comprises guanosine (m 7) G methylated at the guanine 7 position. In some embodiments, such cap 0 structure is linked to RNA by a 5 '-to 5' -triphosphate bond, and is also referred to herein as (m 7) Gppp. In some embodiments, the cap 0 structure comprises a guanosine that is methylated at the 2' position of guanosine ribose. In some embodiments, the cap 0 structure comprises a guanosine that is methylated at the 3' position of guanosine ribose. In some embodiments, the guanosine included in the 5 'cap comprises a methylation ((m ^27,2'-O) G) at the guanine 7 position and at the ribose 2' position. In some embodiments, the guanosine included in the 5 'cap comprises a methylation ((m ^27,3'-O) G) at the guanine 7 position and at the ribose 2' position.

In some embodiments, the cap 1 structure comprises a guanosine that is methylated at the guanine 7 position ((m 7) G) and optionally methylated at the ribose 2' or 3' position, and a first nucleotide that is 2' o-methylated in the RNA ((m ^2'-O)N₁). In some embodiments, the cap 1 structure comprises a guanosine that is methylated at the guanine 7 position ((m 7) G) and methylated at the ribose 3' position, and a first nucleotide that is 2' o-methylated in the RNA ((m ^2'-O)N₁). In some embodiments, the cap 1 structure is linked to the RNA by a 5' -to 5' -triphosphate linkage, and is also referred to herein as, for example ((m 7) Gppp (^2'-O)N₁) or (m ^27,3'-O)Gppp(^2'-O)N₁), wherein N ₁ is as defined and described herein.

In some embodiments, the cap 2 structure comprises a guanosine that is methylated at the guanine 7 position ((m 7) G) and optionally methylated at the ribose 2' or 3' position, and a first nucleotide and a second nucleotide of the RNA that are 2' o methylated ((m ² ^'-O)N₁p(m^2'-O)N₂). In some embodiments, the cap 2 structure comprises a guanosine that is methylated at the guanine 7 position ((m 7) G) and methylated at the ribose 3' position, and a first nucleotide and a second nucleotide of the RNA that are 2' o methylated.

In some embodiments, the 5' cap is a dinucleotide cap structure. In some embodiments, the 5' cap is a dinucleotide cap structure comprising N ₁, wherein N ₁ is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G ^*N₁, wherein N ₁ is as defined above and herein, and G ^* comprises the structure of formula (I):

(I)

or a salt thereof,

Wherein the method comprises the steps of

R ₂ and R ₃ are each-OH or-OCH ₃, and

X is O or S.

In some embodiments, R ₂ is —oh. In some embodiments, R ₂ is-OCH ₃. In some embodiments, R ₃ is —oh. In some embodiments, R ₃ is-OCH ₃. In some embodiments, R ₂ is-OH and R ₃ is-OH. In some embodiments, R ₂ is-OH and R ₃ is-CH ₃. In some embodiments, R ₂ is-CH ₃ and R ₃ is-OH. In some embodiments, R ₂ is-CH ₃ and R ₃ is-CH ₃.

In some embodiments, X is O. In some embodiments, X is S.

In some embodiments, the 5 'cap is a dinucleotide cap0 structure (e.g., ,(m⁷)GpppN₁、(m₂ ^7,2'-O)GpppN₁、(m₂ ^7,3'-O)GpppN₁、(m⁷)GppSpN₁、(m₂ ^7,2'-O)GppSpN₁ or (m ₂ ^7,3'-O)GppSpN₁), wherein N ₁ is as defined and described herein, in some embodiments, the 5' cap is a dinucleotide cap0 structure (e.g., ,(m⁷)GpppN₁、(m₂ ^7,2'-O)GpppN₁、(m₂ ^7,3'-O)GpppN₁、(m⁷)GppSpN₁、(m₂ ^7,2'-O)GppSpN₁ or (m ₂ ^7,3'-O)GppSpN₁), wherein N ₁ is g, in some embodiments, the 5 'cap is a dinucleotide cap0 structure (e.g., ,(m⁷)GpppN₁、(m₂ ^7,2'-O)GpppN₁、(m₂ ^7,3'-O)GpppN₁、(m⁷)GppSpN₁、(m₂ ^7,2'-O)GppSpN₁ or (m ₂ ^7,3'-O)GppSpN₁), wherein N ₁ is A, U or c, in some embodiments, the 5' cap is a dinucleotide cap 1 structure (e.g., ,(m⁷)Gppp(m^2'-O)N₁、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁、(m⁷)GppSp(m^2'-O)N₁、(m₂ ^7,2'-O)GppSp(m^2'-O)N₁ or (m ₂ ^7,3'-O)GppSp(m^2'-O)N₁), wherein N ₁ is as defined and described herein, in some embodiments, the 5 'cap is selected from the following groups ：(m⁷)GpppG ("Ecap0")、(m⁷)Gppp(m^2'-O)G ("Ecap1")、(m₂ ^7,3'-O)GpppG ("ARCA") and (m ₂ ^7,2'-O) GppSpG ("β -S-ARCA"). In some embodiments, the 5' cap is a (m ⁷) gppg ("eca 0") having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ⁷)Gppp(m^2'-O) G ("Ecap 1") having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,2'-O) GpppG ("ARCA") having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,2'-O) GppSpG ("β -S-ARCA") having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is a trinucleotide cap structure. In some embodiments, the 5' cap is a trinucleotide cap structure comprising N ₁pN₂, wherein N ₁ and N ₂ are as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G ^*N₁pN₂, wherein N ₁ and N ₂ are as defined above and herein, and G ^* comprises the structure of formula (I):

(I)

Or a salt thereof, wherein R ₂、R₃ and X are as defined and described herein.

In some embodiments, the 5 'cap is a trinucleotide cap 0 structure (e.g., (m ⁷)GpppN₁pN₂、(m₂ ^7,2'-O)GpppN₁pN₂ or (m ₂ ^7,3'-O)GpppN₁pN₂) where N ₁ and N ₂ are as defined and described herein). In some embodiments, the 5' cap is a trinucleotide cap 1 structure (e.g., ,(m7)Gppp(m^2'-O)N₁pN₂、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁pN₂、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁pN₂), where N ₁ and N ₂ are as defined and described herein). In some embodiments, the 5 'cap is a trinucleotide cap 2 structure (e.g., ,(m7)Gppp(m^2'-O)N₁p(m^2'-O)N₂、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁p(m^2'-O)N₂、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁p(m^2'-O)N₂), where N ₁ and N ₂ are as defined and described herein). In some embodiments, the 5' cap is selected from the following groups ：(m₂ ^7,3'-O)Gppp(m^2'-O)ApG ("CleanCap AG"、"CC413")、(m₂ ^7,3'-O)Gppp(m^2'-O)GpG ("CleanCap GG")、(m7)Gppp(m^2'-O)ApG、(m7)Gppp(m^2'-O)GpG、(m₂ ^7,3'-O)Gppp(m₂ ^6,2'-O)ApG and (m 7) Gppp (m ^2'-O) ApU.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O)Gppp(m^2'-O) ApG ("CLEANCAP AG", "CC 413") having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is a (m ₂ ^7,3'-O)Gppp(m^2'-O) GpG ("CLEANCAP GG") having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m 7) Gppp (m ^2'-O) ApG having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m 7) Gppp (m ^2'-O) GpG having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O)Gppp(m₂ ^6,2'-O) ApG having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m 7) Gppp (m ^2'-O) ApU having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is a tetranucleotide cap structure. In some embodiments, the 5' cap is a tetranucleotide cap structure comprising N ₁pN₂pN₃, wherein N ₁、N₂ and N ₃ are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide cap G ^* N₁pN₂pN₃, wherein N ₁、N₂ and N ₃ are as defined above and herein, and G ^* comprises the structure of formula (I):

(I)

In some embodiments, the 5' cap is a tetranucleotide cap 0 structure (e.g., ,(m⁷)GpppN₁pN₂pN₃、(m₂ ^7,2'-O)GpppN₁pN₂pN₃ or (m ₂ ^7,3'-O)GpppN₁N₂pN₃), where N ₁、N₂ and N ₃ are as defined and described herein). In some embodiments, the 5' cap is a tetranucleotide cap 1 structure (e.g., ,(m⁷)Gppp(m^2'-O)N₁pN₂pN₃、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁pN₂pN₃、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁pN₂N₃), wherein N ₁、N₂ and N ₃ are as defined and described herein, in some embodiments, the 5' cap is a tetranucleotide cap 2 structure (e.g., ,(m⁷)Gppp(m^2'-O)N₁p(m^2'-O)N₂pN₃、(m₂ ^7,2'-O)Gppp(m^2'-O)N₁p(m^2'-O)N₂pN₃、(m₂ ^7,3'-O)Gppp(m^2'-O)N₁p(m^2'-O)N₂pN₃), wherein N ₁、N₂ and N ₃ are as defined and described herein, in some embodiments, the 5' cap is selected from the following groups ：(m₂ ^7,3'-O)Gppp(m^2'-O)Ap(m^2'-O)GpG、(m₂ ^7,3'-O)Gppp(m^2'-O)Gp(m^2'-O)GpC、(m⁷)Gppp(m^2'-O)Ap(m^2'-O)UpA and (m ⁷)Gppp(m^2'-O)Ap(m^2'-O) GpG.

In some embodiments, the 5' cap is a (m ₂ ^7,3'-O)Gppp(m^2'-O)Ap(m^2'-O) GpG having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O)Gppp(m^2'-O)Gp(m^2'-O) GpC having the structure:

Or a salt thereof.

In some embodiments, the 5' cap is (m ⁷)Gppp(m^2'-O)Ap(m^2'-O) UpA having the following structure:

Or a salt thereof.

In some embodiments, the 5' cap is a (m ⁷)Gppp(m^2'-O)Ap(m^2'-O) GpG having the following structure:

Or a salt thereof.

Cap proximal sequence

In some embodiments, a 5' UTR as used in the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, the cap proximal sequence comprises a sequence adjacent to a 5' cap. In some embodiments, the cap proximal sequence comprises nucleotides in RNA polynucleotide positions +1, +2, +3, +4, and/or +5.

In some embodiments, the cap structure comprises one or more polynucleotides of the cap proximal sequence. In some embodiments, the cap structure comprises the m7 guanosine cap and nucleotide +1 (N ₁) of the RNA polynucleotide. In some embodiments, the cap structure comprises the m7 guanosine cap and nucleotide +2 (N ₂) of the RNA polynucleotide. In some embodiments, the cap structure comprises nucleotides +1 and +2 (N ₁ and N ₂) of the m7 guanosine cap and the RNA polynucleotide. In some embodiments, the cap structure comprises nucleotides +1, +2, and +3 (N ₁、N₂ and N ₃) of the m7 guanosine cap and the RNA polynucleotide.

One of skill in the art will appreciate upon reading this disclosure that in some embodiments, one or more residues of the cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in the RNA as already included in the cap entity (e.g., cap 1 structure or cap 2 structure, etc.), or that in some embodiments, at least some of the residues in the cap proximal sequence may be added enzymatically (e.g., by a polymerase such as T7 polymerase). For example, in certain exemplary embodiments utilizing m ₂ ^7,3'-OGppp(m₁ ^2'-O) ApG caps, +1 (i.e., N ₁) and +2 (i.e., N ₂) are capped (m ₁ ^2'-O) a and G residues and +3, +4, and +5 are added by a polymerase (e.g., T7 polymerase).

In some embodiments, the 5 'cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises N ₁ of the 5' cap, wherein N1 is any nucleotide, e.g., A, C, G or U. In some embodiments, the 5 'cap is a trinucleotide cap structure (e.g., as described above and herein), wherein the cap proximal sequence comprises N ₁ and N ₂ of the 5' cap, wherein N ₁ and N ₂ are independently any nucleotide, e.g., A, C, G or U. In some embodiments, the 5 'cap is a tetranucleotide cap structure (e.g., a trinucleotide cap structure as described above and herein), wherein the cap proximal sequence comprises N ₁、N₂ and N ₃ of the 5' cap, wherein N ₁、N₂ and N ₃ are any nucleotide, e.g., A, C, G or U.

In some embodiments, for example, where the 5 'cap is a dinucleotide cap structure, the cap proximal sequence comprises N ₁ of the 5' cap, and N ₂、N₃、N₄ and N ₅, wherein N ₁ to N ₅ correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide. In some embodiments, for example, where the 5 'cap is a trinucleotide cap structure, the cap proximal sequence comprises N ₁ and N ₂, and N ₃、N₄ and N ₅ of the 5' cap, wherein N ₁ to N ₅ correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide. In some embodiments, for example, where the 5 'cap is a four nucleotide cap structure, the cap proximal sequence comprises N ₁、N₂ and N ₃, and N ₄ and N ₅ of the 5' cap, where N ₁ to N ₅ correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

In some embodiments, N ₁ is a. In some embodiments, N ₁ is C. In some embodiments, N ₁ is G. In some embodiments, N ₁ is U. In some embodiments, N ₂ is a. In some embodiments, N ₂ is C. In some embodiments, N ₂ is G. In some embodiments, N ₂ is U. In some embodiments, N ₃ is a. In some embodiments, N ₃ is C. In some embodiments, N ₃ is G. In some embodiments, N ₃ is U. In some embodiments, N ₄ is a. In some embodiments, N ₄ is C. In some embodiments, N ₄ is G. In some embodiments, N ₄ is U. In some embodiments, N ₅ is a. In some embodiments, N ₅ is C. In some embodiments, N ₅ is G. In some embodiments, N ₅ is U. It will be appreciated that the various embodiments described above and herein (e.g., for N ₁ to N ₅) may be employed alone or in combination and/or may be combined with other embodiments of the variables described above and herein (e.g., 5' caps).

5' UTR

In some embodiments, a nucleic acid (e.g., DNA, RNA) as used in the present disclosure comprises a 5' UTR. In some embodiments, the 5' UTR may include a plurality of different sequence components, in some embodiments, such plurality may be or include multiple copies of one or more specific sequence components (e.g., as may be from a particular source or otherwise referred to as functional or signature sequence components). In some embodiments, the 5' UTR comprises a plurality of different sequence components.

The term "untranslated region" or "UTR" is commonly used in the art to refer to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA polynucleotide (e.g., an mRNA molecule). The untranslated region (UTR) may be present 5 '(upstream) of the open reading frame (5' UTR) and/or 3 '(downstream) of the open reading frame (3' UTR). As used herein, the term "five-primer untranslated region" or "5 'UTR" refers to a polynucleic nucleotide sequence between the 5' end of a polynucleic nucleotide (e.g., transcription initiation site) and the start codon of a polynucleic nucleotide coding region. In some embodiments, a "5 'UTR" refers to a polynucleotide sequence that begins at the 5' end of a polynucleotide (e.g., transcription initiation site) and ends one nucleotide (nt) before the start codon (typically AUG) of the polynucleotide coding region, e.g., in its natural context. In some embodiments, the 5' UTR comprises a Kozak sequence. The 5' UTR is downstream of the 5' cap (if present), e.g. directly adjacent to the 5' cap. In some embodiments, a 5' UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein. In some embodiments, the cap proximal sequence comprises a sequence adjacent to a 5' cap.

Exemplary 5' UTRs include human alpha globulin (hAg) 5' UTRs or fragments thereof, TEV 5' UTRs or fragments thereof, HSP70 5' UTRs or fragments thereof, or c-Jun 5' UTRs or fragments thereof.

In some embodiments, an RNA disclosed herein comprises hAg' UTR or a fragment thereof.

In some embodiments, the RNAs disclosed herein comprise 5 'UTRs having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5' UTR having a sequence as shown in AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 49). In some embodiments, the RNA disclosed herein comprises the 5' UTR provided in SEQ ID NO. 49.

PolyA tail

In some embodiments, a polynucleotide (e.g., DNA, RNA) disclosed herein comprises a poly (a) sequence, e.g., as described herein. In some embodiments, the poly (a) sequence is located downstream of the 3 'UTR, e.g., adjacent to the 3' UTR.

As used herein, the term "poly (a) sequence" or "polyA tail" refers to an uninterrupted or intermittent sequence of adenylate residues typically located at the 3' end of an RNA polynucleotide. poly (a) sequences are known to those skilled in the art and may follow the 3' UTR in the RNAs described herein. The uninterrupted poly (A) sequence is characterized by contiguous adenylate residues. In nature, uninterrupted poly (A) sequences are typical. In some embodiments, a polynucleotide disclosed herein comprises an uninterrupted poly (a) sequence. In some embodiments, a polynucleotide disclosed herein comprises a discontinuous poly (a) sequence. In some embodiments, the RNAs disclosed herein can have a poly (a) sequence that is linked to the free 3' end of the RNA by a template-independent RNA polymerase after transcription, or a poly (a) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

A poly (a) sequence of about 120 a nucleotides has been shown to have a beneficial effect on RNA levels in transfected eukaryotic cells, as well as on protein levels translated by open reading frames present upstream (5') of the poly (a) sequence (Holtkamp et al, 2006, blood, volume 108, pages 4009-4017, incorporated herein by reference).

In some embodiments, the poly (A) sequence as described in the present disclosure is not limited to a particular length, and in some embodiments, the poly (A) sequence is any length. In some embodiments, the poly (a) sequence comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 a nucleotides, and in particular about 120 a nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the poly (a) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% by number of the nucleotides in the poly (a) sequence are a nucleotides, but the remaining nucleotides are allowed to be nucleotides other than a nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid) or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly (a) sequence, i.e. 100% by number of nucleotides in the poly (a) sequence are a nucleotides. The term "a nucleotide" or "a" refers to an adenylate.

In some embodiments, the poly (a) sequence is ligated during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylates) in the strand complementary to the coding strand. The DNA sequence (coding strand) encoding a poly (A) sequence is referred to as a poly (A) cassette.

In some embodiments, the poly (a) cassette present in the DNA coding strand consists essentially of dA nucleotides, but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cartridge is disclosed in WO2016/005324, which is hereby incorporated by reference. Any poly (A) cassette disclosed in WO2016/005324 may be used in accordance with the present disclosure. Poly (a) cassettes are contemplated which consist essentially of dA nucleotides but are interrupted by random sequences having an equivalent distribution of four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5 to 50 nucleotides, show constant amplification of plasmid DNA in e.coli (e.coli) at the DNA level, and are still associated with beneficial properties on the RNA level with respect to supporting RNA stability and translation efficiency. In some embodiments, the poly (a) sequence contained in the RNA polynucleotides described herein consists essentially of a nucleotides, but is interrupted by a random sequence of four nucleotides (A, C, G, U). Such random sequences may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

In some embodiments, no nucleotide other than the a nucleotide flanks the 3 'end of the poly (a) sequence, i.e., the poly (a) sequence is not obscured at its 3' end by or is followed by a nucleotide other than a.

In some embodiments, the poly (a) sequence can comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly (a) sequence can consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly (a) sequence can consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly (a) sequence comprises at least 100 nucleotides. In some embodiments, the poly (a) sequence comprises about 150 nucleotides. In some embodiments, the poly (a) sequence comprises about 120 nucleotides.

In some embodiments, the polyA tail comprises a specific amount of adenosine, such as about 50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120 or about 150 or about 200. In some embodiments, the polyA tail of the string construct may comprise 200 a residues or less. In some embodiments, the polyA tail of the string construct may comprise about 200 a residues. In some embodiments, the polyA tail of the string construct may comprise 180 a residues or less. In some embodiments, the polyA tail of the string construct may comprise about 180 a residues. In some embodiments, the polyA tail may comprise 150 residues or less.

In some embodiments, the RNA comprises a poly (A) sequence comprising a AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 50) nucleotide sequence, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of SEQ ID NO. 50. In some embodiments, the poly (A) tail comprises the nucleotide sequence as set forth in SEQ ID NO. 50.

In some embodiments, the polyA tail comprises a plurality of a residues interrupted by a linker peptide. In some embodiments, the connecting peptide comprises nucleotide sequence GCAUAUGACU (SEQ ID NO: 40).

3' UTR

In some embodiments, an RNA as used in the present disclosure comprises a 3' UTR. As used herein, the term "triple-primer untranslated region", "3 'untranslated region" or "3' UTR" refers to an mRNA molecule sequence that begins after the stop codon of the open reading frame sequence coding region. In some embodiments, the 3' UTR begins immediately after the stop codon of the coding region of the open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after the stop codon of the coding region of the open reading frame sequence, e.g., in its natural context. The term "3' UTR" preferably excludes poly (A) sequences. Thus, the 3' UTR is upstream of, if present, the poly (A) sequence, e.g., immediately adjacent to the poly (A) sequence.

In some embodiments, the RNAs disclosed herein comprise a 3' UTR comprising an F-component and/or an I-component. In some embodiments, the 3' UTR or proximal sequence thereof comprises a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an XhoI site.

In some embodiments, the RNA construct comprises an F module. In some embodiments, the F module sequence is the 3' UTR of the amino terminal split enhancer (AES).

In some embodiments, the RNAs disclosed herein comprise 3 'UTRs having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to 3' UTRs having a sequence as shown in CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 51). In some embodiments, the RNA disclosed herein comprises the 3' UTR provided in SEQ ID NO: 51.

In some embodiments, the 3' utr is an FI component as described in WO2017/060314, which is incorporated herein by reference in its entirety.

RNA forms

At least three different forms have been developed that can be used in RNA compositions (e.g., pharmaceutical compositions), namely unmodified uridine-containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplified mRNA (saRNA). Each of these types of platforms exhibits unique characteristics. In general, in all three forms, the RNA is capped, contains an Open Reading Frame (ORF) flanked by untranslated regions (UTRs), and has a polyA tail at the 3' end. The ORF of uRNA and modRNA vectors encodes the incretins. saRNA has multiple ORFs.

In some embodiments, the RNAs described herein can have modified nucleosides. In some embodiments, the RNA comprises at least one (e.g., each) modified nucleoside that replaces uridine.

As used herein, the term "uracil" describes every nucleobase that can be present in an RNA nucleic acid. The uracil has the structure:

。

As used herein, the term "uridine" describes every nucleoside that can be present in RNA. The structure of uridine is:

。

UTP (uridine 5' -triphosphate) has the following structure:

。

pseudo-UTP (pseudouridine 5' -triphosphate) has the following structure:

。

"pseudouridine" is an example of a modified nucleoside that is an isomer of uridine, wherein uracil is attached to the pentose ring through a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methyl-pseudouridine (m 1 ψ), which has the following structure:

。

N1-methyl-pseudo-UTP has the following structure:

。

another exemplary modified nucleoside is 5-methyl-uridine (m 5U), which has the structure:

。

in some embodiments, one or more uridine in the RNAs described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

In some embodiments, the RNA comprises at least one modified nucleoside that replaces uridine. In some embodiments, the RNA comprises modified nucleosides that replace each uridine.

In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m 5U). In some embodiments, the RNA may comprise more than one type of modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m 1 ψ) and 5-methyl-uridine (m 5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U).

In some embodiments, the modified nucleoside that replaces one or more (e.g., all) uridine in the RNA can be any one or more of 3-methyl-uridine (m 3U), 5-methoxy-uridine (mo 5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2U), 4-thio-uridine (s 4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5U), Uridine 5-oxo-acetic acid methyl ester (mcmo U), 5-carboxymethyl-uridine (cm 5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm U), 5-methoxycarbonylmethyl-uridine (mcm 5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5s 2U), 5-aminomethyl-2-thio-uridine (nm 5s 2U), 5-methylaminomethyl-uridine (mcm 5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mcm 5s 2U), 5-methylaminomethyl-2-seleno-uridine (mm 5se 2U), 5-carbamoylmethyl-uridine (ncm U), 5-carboxymethylaminomethyl-uridine (cmnm U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm s 2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (τm5U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uridine (τm5s 2U), 1-taurine methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m 5s 2U), 1-methyl-4-thio-pseudouridine (m 1s 4. Phi.), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3. Phi.), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine (m 5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine (acp 3U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp 3. Phi.), 5- (isopentenylaminomethyl) uridine (mm 5U), 5- (isopentenylaminomethyl) -2-thio-uridine (mm 5s 2U), alpha-thio-uridine, 2 '-O-methyl-uridine (Um), 5,2' -O-dimethyl-uridine (m 5 Um), 2 '-O-methyl-pseudouridine (. Phi.,) 2-thio-2' -O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2 '-O-methyl-uridine (mm 5 Um), alpha-thio-uridine, 2' -O-methyl-uridine (mm 5 Um), 5-carbamoylmethyl-2 ' -O-methyl-uridine (ncm Um), 5-carboxymethylaminomethyl-2 ' -O-methyl-uridine (cmnm Um), 3,2' -O-dimethyl-uridine (m 3 Um), 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (mm 5 Um), 1-thio-uridine, deoxythymidine, 2' -F-arabino-uridine, 2' -F-uridine, 2' -OH-arabino-uridine, 5- (2-carbomethoxyvinyl) uridine, 5- [3- (1-E-propenyl) amino) uridine, or any other modified uridine known in the art.

In some embodiments, the RNA comprises other modified nucleosides or comprises further modified nucleosides, such as modified cytidine. For example, in some embodiments, 5-methylcytidine is partially or completely, preferably completely, substituted for cytidine in the RNA. In some embodiments, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U). In some embodiments, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1ψ) in place of each uridine.

In some embodiments of the disclosure, the RNA is a "replicon RNA" or simply "replicon," in particular a "self-replicating RNA" or a "self-amplifying RNA. In a particularly preferred embodiment, the replicon or self-replicating RNA is derived from or comprises a component derived from a single-stranded (ss) RNA virus, particularly a positive-stranded ssRNA virus such as an alpha virus. Alpha viruses are typically representative of positive-stranded RNA viruses. The alpha virus replicates in the cytoplasm of infected cells (for a review of the alpha virus lifecycle, see Jos e et al, future microbiol., 2009, volume 4, pages 837-856, which is incorporated herein by reference in its entirety). The total genomic length of many alpha viruses is typically in the range of 11,000 to 12,000 nucleotides, and genomic RNAs typically have a 5 'cap and a 3' poly (a) tail. The genome of alpha viruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA, and protein modification) and structural proteins (forming viral particles). There are typically two Open Reading Frames (ORFs) in the genome. The four nonstructural proteins (nsP 1-nsP 4) are usually encoded together by a first ORF near the 5 'end of the genome, while the alpha virus structural proteins are encoded together by a second ORF found downstream of the first ORF and extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, in a ratio of about 2:1. In cells infected with an alpha virus, only the nucleic acid sequence encoding the non-structural protein is translated from the genomic RNA, while the genetic information encoding the structural protein is translated from a subgenomic transcript, which is an RNA molecule resembling eukaryotic messenger RNA (mRNA; gould et al 2010, anti Res., volume 87, pages 111-124). After infection, i.e., at an early stage of the viral life cycle, (+) strand genomic RNA is used directly like messenger RNA to translate the open reading frame encoding the non-structural polyprotein (nsP 1234).

Alpha virus-derived vectors have been proposed for delivering foreign genetic information into target cells or organisms. In a simple approach, the first ORF encodes an alpha virus-derived RNA-dependent RNA polymerase (replicase), which mediates self-amplification of RNA after translation. The second ORF encoding the structural protein of the alpha virus is replaced with an open reading frame encoding the protein of interest (e.g., an incretin). An alpha virus-based trans-replication system relies on an alpha virus nucleotide sequence component on two separate nucleic acid molecules, one nucleic acid molecule encoding a viral replicase and the other nucleic acid molecule being capable of trans-replication by the replicase (hence the name trans-replication system). Trans-replication requires the presence of two such nucleic acid molecules in a given host cell. Nucleic acid molecules capable of trans-replication by replicases must contain certain alpha viral sequence components that allow for alpha viral replicase recognition and RNA synthesis.

Characteristics of an unmodified uridine platform can include, for example, one or more inherent adjuvant effects as well as good tolerance and safety. Characteristics of a modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect, inactivated immune innate immunosensor activation ability, and thus good tolerance and safety obtained. Features of the self-amplification platform may include, for example, long duration of protein expression, good tolerance and safety, higher efficacy potential at very low vaccine doses.

The present disclosure provides optimized specific RNA constructs, e.g., for improved manufacturability, encapsulation, expression levels (and/or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein.

Codon optimization and GC enrichment

As used herein, the term "codon optimized" refers to altering codons in the coding region of a nucleic acid molecule (e.g., a polyribonucleotide) to reflect typical codon usage of the host organism (e.g., an individual receiving the polyribonucleotide), preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of the present disclosure, in certain embodiments, the coding region is codon optimized to achieve optimal expression in a subject treated with an RNA molecule described herein. In some embodiments, codon optimization can be performed, i.e., those codons with common tRNA supply are replaced with "rare codons". In some embodiments, codon optimization may include increasing the guanosine/cytosine (G/C) content of the RNA coding regions described herein compared to the corresponding coding sequence of the wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably unmodified compared to the amino acid sequence.

In some embodiments, the coding sequence (also referred to as a "coding region") is codon optimized for expression in an individual (e.g., a human) to whom the composition (e.g., a pharmaceutical composition) is to be administered. Thus, in some embodiments, the sequence in the polynucleotide (e.g., a polyribonucleotide) may be different from the wild-type sequence encoding the relevant incretin agent, even when the amino acid sequence of the incretin agent is wild-type.

In some embodiments, codon optimization strategies for expression in an individual of interest (e.g., a human) and even in some cases for expression in a particular cell or tissue.

Certain codons for a particular amino acid are expressed by various species with particular preference. Without wishing to be bound by any one theory, codon preference (codon usage difference between organisms) is generally related to the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend inter alia on the nature of the codons translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of the selected tRNA in the cell can generally reflect codons that are most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. A codon usage table is available, for example, at www.kazusa.orjp/codon/a "codon usage database", and this type of table can be adapted in various ways. Computer algorithms for codon optimization of specific sequences for expression in specific individuals or cells thereof are also available, such as Gene force (Aptagen; jacobus, pa.).

In some embodiments, polynucleotides (e.g., polyribonucleotides) of the present disclosure are codon optimized, wherein codons in the polynucleotide (e.g., polyribonucleotides) are suitable for human codon (referred to herein as "human codon optimized polynucleotide") use. Codons encoding the same amino acid occur at different frequencies in an individual (e.g., a human). Thus, in some embodiments, the coding sequences of the polynucleotides of the present disclosure are modified such that the frequency of codons encoding the same amino acid corresponds to the naturally occurring frequency of codons according to human codon usage, e.g., as shown in table 13. For example, in the case of amino acid Ala, the wild-type coding sequence is preferably adjusted in such a way that codon "GCC" is used at a frequency of 0.40, codon "GCT" is used at a frequency of 0.28, codon "GCA" is used at a frequency of 0.22 and codon "GCG" is used at a frequency of 0.10, etc. (see Table 13). Thus, in some embodiments, such a procedure (as exemplified for Ala) is applied to each amino acid encoded by the coding sequence of the polynucleotide to obtain a sequence suitable for human codon use.

TABLE 13 eukaryotic codon usage tables containing the frequency of use for each amino acid.

Certain strategies for codon optimization and/or G/C enrichment for human expression are described in WO2002/098443, which is incorporated herein by reference in its entirety. In some embodiments, a multi-parameter optimization strategy may be used to optimize the coding sequence. In some embodiments, the optimization parameters may include parameters that affect protein expression, which may affect, for example, transcription levels, mRNA levels, and/or translation levels. In some embodiments, exemplary optimization parameters include, but are not limited to, transcription level parameters (including, for example, GC content, consensus splice sites, recessive splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof), mRNA level parameters (including, for example, RNA instability motifs, ribosome entry sites, repeat sequences, and combinations thereof), translation level parameters (including, for example, codon usage, premature poly (a) sites, ribosome entry sites, secondary structures, and combinations thereof), or combinations thereof. In some embodiments, the coding sequence may be optimized by the GeneOptimaizer algorithm as described in Fath et al "Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression" PloS ONE 6(3): e17596;Rabb et al , "The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization" Systems and Synthetic Biology (2010) 4:215-225; and Graft et al "Codon-optimized genes that enable increased heterologous expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA" Methods Mol Med (2004) 94:197-210,, each of which is incorporated herein in its entirety for the purposes described herein. In some embodiments, the coding sequence may be optimized by the adaptation and optimization algorithm "GENEius" of Eurofins, as compared to other optimization algorithms, as described in Eurofins 'Application Notes: eurofins' adaption) and optimization software "GENEius", the entire contents of which are incorporated by reference for the purposes described herein.

In some embodiments, the coding sequences as used in the present disclosure have an increased G/C content as compared to the wild-type coding sequence of the relevant incretin agent. In some embodiments, the guanosine/cytidine (G/C) content of the coding region is modified relative to the wild-type coding sequence of the relevant incretin agent, but the amino acid sequence encoded by the polyribonucleotide is unmodified.

Without wishing to be bound by any particular theory, it is proposed that GC enrichment can improve translation of the payload sequence. In general, sequences with increased G (guanosine)/C (cytidine) content are more stable than sequences with increased a (adenosine)/U (uridine) content. With respect to the fact that several codons encode one and the same amino acid (so-called degeneracy of the genetic code), the most advantageous codons for stability (so-called alternative codon usage) can be determined. Depending on the amino acids encoded by the polyribonucleotides, there are various possibilities for modifying the ribonucleic acid sequence compared to its wild-type sequence. In particular, codons containing a and/or U nucleosides can be modified by replacing such codons with other codons encoding the same amino acid but not containing a and/or U or containing lower a and/or U nucleoside content.

In some embodiments, the G/C content of the polynucleotide coding region described herein is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6% or even more compared to the G/C content of the coding region of, for example, a wild-type RNA prior to codon optimization. In some embodiments, the G/C content of the polynucleotide coding region described herein is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6% or even more compared to the G/C content of the coding region of, for example, a wild-type RNA prior to codon optimization.

In some embodiments, the stability and translation efficiency of a polyribonucleotide may incorporate one or more components established to facilitate the stability and/or translation efficiency of a polyribonucleotide, exemplary such components being described, for example, in WO2007/036366, which is incorporated herein by reference. In some embodiments, to increase expression of a polyribonucleotide as used in the present disclosure, the polyribonucleotide may be modified within the coding region (i.e., the sequence encoding the expressed peptide or protein) without altering the sequence of the expressed peptide or protein, e.g., increasing GC content to increase mRNA stability and/or for codon optimization, and thus enhance translation in a cell.

Encoding incretins exemplary polyribonucleotides

In some embodiments, the polyribonucleotide comprises a ribonucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the sequences shown in table 14 below. In addition to the sequences encoding the incretins and signal peptide, the polyribonucleotides include the exemplary polyribonucleotide features described herein, including cap proximal sequence (AAUA), 5 'UTR sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 49), polyA tail sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 50)、 and 3' UTR sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 51).

TABLE 14 exemplary polyribonucleotides encoding incretins wherein examples x2 and x4 include the linking peptide between the various repeat units and the furin cleavage site

RNA delivery techniques

The provided polyribonucleotides can be delivered for therapeutic applications described herein using any suitable method known in the art, including, for example, as naked RNA delivery, or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and/or peptide-based vectors. See, for example Wadhwa et al, "reports AND CHALLENGES IN THE DELIVERY of mRNA-Based Vaccines" pharmaceuticals (2020) 102 (page 27), the contents of which are incorporated herein by reference for information regarding various methods that can be used to deliver the polyribonucleotides described herein.

In some embodiments, one or more polyribonucleotides can be formulated with the lipid nanoparticle for delivery (e.g., administration).

In some embodiments, the lipid nanoparticle may be designed to protect the polyribonucleotide from extracellular rnases and/or engineered for systemic delivery of RNA to target cells (e.g., liver, intestinal, or pancreatic cells). In some embodiments, such lipid nanoparticles may be particularly useful for delivering a polyribonucleotide when the polyribonucleotide is administered intraperitoneally, intravenously, or intramuscularly to an individual.

Particles for delivery of at least one polyribonucleotide

The polyribonucleotides provided herein can be delivered by particles. In the context of the present disclosure, the term "particle" refers to a structured entity formed by a molecule or a complex of molecules. In some embodiments, the term "particle" refers to a microscale or nanoscale structure, such as a microscale or nanoscale compact structure, dispersed in a medium. In some embodiments, the particle is a nucleic acid-containing particle, such as a particle comprising a polyribonucleotide.

Electrostatic interactions between positively charged molecules (such as polymers and lipids) and negatively charged nucleic acids (e.g., polyribonucleotides) are involved in particle formation. This results in the complexing and spontaneous formation of nucleic acid particles (e.g., ribonucleic acid particles). In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) are nanoparticles.

A "nucleic acid particle" (e.g., ribonucleic acid particle) is a particle that encompasses or contains a nucleic acid and is used to deliver a nucleic acid (e.g., a polyribonucleotide) to a target site of interest (e.g., a cell, tissue, organ, and the like). The nucleic acid particles (e.g., ribonucleic acid particles) can be formed from (i) at least one cationic or cationically ionizable lipid or lipid-like material, (ii) at least one cationic polymer (e.g., protamine), or a mixture of (i) and (ii), and (iii) a nucleic acid (e.g., a polyribonucleotide). Nucleic acid particles (e.g., ribonucleic acid particles) include a plurality of lipid nanoparticles (single lipid nanoparticles) and lipid complexes (LPX).

In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) comprise more than one type of nucleic acid molecule (e.g., polyribonucleotides), wherein the molecular parameters of the nucleic acid molecules may be similar or different from each other, such as with respect to molar mass or basic structural components, such as molecular architecture, capping, coding regions, or other features.

In some embodiments, provided nucleic acid particles (e.g., ribonucleic acid particles) can comprise lipid nanoparticles. As used in this disclosure, "nanoparticle" refers to particles having an average diameter suitable for parenteral administration. In various embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, lipid nanoparticles as described in the present disclosure may have an average size (e.g., average diameter) of about 50 nm to about 100 nm. In some embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 50 nm to about 150 nm. In some embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 60 nm to about 120 nm. In some embodiments, lipid nanoparticles as described in the present disclosure may have an average size (e.g., average diameter) of about 30 nm、35 nm、40 nm、45 nm、50 nm、55 nm、60 nm、65 nm、70 nm、75 nm、80 nm、85 nm、90 nm、95 nm、100 nm、105 nm、110 nm、115 nm、120 nm、125 nm、130 nm、135 nm、140 nm、145 nm or 150 nm.

The nucleic acid particles (e.g., ribonucleic acid particles) described herein can exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. For example, the nucleic acid particles (e.g., ribonucleic acid particles) can exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

The nucleic acid particles (e.g., ribonucleic acid particles) described herein can be characterized by an "N/P ratio," which is the molar ratio of cationic (nitrogen) groups (N "in N/P) in the cationic polymer to anionic (phosphate) groups (P" in N/P) in the RNA. It is understood that a cationic group is a group in the cationic form (e.g., N ⁺), or a group that can ionize to become cationic. The use of a single number in an N/P ratio (e.g., an N/P ratio of about 5) is intended to mean that the number exceeds 1, e.g., an N/P ratio of about 5 is intended to mean 5:1. In some embodiments, a nucleic acid particle (e.g., ribonucleic acid particle) described herein has an N/P ratio of greater than or equal to 5. In some embodiments, a nucleic acid particle (e.g., ribonucleic acid particle) described herein has an N/P ratio of about 5, 6, 7, 8, 9, or 10. In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) described herein have an N/P ratio of about 10 to about 50. In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) described herein have an N/P ratio of about 10 to about 70. In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) described herein have an N/P ratio of about 10 to about 120.

The nucleic acid particles (e.g., ribonucleic acid particles) described herein can be prepared using a variety of methods that can involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer and mixing the colloid with a nucleic acid to obtain a nucleic acid particle.

As used herein, the term "colloid" refers to a type of homogeneous mixture in which the dispersed particles do not settle. The insoluble particles in the mixture may be microscopic, with a particle size between 1 and 1000 nanometers. The mixture may be referred to as a colloid or colloid suspension. Sometimes the term "colloid" refers only to the particles in the mixture and not the entire suspension.

The term "average diameter" or "average diameter" refers to the average hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS), wherein data analysis is performed using a so-called cumulant algorithm, the result of which provides a so-called Z-average and dimensionless Polydispersity Index (PI) with a length dimension (Koppel, d., j. Chem. Phys. 57, 1972, pages 4814-4810, ISO 13321, which is incorporated herein by reference). Herein, "average diameter (AVERAGE DIAMETER)", "average diameter (MEAN DIAMETER)", "diameter" or "size" of the particles are used synonymously with the value of Z-average.

The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis as mentioned in the definition of "average diameter". Under certain preconditions, it may be considered a measure of the size distribution of a collection of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

Different types of nucleic acid particles have been previously described as suitable for delivering nucleic acids in particulate form (e.g., kaczmarek et al, 2017, genome Medicine 9, 60, which is incorporated herein by reference). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids physically protects the nucleic acids from degradation and, depending on the particular chemistry, can facilitate cellular uptake and endosomal escape.

The present disclosure describes particles comprising nucleic acids (e.g., polyribonucleotides), at least one cationic or cationically ionizable lipid or lipid-like material, and/or at least one cationic polymer that binds to nucleic acids (e.g., polyribonucleotides) to form nucleic acid particles (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles), and compositions comprising such particles. Nucleic acid particles (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) can comprise nucleic acids (e.g., polyribonucleotides) that are complexed with the particles in different forms through non-covalent interactions. The particles described herein are not viral particles, in particular, they are not infectious viral particles, i.e. they are not capable of virally infecting cells.

Some embodiments described herein relate to compositions, methods, and uses involving more than one, e.g., 2, 3, 4, 5, 6, or even more nucleic acid species (e.g., polyribonucleotide species).

In nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations, it is possible to formulate each nucleic acid species (e.g., polyribonucleotide species) individually as a single nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation. In this case, each single nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation will comprise one nucleic acid species (e.g., a polynucleotide species). The single nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation can exist as a separate entity, e.g., in a separate container. Such formulations may be obtained by separately providing each nucleic acid species (e.g., polyribonucleotide species), typically each in the form of a solution containing the nucleic acid, and a particle forming reagent, thereby allowing the formation of particles. The corresponding particles will exclusively contain the particular nucleic acid species (e.g. polyribonucleotide species) provided at the time of particle formation (single particle formulation).

In some embodiments, a composition (e.g., a pharmaceutical composition) comprises more than one single nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation. The corresponding pharmaceutical compositions are referred to as "mixed microparticle formulations". The mixed microparticle formulation according to the present invention can be obtained by separately forming a single-nucleic-acid-particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation as described above, followed by a step of mixing the single-nucleic-acid-particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation. By means of the mixing step, a formulation comprising a mixed population of nucleic acid-containing particles can be obtained. A population of single nucleic acid particles (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) can be together in a container comprising a mixed population of single nucleic acid particle (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) formulations.

Or different nucleic acid species (e.g., polyribonucleotide species) may be formulated together as a "combined microparticle formulation". Such formulations may be obtained by providing a combined preparation (typically a combined solution) of different nucleic acid species (e.g., polyribonucleotide species) and particle forming reagents, thereby allowing the formation of particles. In contrast to the "mixed microparticle formulation," a "combined microparticle formulation" will typically comprise particles comprising more than one nucleic acid species (e.g., a polynucleic acid species) species. In a combined microparticle composition, different nucleic acid species (e.g., polyribonucleotide species) are typically present together in a single particle.

In certain embodiments, the nucleic acid (e.g., polyribonucleotide) is resistant to degradation by nucleases in aqueous solution when present in a provided nucleic acid particle (e.g., ribonucleic acid particle, e.g., lipid nanoparticle).

In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) are lipid nanoparticles. In some embodiments, the lipid nanoparticle is a liver-targeting lipid nanoparticle. In some embodiments, the lipid nanoparticle is a cationic lipid nanoparticle comprising one or more cationic lipids (e.g., lipids described herein). In some embodiments, the cationic lipid nanoparticle may comprise at least one cationic lipid, at least one polymer-coupled lipid, and at least one helper lipid (e.g., at least one neutral lipid).

Cationic polymer material

Cationic polymers have been considered useful in the development of particulate delivery vehicles, as reported in PCT application publication No. WO2021/001417, the entire contents of which are incorporated herein by reference. As used herein, the term "polymer" refers to a composition comprising one or more molecules comprising repeating units of one or more monomers. As used herein, "polymer," "polymeric material," and "polymer composition" are used interchangeably and refer to a composition of polymer molecules unless otherwise specified. Those skilled in the art will appreciate that the polymer composition comprises polymer molecules having molecules of different lengths (e.g., comprising different amounts of monomers). The polymer compositions described herein are characterized by one or more of normalized molecular weight (Mn), weight average molecular weight (Mw), and/or polydispersity index (PDI). In some embodiments, such repeat units may all be identical ("homopolymers"), or in some cases, more than one type of repeat unit ("heteropolymers" or "copolymers") may be present within the polymeric material. In some cases, the polymer is bio-derived, e.g., a biopolymer, such as a protein. In some cases, additional moieties may also be present in the polymeric material, such as targeting moieties, such as those described herein.

In some embodiments, the polymer as used in the present disclosure may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, in some embodiments, the repeating units may be arranged in a random order, alternatively or additionally, in some embodiments, the repeating units may be arranged in an alternating order or as a "block" copolymer, e.g., the "block" copolymer comprising one or more regions each comprising a first repeating unit (e.g., a first block), and one or more regions each comprising a second repeating unit (e.g., a second block), etc. The block copolymer may have two (diblock copolymer), three (triblock copolymer) or a greater number of different blocks.

In certain embodiments, the polymeric materials as used in the present disclosure are biocompatible. In certain embodiments, the biocompatible material is biodegradable, e.g., capable of being chemically and/or biologically degraded within a physiological environment (e.g., in vivo).

In certain embodiments, the polymeric material may be or comprise protamine or polyalkyleneimine.

As will be appreciated by those skilled in the art, the term "protamine" is generally used to refer to any of a variety of strongly basic proteins of relatively low molecular weight that are rich in arginine and found to be particularly relevant to DNA, replacing the somatic histones in sperm cells of various animals (e.g., fish). In particular, the term "protamine" is generally used to refer to a protein found in fish sperm that is strongly alkaline, soluble in water, does not condense by heat, and produces primarily arginine upon hydrolysis. In purified form, protamine is used in long acting formulations of insulin and neutralizes the anticoagulant effect of heparin.

In some embodiments, the term "protamine" as used herein refers to protamine amino acid sequences obtained from or derived from natural or biological sources, including fragments thereof and/or multimeric forms of the amino acid sequences or fragments thereof, as well as (synthetic) polypeptides that are artificial and specifically designed for a particular purpose and cannot be isolated from natural or biological sources.

In some embodiments, the polyalkyleneimine comprises Polyethyleneimine (PEI) and/or polypropyleneimine. In some embodiments, the preferred polyalkyleneimine is Polyethyleneimine (PEI). In some embodiments, the average molecular weight of the PEI is preferably from 0.75 ∙ to 107 Da, preferably from 1000 to 105 Da, more preferably from 10000 to 40000 Da, more preferably from 15000 to 30000 Da, even more preferably from 20000 to 25000 Da.

Cationic materials contemplated for use herein (e.g., polymeric materials, including polycationic polymers) include those materials capable of electrostatically binding nucleic acids. In some embodiments, the cationic polymeric materials contemplated for use herein include any cationic polymeric material to which nucleic acids can bind (e.g., by forming a complex with a nucleic acid or forming vesicles in which a nucleic acid is blocked or encapsulated).

In some embodiments, the particles described herein may comprise polymers other than cationic polymers, such as non-cationic polymeric materials and/or anionic polymeric materials. In summary, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials.

Lipid composition

Lipid and lipid-like material

Lipids and lipid-like materials have also been considered useful in the development of particulate delivery vehicles. The terms "lipid" and "lipid-like material" are defined broadly herein as molecules comprising one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising a hydrophobic moiety and a hydrophilic moiety are also commonly referred to as amphiphiles. Lipids are generally poorly soluble in water. In an aqueous environment, amphiphilic properties allow molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as it is present in vesicles, multilamellar/unilamellar liposomes or membranes in an aqueous environment. Hydrophobicity may be imparted by including non-polar groups including, but not limited to, long chain saturated and unsaturated aliphatic hydrocarbyl groups and such groups substituted with one or more aromatic, cycloaliphatic, or heterocyclic groups. Hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphates, carboxyl groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

Typically, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water and the non-polar moiety is insoluble in water. In addition, the polar moiety may bear a formal positive or formal negative charge. Or the polar moiety may bear both formal positive and negative charges and be zwitterionic or an internal salt. For the purposes of this disclosure, amphiphilic compounds may be, but are not limited to, one or a negative number of natural or unnatural lipids and lipid-like compounds.

"Lipid-like material" is a substance that is structurally and/or functionally related to lipids, but may not be considered a lipid in a strict sense. For example, the term includes compounds that are capable of forming an amphiphilic layer when present in vesicles, multilamellar/unilamellar liposomes or membranes in an aqueous environment, and includes surfactants or synthetic compounds having both hydrophilic and hydrophobic moieties. In general, the term refers to molecules comprising hydrophilic and hydrophobic portions with different structural organization, which may or may not be similar to the structural organization of lipids.

Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, amino lipids, and sphingolipids.

In general, lipids can be classified into eight classes, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from condensation of ketoacyl subunits), sterols, and prenyl alcohol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fat, fat is a subset of lipids known as triglycerides. Lipids also encompass molecules such as fatty acids and derivatives thereof (including triglycerides, diglycerides, monoglycerides and phospholipids), as well as metabolites containing sterols such as cholesterol.

Fatty acids are a group of different molecules made up of hydrocarbon chains terminated with carboxylic acid groups, the arrangement being such that the molecules have a polar hydrophilic end and a non-polar hydrophobic end that is insoluble in water. The carbon chain, which is typically between four and 24 carbons in length, may be saturated or unsaturated and may be linked to functional groups containing oxygen, halogen, nitrogen and sulfur. If the fatty acid contains a double bond, cis or trans geometric isomerism may exist, which significantly affects the configuration of the molecule. Cis double bonds cause bending of the fatty acid chains, an effect that complexes with more double bonds in the chain. Other major lipid classes in the fatty acid class are fatty esters and fatty amides.

Glycerolipids consist of mono-, di-and tri-substituted glycerins, the most notable of which are fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerols" is sometimes used synonymously with "triglycerides". In this class of compounds, the three hydroxyl groups of glycerol are typically each esterified with a different fatty acid. Other subclasses of glycerolipids are represented by glycosylglycerols, characterized by the presence of one or more sugar residues linked to glycerol by glycosidic bonds.

Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked by an ester linkage to two fatty acid-derived "tails" and by a phosphate ester linkage to one "head" group. Examples of glycerophospholipids commonly known as phospholipids (although sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

Sphingolipids are members of a family of complex compounds that share a common structural feature, namely a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramide (N-acyl-sphingoid base) is a major subset of sphingoid base derivatives with amide linked fatty acids. Fatty acids are generally saturated or monounsaturated and have a chain length of 16 to 26 carbon atoms. The main sphingomyelin of mammals is sphingomyelin (ceramide phosphorylcholine), whereas insects mainly contain ceramide phosphorylethanolamine, and fungi have phytoceramide phosphorylinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules consisting of one or more sugar residues linked to a sphingoid base by glycosidic linkages. Examples of such substances are simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

Sterols such as cholesterol and its derivatives, or tocopherols and its derivatives, and glycerophospholipids and sphingomyelins are important components of membrane lipids.

Glycolipids are compounds in which fatty acids are directly linked to the sugar backbone, forming a structure compatible with membrane bilayers. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar glycolipids are the acylated glucosamine precursors of the lipid A component of lipopolysaccharide in Gram-negative bacteria (Gram-negative bacteria). A typical lipid a molecule is a disaccharide of glucosamine, which is derivatized with up to seven fatty acyl chains. The smallest lipopolysaccharide required for growth in E.coli is Kdo 2-lipid A, which is the hexaacylated disaccharide of glucosamine formed by glycosylation of two 3-deoxy-D-manno-octanoonic acid (Kdo) residues.

Polyketides are synthesized by classical enzymes and iterative and multi-modular enzymes sharing mechanical characteristics with fatty acid synthases polymerize acetyl and propionyl subunits. Polyketides contain a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources and have a vast structural diversity. Many polyketides are cyclic molecules whose backbone is typically further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

Lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

In some embodiments, suitable lipids or lipid-like materials for use in the present disclosure include those described in WO2020/128031 and US2020/0163878, the entire contents of which are incorporated herein by reference for the purposes described herein.

Cationic or cationically ionizable lipid and lipid-like materials

In some embodiments, the cationic or cationically ionizable lipid or lipid-like material contemplated for use herein includes any cationic or cationically ionizable lipid or lipid-like material capable of electrostatically binding nucleic acids. In one embodiment, the cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein may be associated with a nucleic acid, e.g., by forming a complex with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

Cationic lipids or lipid-like materials are characterized by having a net positive charge (e.g., at an associated pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl group, or multiple acyl chains, and the head group of the lipid typically carries a positive charge.

In certain embodiments, the cationic lipid or lipid-like material has a net positive charge only at certain pH, particularly acidic pH, and preferably has no net positive charge, preferably no charge, i.e., is neutral, at a different, preferably higher pH, such as physiological pH. This ionisable behaviour is believed to enhance efficacy by helping endosomes escape and reducing toxicity compared to particles that remain cationic at physiological pH.

In some embodiments, the cationic or cationically ionizable lipid or lipid-like material comprises a head group that includes at least one nitrogen atom (N) that is positively charged or capable of protonation.

Examples of cationic lipids include, but are not limited to, 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP), N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), 1, 2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3- (N- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol), dimethyl Dioctadecyl Ammonium (DDAB), 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP), 1, 2-dioleoyl-3-dimethylammonium propane, 1, 2-dialkyloxy-3-dimethylammonium propane, dioctadecyl dimethylammonium chloride (DODAC), 1, 2-distearyloxy-N, N-dimethyl-3-aminopropane (DSDMA), 2, 3-di (tetradecyloxy) propyl- (2-hydroxyethyl) -Dimethylazenium (DMRIE), 1, 2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l, 2-dimyristoyl-3-trimethylammoniopropane (DMTAP), 1, 2-dioleyloxy propyl-3-dimethyl-hydroxyethylammonium bromide (DORIE) and 2, 3-dioleyloxy-N- [2 (spermidine) ethyl ] -N, N-dimethyl-l-propylammonium (DOSPA) trifluoroacetate, 1, 2-Dioleoyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-Dilinoleyloxy-N, N-dimethylaminopropane (DLenDMA), dioctadecylamido Gan Anxian-spermine (DOGS), 3-dimethylamino-2- (cholest-5-en-3- β -oxybuty-4-yloxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (CLinDMA), 2- [5'- (cholest-5-en-3- β -oxy) -3' -oxapentoxy) -3-dimethyl-1- (cis, cis-9 ',12' -octadecadienyloxy) propane (CpLinDMA), N, N-dimethyl-3, 4-dioleyloxybenzyl amine (DMOBA), 1,2-N, N '-dioleylaminoformyl-3-dimethylaminopropane (DOcarbDAP), 2, 3-dioleyloxyn-N, N-dimethylpropylamine (DLinDAP), 1,2-N, N' -dioleylaminoformyl-3-dimethylaminopropane (DLincarbDAP), 1, 2-dioleylaminoformyl-3-dimethylaminopropane (DLinCDAP), 2-dioleylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), 2, 2-Di-lino-4-dimethylaminoethyl- [1,3] -dioxolane (DLin-K-XTC 2-DMA), 2-Di-lino-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), thirty-seven carbon-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate (DLin-MC 3-DMA), N- (2-hydroxyethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanium bromide (DMRIE), (+ -) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (cis-9-tetradecyloxy) -1-propanium bromide (GAP-DMOS), (±) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (dodecyloxy) -1-propanenitrile (GAP-DLRIE), (±) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanenitrile (GAP-dmriie), N- (2-aminoethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propanenitrile (βae-dmriie), N- (4-carboxybenzyl) -N, N-dimethyl-2, 3-bis (oleoyloxy) propan-1-ammonium (DOBAQ), 2- ({ 8- [ (3β) -cholest-5-en-3-yloxy ] octyl } oxy) -N, N-dimethyl-3- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] propan-1-amine (octyl-CLinDMA), 1, 2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1, 2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1- [2- ((1S) -1- [ (3-aminopropyl) amino ] -4- [ bis (3-amino-propyl) amino ] butylcarboxamido) ethyl ] -3, 4-di [ oleoyl ] -benzamide (MVL 5), 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2, 3-bis (dodecyloxy) -N- (2-hydroxyethyl) -N, N-dimethylpropan-1-ammonium bromide (DLRIE), N- (2-aminoethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) propan-1-ammonium bromide (dmo), 8' - (((((2 (dimethylamino) ethyl) thio) carbonyl) azanediyl) dioctanoic acid di ((Z) -non-2-en-1-yl) ester (ATX), N-dimethyl-2, 3-bis (dodecyloxy) propan-1-amine (DLDMA), N, N-dimethyl-2, 3-bis (tetradecyloxy) propan-1-amine (DMDMA), di ((Z) -non-2-en-1-yl) -9- ((4- (dimethylaminobutyryl) oxy) heptadecanedioate (L319), N-dodecyl-3- ((2-dodecylcarbamoyl-ethyl) - {2- [ (2-dodecylcarbamoyl-ethyl) -2- { (2-dodecylcarbamoyl-ethyl) - [2- (2-dodecylcarbamoyl-ethylamino) -ethyl ] -amino } -ethylamino) propanamide (lipid 98N 12-5), 1- [2- [ bis (2-hydroxydodecyl) amino ] ethyl- [2- [4- [2- [ bis (2 hydroxydodecyl) amino ] ethyl ] pyrazin-1-yl ] ethyl ] amino ] dodecyl-2-ol (lipid C12-200), LIPOFECTIN (commercially available cationic lipid comprising DOTMA and 1, 2-dioleoyl-sn-3 phosphoethanolamine (DOPE), from GIBCO/BRL, grand Island, N.Y.), LIPOFECTAMINE (commercially available cationic lipid comprising N- (1- (2, 3 dioleyloxy) propyl) -N- (2- (spermimidoyl) ethyl) -N, N-dimethyltrifluoro ammonium acetate (DOSPA) and (DOPE), from GIBCO/BRL), and TRANSFECTAM (commercially available cationic lipid comprising ethanol dioctadecyl amido Gan Anxian carboxyspermine (DOGS), from Promega Corp., madison, wis.), or any combination of any of the foregoing. Other suitable cationic lipids for use in the present disclosure include those described in WO2020/128031 and US2020/0163878, the entire contents of which are incorporated herein by reference for the purposes described herein. Other suitable cationic lipids for use in the present disclosure include those described in WO2010/053572 (including C12-200 described in paragraph [00225 ]) and WO2012/170930, which patents are incorporated herein by reference for the purposes described herein. Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US2015/0140070, which is incorporated herein by reference in its entirety).

In some embodiments, a formulation useful in pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) compositions as described herein can comprise at least one cationic lipid. Representative cationic lipids include, but are not limited to, 1, 2-dioleoyl-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dioleoyl-3 morpholinopropane (DLin-MA), 1, 2-dioleoyl-3-dimethylaminopropane (DLinDAP), 1, 2-dioleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyloxy-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleyloxy-3-trimethylaminopropane chloride salt (DLin-tma. Ci), 1, 2-dioleoyl-3-trimethylaminopropane chloride salt (DLin-tap. Ci), 1, 2-dioleoyl-3- (N-methylpyrazinyl) propane (DLin-MPZ), 3- (N, N-dioleyloxy) -1, 2-propanediol (N-dioleyloxy) -1, 2-dioleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleyloxy-3-trimethylaminopropane chloride salt (DLin-tap. Ci), 1, 2-dioleyloxy-3- (N-methylpyrazinyl) propane (DLin-MPZ), 3- (N, N-dioleyloxy) -3-dimethylaminopropane (DLin-2-propanediol) and 2- [ dioleoyl-3-trimethylaminopropane chloride salt (DLin-TAP) 2, 2-Di-lino-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), di-lino-methyl-4-dimethylaminobutyrate (DLin-MC 3-DMA), MC3 (US 2010/0325420, which is incorporated herein by reference in its entirety).

In some embodiments, the amino or cationic lipids useful as described herein have at least one protonatable or deprotonated group such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably at or above physiological pH. It will of course be appreciated that the addition or removal of protons changes with pH to an equilibrium process, and that reference to charged or neutral lipids refers to the nature of the main species and does not require that all lipids must be present in charged or neutral form. Lipids having more than one protonatable or deprotonated group or being zwitterionic are not excluded and are equally applicable in the context of the present invention.

In some embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

In some embodiments, in lipid compositions as used in the present disclosure, the cationic lipid may comprise from about 10 mol% to about 100 mol%, from about 20 mol% to about 100 mol%, from about 30 mol% to about 100 mol%, from about 40 mol% to about 100 mol%, or from about 50 mol% to about 100 mol% of the total lipid.

Additional lipids or lipid-like materials

In some embodiments, a formulation as used in the present disclosure may comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non-cationic ionizable lipids or lipid-like materials). In summary, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. In some embodiments, optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties (such as cholesterol and lipids) in addition to the ionizable/cationic lipid or lipid-like substance may, for example, enhance particle stability and efficacy of nucleic acid delivery.

In some embodiments, lipids or lipid-like materials may be incorporated that may or may not affect the overall charge of the particle. In certain embodiments, such lipid or lipid-like material is a non-cationic lipid or lipid-like material.

In some embodiments, the non-cationic lipid may comprise, for example, one or more anionic lipids and/or neutral lipids. An "anionic lipid" is negatively charged (e.g., at a selected pH).

The "helper lipid" exists in the form of an uncharged neutral zwitterionic (e.g., at a selected pH), or in some embodiments, in a form that has a cationic or positive charge at physiological pH. In some embodiments, the formulation comprises one of (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof, in combination with a lipid component. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholestanone, cholestanol, cholestyl-2 '-hydroxyethyl ether, cholestyl-4' -hydroxybutyl ether, tocopherols and derivatives thereof, and mixtures thereof.

Specific exemplary phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. In particular, such phospholipids include diacyl phosphatidylcholine, such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), eicosapentaenoyl phosphatidylcholine, dilauroyl phosphatidylcholine, dipalmitoyl phosphatidylcholine (DPPC), dicapranoyl phosphatidylcholine (DAPC), dibbehenyl phosphatidylcholine (DBPC), ditrianoyl phosphatidylcholine (DTPC), bistetracosanoyl phosphatidylcholine (DLPC), palmitoyl oleoyl-phosphatidylcholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 diether PC), 1-oleoyl-2-cholesterol-pessanoyl-sn-glycero-3-phosphorylcholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphorylcholine (C16 Lyso PC) and phosphatidylethanolamine, in particular diacyl phosphatidylethanolamine, such as dioleoyl phosphatidylethanolamine (dopolyethanolamine), stearoyl phosphatidylethanolamine (pe), and phosphatidylethanolamine (DPPE-phosphatidylethanolamine (DPPE), phosphatidylethanolamine (DPPE-phosphatidylethanolamine) have the same hydrophobe as the other phosphatidyl group.

In certain embodiments, a formulation as used in the present disclosure includes DSPC or DSPC and cholesterol.

In some embodiments, a formulation as used in the present disclosure comprises an ionizable or cationic auxiliary lipid.

In certain embodiments, the formulation as used in the present disclosure includes both cationic lipids and additional (non-cationic) lipids.

In some embodiments, the formulations herein include a polymer-coupled lipid, such as a pegylated lipid. "PEGylated lipids" or "PEG conjugated lipids" comprise both a lipid moiety and a polyethylene glycol moiety. Pegylated lipids are known in the art.

Without wishing to be bound by theory, the amount of (total) cationic lipids may affect important characteristics such as charge, particle size, stability, tissue selectivity, and biological activity of the nucleic acid as compared to the amount of other lipids in the formulation. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

Lipid complex particles

In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipid complex particles.

The "RNA lipid complex particles" contain lipids (in particular cationic lipids) and RNA. Electrostatic interactions between positively charged lipid plasmids and negatively charged RNAs lead to the complexation and spontaneous formation of RNA lipid complex particles. Positively charged lipid plasmids can generally be synthesized using cationic lipids (e.g., DOTMA) and additional lipids (e.g., DOPE). In one embodiment, the RNA lipid complex particles are nanoparticles.

In certain embodiments, the RNA lipid complex particles include both cationic lipids and additional lipids. In one exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In particular embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In one exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

In some embodiments, the RNA lipid complex particles have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm in one embodiment. In specific embodiments, the RNA lipid complex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In one embodiment, the RNA lipid complex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipid complex particles have an average diameter in the range of about 300 nm to about 500 nm. In one exemplary embodiment, the RNA lipid complex particles have an average diameter of about 400 nm.

The RNA lipid complex particles and compositions comprising the RNA lipid complex particles described herein are useful for delivering RNA to a target tissue following parenteral administration, particularly following intravenous administration. RNA lipid complex particles can be prepared using a lipid plasmid, which can be obtained by injection of an alcoholic solution of the lipid into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises, for example, about 5 mM amounts of acetic acid. The lipid plasmid can be used to prepare RNA lipid complex particles by mixing the lipid plasmid with RNA. In one embodiment, the lipid plasmid and RNA lipid complex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1, 2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP). In one embodiment, the at least one additional lipid comprises 1, 2-di- (9Z-octadecenoyl) -sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol (Chol), and/or 1, 2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC). In one embodiment, the at least one cationic lipid comprises 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA) and the at least one additional lipid comprises 1, 2-di- (9Z-octadecenoyl) -sn-glycerol-3-phosphate ethanolamine (DOPE). In one embodiment, the lipid plasmid and RNA lipid complex particles comprise 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA) and 1, 2-di- (9Z-octadecenoyl) -sn-glycero-3-phosphoethanolamine (DOPE).

Spleen-targeting RNA lipid complex particles are described in WO2013/143683, which is incorporated herein by reference. RNA lipid complex particles with a net negative charge have been found to be useful for preferentially targeting spleen tissue or spleen cells, such as antigen presenting cells, particularly dendritic cells. Thus, RNA accumulation and/or RNA expression occurs in the spleen after administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lung and/or liver following administration of the RNA lipid complex particles. In one embodiment, RNA accumulation and/or RNA expression occurs in antigen presenting cells (e.g., professional antigen presenting cells in the spleen) after administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.

Lipid Nanoparticles (LNP)

In some embodiments, the nucleic acids (e.g., RNAs) described herein are administered in the form of Lipid Nanoparticles (LNPs). In some embodiments, the LNP may comprise any lipid capable of forming a particle to which one or more nucleic acid molecules are attached, or in which one or more nucleic acid molecules are encapsulated.

In some embodiments, the LNP comprises one or more cationic lipids and one or more stabilizing lipids. Stabilizing lipids include neutral lipids, helper lipids, and pegylated lipids.

In some embodiments, the LNP comprises a cationic lipid, a helper lipid, a sterol, a polymer-conjugated lipid, and an RNA encapsulated within or bound to the lipid nanoparticle.

In some embodiments, the helper lipid is phosphatidylcholine, such as 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), l, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), phosphatidylethanolamine, such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM). In some embodiments, the helper lipid is 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1- α -Phosphatidylserine (PS), or DOPE. In some embodiments, the helper lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, DOTAP, PS and SM. In some embodiments, the helper lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOTAP, PS and SM. In some embodiments, the helper lipid is DSPC. In some embodiments, the helper lipid is DOTAP. In some embodiments, the helper lipid is DOPE. In some embodiments, the helper lipid is PS.

In some embodiments, the sterol is cholesterol.

In some embodiments, the polymer-coupled lipid is a pegylated lipid (PEG lipid). In some embodiments, the PEG lipid is selected from the group consisting of PEGylated diacylglycerols (PEG-DAG), such as l- (monomethoxy-polyethylene glycol) -2, 3-dimyristoyl glycerol (PEG-DMG) (e.g., 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (PEG 2000-DMG)), PEGylated phosphatidylethanolamine (PEG-PE), PEGylated diacylglycerols (PEG-S-DAG), such as 4-O- (2 ',3' -di (tetradecyloxy) propyl-1-O- (omega-methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEGylated dialkoxypropyl carbamates, such as omega-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecyloxy) propyl) carbamate and 2, 3-di (tetradecyloxy) propyl 1-N- (omega methoxy (polyethoxy) ethyl) carbamate. The pegylated lipids have the following structure:

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

R ₁₂ and R ₁₃ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds, and w has an average value in the range from 30 to 60. In some embodiments, R ₁₂ and R ₁₃ are each independently a straight saturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R ₁₂ and R ₁₃ are each independently a straight saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

In some embodiments, the pegylated lipid is DMG-PEG 2000, e.g., having the following structure:

。

In some embodiments, the pegylated lipid is or comprises 2- [ (polyethylene glycol) -2000] -N, N-bitetradecylacetamide having a chemical structure as shown below:

or a pharmaceutically acceptable salt thereof, wherein n' is an integer from 45 to 50

In some embodiments, the cationic lipid component of the LNP has the structure of formula (III):

(III)

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

One of L ¹ or L ² is -O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NRaC (=o) O-, and the other of L ¹ or L ² is -O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa- or-NRaC (=o) O-, or a direct bond;

Each of G ¹ and G ² is independently unsubstituted C ₁-C₁₂ alkylene or C ₁-C₁₂ alkenylene;

G ³ is C ₁-C₂₄ alkylene, C ₁-C₂₄ alkenylene, C ₃-C₈ cycloalkylene, C ₃-C₈ cycloalkenyl;

r ^a is H or C ₁-C₁₂ alkyl;

R ¹ and R ² are each independently C ₆-C₂₄ alkyl or C ₆-C₂₄ alkenyl;

R ³ is H, OR ⁵、CN、-C(=O)OR⁴、-OC(=O)R⁴ or-NR ⁵C(=O)R⁴;

R ⁴ is C ₁-C₁₂ alkyl;

R ⁵ is H or C ₁-C₆ alkyl, and

X is 0,1 or 2.

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

Or (b)

Wherein:

a is a3 to 8 membered cycloalkyl or cycloalkylene ring;

R ⁶ is independently at each occurrence H, OH or C ₁-C₂₄ alkyl;

n is an integer in the range of 1 to 15.

In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID):

Or (b)

Wherein y and z are each independently integers in the range of 1 to 12.

In any of the foregoing embodiments of formula (III), one of L ¹ or L ² is-O (c=o) -. For example, in some embodiments, each of L ¹ and L ² is-O (c=o) -. In some different embodiments shown in any of the foregoing, L ¹ and L ² are each independently- (c=o) O-or-O (c=o) -. For example, in some embodiments, each of L ¹ and L ² is- (c=o) O-.

In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

Or (b) 。

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

;;

Or (b) 。

In some of the foregoing embodiments of formula (III), n is an integer in the range of 2 to 12, e.g., 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some other of the foregoing embodiments of formula (III), y and z are each independently an integer in the range of 2 to 10. For example, in some embodiments, y and z are each independently integers in the range of 4 to 9 or 4 to 6.

In some of the foregoing embodiments of formula (III), R ⁶ is H. In other preceding embodiments, R ⁶ is C ₁-C₂₄ alkyl. In other embodiments, R ⁶ is OH.

In some embodiments of formula (III), G ³ is unsubstituted. In other embodiments, G ³ is substituted. In various embodiments, G ³ is a linear C ₁-C₂₄ alkylene or linear C ₁-C₂₄ alkenylene.

In some other of the foregoing embodiments of formula (III), R ¹ or R ², or both, are C ₆-C₂₄ alkenyl. For example, in some embodiments, R ¹ and R ² each independently have the following structure:

,

Wherein:

R ^7a and R ^7b are independently at each occurrence H or C ₁-C₁₂ alkyl, and

A is an integer of 2 to 12,

Wherein R ^7a、R^7b and a are each selected such that R ¹ and R ² each independently contain from 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

In some of the foregoing embodiments of formula (III), at least one occurrence of R ^7a is H. For example, in some embodiments, R ^7a is H at each occurrence. In various other embodiments of the foregoing, at least one occurrence of R ^7b is C ₁-C₈ alkyl. For example, in some embodiments, the C ₁-C₈ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, n-hexyl, or n-octyl.

In various embodiments of formula (III), R ¹ or R ², or both, have one of the following structures:

;;;;;;;;;。

In some of the foregoing embodiments of formula (III), R ³ is OH, CN, -C (=o) OR ⁴、-OC(=O) R⁴, OR-NHC (=o) R ⁴. In some embodiments, R ⁴ is methyl or ethyl.

In various embodiments, the cationic lipid of formula (III) has one of the structures listed in table 15 below.

TABLE 15 exemplary cationic lipid Structure of formula (III)

In various embodiments, the cationic lipid has one of the structures listed in table 16 below.

TABLE 16 exemplary cationic lipid structures of formulas A-F

In some embodiments, the LNP comprises a cationic lipid as the ionizable lipid-like material (lipid). In some embodiments, the cationic lipid has one of the following structures as described in Melamed et al, SCIENCE ADVANCES, 2023, 9, eade1444 (the entire contents of which are incorporated herein by reference):

X-1

X-2

X-3

X-4。

In some embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, lipid nanoparticles as described in the present disclosure may have an average size (e.g., average diameter) of about 50 nm to about 100 nm. In some embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 50 nm to about 150 nm. In some embodiments, the lipid nanoparticle may have an average size (e.g., average diameter) of about 60 nm to about 120 nm. In some embodiments, lipid nanoparticles as described in the present disclosure may have an average size (e.g., average diameter) of about 30 nm、35 nm、40 nm、45 nm、50 nm、55 nm、60 nm、65 nm、70 nm、75 nm、80 nm、85 nm、90 nm、95 nm、100 nm、105 nm、110 nm、115 nm、120 nm、125 nm、130 nm、135 nm、140 nm、145 nm or 150 nm. The term "average diameter (AVERAGE DIAMETER)" or "average diameter (MEAN DIAMETER)" refers to the average hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS), wherein data analysis is performed using a so-called cumulant algorithm, the result of which provides a so-called Z-average with length dimension and dimensionless Polydispersity Index (PI) (Koppel, chem. Phys. 57, 1972, pp 4814-4810, ISO 13321, which is incorporated herein by reference). Here, "average diameter (AVERAGE DIAMETER)", "average diameter (MEAN DIAMETER)", "diameter" or "size" of the particles are used synonymously with the value of Z-average.

In some embodiments, the lipid nanoparticles described herein can exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. For example, the lipid nanoparticle may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3. The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis as mentioned in the definition of "average diameter". Under certain preconditions, it may be considered a measure of the size distribution of a collection of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

The lipid nanoparticles described herein may be characterized by an "N/P ratio", which is the molar ratio of cationic (nitrogen) groups (N "in N/P) to anionic (phosphate) groups (P" in N/P) in RNA in a cationic polymer. It is understood that a cationic group is a group in the cationic form (e.g., N ⁺), or a group that can ionize to become cationic. The use of a single number in an N/P ratio (e.g., an N/P ratio of about 5) is intended to mean that the number exceeds 1, e.g., an N/P ratio of about 5 is intended to mean 5:1. In some embodiments, the lipid nanoparticles described herein have an N/P ratio of greater than or equal to 5. In some embodiments, the lipid nanoparticles described herein have an N/P ratio of about 5, 6, 7, 8, 9, or 10. In some embodiments, the lipid nanoparticle described herein has an N/P ratio of about 10 to about 50. In some embodiments, the lipid nanoparticle described herein has an N/P ratio of about 10 to about 70. In some embodiments, the lipid nanoparticle described herein has an N/P ratio of about 10 to about 120.

In some embodiments, the formulations described herein comprising lipid nanoparticles are formulated for intramuscular (i.m.) or intravenous (i.v.) delivery, and the lipid nanoparticles comprise i) about 30 to about 50 mol% cationic lipid, ii) about 1 to about 5 mol% PEG-conjugated lipid, iii) about 5 to about 15 mol% auxiliary lipid, and iv) about 30 to about 50 mol% steroid.

In some embodiments, the formulations described herein comprising lipid nanoparticles are formulated for intraperitoneal (i.p.) delivery, and the lipid nanoparticles comprise i) about 30 mol% to about 50 mol% cationic lipid, ii) about 1 mol% to 5 mol% PEG conjugated lipid, iii) about 30 mol% to about 50 mol% auxiliary lipid, and iv) about 20 mol% to about 40 mol% cholesterol. Without wishing to be bound by any theory, it is expected that intraperitoneal (i.p.) delivery of such formulations will result in enhanced delivery of lipid nanoparticles to pancreatic β cells and enhanced in vivo expression of the encoded incretin agent in pancreatic β cells.

In some embodiments, the formulation for i.p. delivery comprises a lipid nanoparticle, wherein the lipid nanoparticle comprises about 35 mol% cationic lipid, about 40 mol% helper lipid, about 22.5 mol% cholesterol, and about 2.5 mol% PEG conjugated lipid. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-2, X-3, or X-4, about 40 mol% DOTAP, DOPE, or PS, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-2, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-3, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-4, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-2, about 40. 40 mol% DOPE, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-3, about 40. 40 mol% DOPE, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-4, about 40. 40 mol% DOPE, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-2, about 40. 40 mol% PS, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-3, about 40. 40 mol% PS, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticle comprises about 35 mol% cationic lipid X-4, about 40. 40 mol% PS, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000. without wishing to be bound by any theory, it is expected that such formulations will result in delivery of lipid nanoparticles to pancreatic β cells and enhanced in vivo expression of encoded incretin agents in pancreatic β cells, particularly when administered by intraperitoneal (i.p.) delivery.

Exemplary methods of manufacturing lipid nanoparticles

Lipids and lipid nanoparticles comprising nucleic acids and methods of making the same are known in the art, including, for example, us patent number 8,569,256, 5,965,542, and us patent publication number 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, 2013/0323369, 2013/024567, 2013/0195920, 2013/01233338, 2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411, 2012/0027803, 2012/0058188, 2011/1583, 2011/0311582, 2011/0262527, 2011/0110125/2011, 2011/011016625, etc. the present invention also includes the compositions of the present invention. 2011/0091525, 2011/007035, 2011/0060032, 2010/0130588, 2007/0042031, 2006/0243493, 2006/0083780, 2006/0008910, 2005/0175682, 2005/017054, 2005/010110253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076, and PCT publications WO99/39741, WO2018/081480, WO2017/004143, WO2017/075531, WO2015/199952, WO2014/008334, WO2013/086373, WO2013/086322, WO 2013/058, WO2013/086373, W11/075 and WO2001/07548, the entire disclosure of which is incorporated herein by reference in its entirety for the purposes described herein.

For example, in some embodiments, the cationic lipid, the helper lipid (e.g., DSPC and/or cholesterol), and the polymer-coupled lipid may be dissolved in ethanol at a predetermined molar ratio (e.g., the molar ratios described herein). In some embodiments, the plurality of lipid nanoparticles (single lipid nanoparticle) are prepared at a total lipid to polyribonucleotide weight ratio of about 10:1 to 30:1. In some embodiments, such polyribonucleotides can be diluted to 0.2 mg/mL in acetate buffer.

In some embodiments, using ethanol injection techniques, colloidal lipid dispersions comprising polyribonucleotides can be formed by injecting an ethanol solution comprising lipids (such as cationic lipids, helper lipids, and polymer-coupled lipids) into an aqueous solution comprising polyribonucleotides (e.g., those described herein).

In some embodiments, the lipid and polynucleic acid solutions can be mixed at room temperature by pumping each solution into a mixing unit at a controlled flow rate (e.g., using a piston pump). In some embodiments, the flow rates of the lipid solution and the RNA solution into the mixing unit are maintained at a ratio of 1:3. After mixing, nucleic acid-lipid particles are formed as the ethanol lipid solution is diluted with aqueous polyribonucleotides. Lipid solubility decreases, while positively charged cationic lipids interact with negatively charged RNAs.

In some embodiments, the solution comprising RNA-encapsulated lipid nanoparticles may be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.

In some embodiments, the RNA-encapsulated lipid nanoparticle may be processed by filtration.

In some embodiments, the particle size and/or internal structure of the lipid nanoparticle (with or without RNA) can be monitored by suitable techniques such as small angle X-ray scattering (SAXS) and/or transmission electron cryomicroscopy (CryoTEM).

Pharmaceutical composition

The present disclosure provides compositions, e.g., pharmaceutical compositions, comprising one or more polyribonucleotides described herein. The pharmaceutical formulation may additionally comprise pharmaceutically acceptable excipients, as used herein, which include any and all solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as appropriate for the particular dosage form desired. Remington' S THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, gennaro (Lippincott, williams & Wilkins, baltimore, MD, 2006; which is incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any known excipient medium is incompatible with the substance or derivative thereof, such as by producing any undesirable biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated as falling within the scope of the present disclosure.

In some embodiments, the excipient is approved for human and for veterinary use. In some embodiments, the excipient is approved by the U.S. food and drug administration (United States Food and Drug Administration). In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), the European Pharmacopeia (EP), the british pharmacopeia, and/or the international pharmacopeia.

Pharmaceutically acceptable excipients for the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulating agents, surfactants and/or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents and/or fragrances may be present in the composition at the discretion of the formulator.

General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington, THE SCIENCE AND PRACTICE of Pharmacy 21 st edition, lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference).

In some embodiments, the pharmaceutical compositions provided herein may be formulated according to known techniques (such as those described in Remington: THE SCIENCE AND PRACTICE of Pharmacy 21 st edition, lippincott Williams & Wilkins, 2005, which is incorporated herein by reference) with one or more pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients.

The pharmaceutical compositions described herein may be administered by any suitable method known in the art. As will be appreciated by one of skill in the art, the route and/or mode of administration may depend on a variety of factors including, for example, but not limited to, stability and/or pharmacokinetics and/or pharmacodynamics of the pharmaceutical compositions described herein.

In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration, including modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intraperitoneal, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular or intra-articular injection and infusion. In preferred embodiments, the pharmaceutical compositions described herein are formulated for intraperitoneal, intravenous, intramuscular, or subcutaneous administration.

In some embodiments, the pharmaceutical compositions described herein are formulated for intraperitoneal administration. In some embodiments, pharmaceutically acceptable excipients useful for intraperitoneal administration include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.

In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable excipients that may be used for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, lipid nanoparticles, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of surfactants. In many cases, it is preferable to include an isotonic agent, for example, a sugar, a polyalcohol (e.g., mannitol, sorbitol) or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition agents which delay absorption (e.g., monostearates and gelatins).

Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and/or microfiltration. In some embodiments, the pharmaceutical compositions can be prepared as described herein and/or by methods known in the art.

Such compositions may also contain adjuvants, such as preserving, wetting, emulsifying and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures and by inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, into the pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts or developed hereafter. Generally, such methods of preparation include the steps of mixing the active agent with a diluent or another excipient and/or one or more other auxiliary ingredients, and then shaping and/or packaging the product into the desired single or multi-dose unit, if necessary and/or desired.

Pharmaceutical compositions as described in the present disclosure may be prepared, packaged and/or sold in bulk, as single unit doses and/or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using the systems and/or methods described herein.

The relative amount of polyribonucleotides encapsulated in the lipid nanoparticle, pharmaceutically acceptable excipient and/or any additional ingredients in the pharmaceutical composition can vary depending on the individual, target cell, disease or disorder to be treated, and can also further depend on the route of administration of the composition.

In some embodiments, the pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient (e.g., the polyribonucleotides encapsulated in the lipid nanoparticles) in the pharmaceutical compositions described herein can be varied in order to obtain an amount of the active ingredient 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 on various pharmacokinetic factors including the activity of the particular compositions of the present disclosure 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 combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The effective amount of the desired pharmaceutical composition can be readily determined and prescribed by a physician of ordinary skill in the art. For example, a physician may begin the dosage of the active ingredient employed in the pharmaceutical composition (e.g., the polyribonucleotides encapsulated in the lipid nanoparticle) at a level below that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

In some embodiments, the pharmaceutical compositions described herein are formulated (e.g., without limitation, for intravenous, intramuscular, or subcutaneous administration) to deliver an active dose that confers an incretin plasma concentration encoded by at least one polyribonucleotide (e.g., those described herein) that mediates pharmacological activity through its primary mode of action (agonists of GLP1 and/or GIP).

In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additives, e.g., which may enhance the stability of such compositions under certain conditions in some embodiments. Examples of additives may include, but are not limited to, salts, buffer substances, preservatives, and carriers. For example, in some embodiments, the pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and/or an aqueous buffer solution, which in some embodiments may comprise one or more salts, including, for example, alkali metal salts or alkaline earth metal salts, such as, for example, sodium, potassium, and/or calcium salts.

In some embodiments, the pharmaceutical compositions described herein may further comprise one or more active agents other than at least one polyribonucleotide encoding an incretin agent. For example, in some embodiments, such other active agents may be or comprise obesity or another known treatment of a disorder or disease associated with obesity. In some embodiments, an exemplary treatment may be a treatment included in table 1 herein.

The present disclosure provides the recognition that incretins may be combined with the polyribonucleotides and/or compositions provided herein, e.g., useful in the treatment or prevention of obesity and obesity-related diseases or disorders. Exemplary incretins that can be used with the compositions described herein include, but are not limited to, those provided in table 1, fragments thereof, or combinations thereof.

The present disclosure further provides the insight that incretins, combinations of incretins and incretin mimetics can be encoded in polyribonucleotides. Delivery of one or more incretins by delivery of a polyribonucleotide may achieve the same or substantially the same efficacy as known incretins and incretin mimics, which are peptide-based products (e.g., those in table 1), but with a lower injection volume per administration.

In some embodiments, the pharmaceutical compositions provided herein are preservative-free, sterile RNA-lipid nanoparticle dispersions in an aqueous buffer for intravenous or intramuscular administration.

Although the description of pharmaceutical compositions provided herein relates primarily to pharmaceutical compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to a variety of animals. Modifications to pharmaceutical compositions suitable for administration to humans in order to adapt the composition for administration to various animals are well known, and ordinary skilled veterinary physicists can design and/or make such modifications by ordinary experimentation alone, if any.

Patient population

The techniques provided herein may be used to treat and/or prevent obesity or diseases or disorders associated with obesity. As described herein, the technology includes polyribonucleotides encoding an incretin agent, an immunoglobulin chain thereof, or a fragment thereof. Accordingly, the present disclosure provides pharmaceutical compositions for the treatment and/or prevention of obesity and obesity-related diseases or disorders (e.g., type 2 diabetes (T2D), early T1D (e.g., within 3 months after T1D diagnosis), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease, or kidney disease). In some embodiments, the pharmaceutical composition comprises a polyribonucleotide as described herein.

In some embodiments, the individual is an individual suffering from and/or susceptible to obesity or a disease or disorder associated with obesity. In some embodiments, an individual may be defined by one or more criteria such as age group, gender, genetic background, pre-existing clinical conditions, and/or prior exposure to therapy.

In some embodiments, individuals may be determined to be classified as in need of the pharmaceutical compositions described herein based on screening tools for obesity and obesity-related diseases and disorders. For example, in some embodiments, the classification of an individual as in need of a pharmaceutical composition described herein may be determined based on results obtained in an Enzyme Immunoassay (EIA), a western blot and/or PCR test, and/or body weight, and/or waist circumference, and/or body mass index.

In some embodiments, the individual is a model organism. In a preferred embodiment, the individual is a human. In some embodiments, the individual is between 18-65 years old. In some embodiments, the age of the individual is in the range of about 0 month to about 6 months, about 6 to about 12 months, about 6 to about 18 months, about 18 to about 36 months, about 1 to about 5 years, about 5 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, about 20 to about 25 years, about 25 to about 30 years, about 30 to about 35 years, about 35 to about 40 years, about 40 to about 45 years, about 45 to about 50 years, about 50 to about 55 years, about 55 to about 60 years, about 60 to about 65 years, about 65 to about 70 years, about 70 to about 75 years, about 75 to about 80 years, about 80 to about 85 years, about 85 to about 90 years, about 90 to about 95 years, or about 95 to about 100 years.

In some embodiments, the individual is a human infant. In some embodiments, the subject is a human infant. In some embodiments, the individual is a human child. In some embodiments, the individual is a human adult. In some embodiments, the individual is an elderly human.

In some embodiments, the individual is not currently considered obese. In some embodiments, the individual is at risk of developing obesity.

In some embodiments, the individual suffers from and/or is susceptible to obesity, pre-diabetes, type 2 diabetes (T2D, and complications thereof), early T1D, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease (e.g., characterized by, for example, major cardiovascular events (MACEs), including CV death, non-fatal myocardial infarction, non-fatal stroke, heart failure with preserved ejection fraction (HFpEF)), kidney disease, or an elevated risk of premature death.

In some embodiments, the individual has and/or is susceptible to additional co-morbidities associated or not associated with obesity, including any of prediabetes, T2D, early T1D, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease (e.g., characterized by major cardiovascular events (MACE), including CV death, non-fatal myocardial infarction, non-fatal stroke, heart failure with preserved ejection fraction (HFpEF)), kidney disease, and an elevated risk of premature death.

In some embodiments, the individual has not previously been treated for obesity or a disease associated with obesity.

In some embodiments, an individual suffering from and/or susceptible to obesity or a disease associated with obesity may have received or is currently receiving other therapies for obesity. In some embodiments, the individual is currently receiving or has received one or more of the treatments listed in table 1. In some embodiments, an individual suffering from and/or susceptible to obesity or an obesity-related disorder may have received or is currently receiving lifestyle intervention, such as reduced calorie intake and/or increased physical activity.

In some embodiments, the individual has received one or more of the treatments listed in table 1 for greater than 1 week, greater than 2 weeks, greater than 3 weeks, greater than 4 weeks, greater than 5 weeks, greater than 6 weeks, greater than 7 weeks, greater than 8 weeks, greater than 12 weeks, greater than 4 months, greater than 5 months, greater than 6 months, greater than 7 months, greater than 8 months, greater than 9 months, greater than 10 months, or greater than 1 year. In some embodiments, the subject is responsive to another treatment upon administration of a polyribonucleotide, composition, or pharmaceutical composition described herein. In some embodiments, the subject is non-responsive to another treatment upon administration of a polyribonucleotide, composition, or pharmaceutical composition described herein.

In some embodiments, the individual has previously received one or more treatments for treating obesity or a disease associated with obesity (e.g., one or more treatments listed in table 1). In some embodiments, the individual receives a prior treatment for treating obesity for greater than 1 week, greater than 2 weeks, greater than 3 weeks, greater than 4 weeks, greater than 5 weeks, greater than 6 weeks, greater than 7 weeks, greater than 8 weeks, greater than 12 weeks, greater than 4 months, greater than 5 months, greater than 6 months, greater than 7 months, greater than 8 months, greater than 9 months, greater than 10 months, or greater than 1 year. In some embodiments, the individual is responsive to prior treatment. In some embodiments, the individual is not responsive to prior treatment.

In some embodiments, the individual is characterized by any of a BMI of 30 or more, a BMI of 40 or more, a waist circumference of greater than 35 inches (89 cm) (in females) or greater than 40 inches (102 cm) (in males), high blood pressure, high glucose levels and/or high cholesterol, high HbA1c levels, hypothyroidism, liver problems, and/or diabetes in the blood sample.

In some embodiments, the individual has not received additional treatment for treating obesity or obesity-related diseases for the last month. In some embodiments, the individual has not received additional treatment for treating obesity or obesity-related diseases for the past year. In some embodiments, the individual has not received additional treatment for treating obesity or obesity-related diseases for the past 2 years.

Therapeutic method

In some embodiments, the pharmaceutical compositions described herein are ingestible by cells for producing encoded incretins at therapeutically relevant serum concentrations. Accordingly, the present disclosure provides methods of using the pharmaceutical compositions described herein. For example, in some embodiments, the methods provided herein comprise administering to an individual a pharmaceutical composition described herein.

As used herein, the term "administration" or "administration" generally refers to the administration of a composition to an individual to deliver an agent (e.g., at least one polyribonucleotide encoding an incretin agent described herein) that is or is included in the composition to a target site or site to be treated. One of ordinary skill in the art will recognize that various routes of administration may be utilized to an individual (e.g., human) under appropriate circumstances. Administration may be by, for example, bronchial (e.g., by bronchial instillation), buccal, transdermal (which may be or include, for example, one or more of intradermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In preferred embodiments, administration may be intramuscular, intraperitoneal, intravenous or subcutaneous.

In some embodiments, administration of the pharmaceutical composition results in delivery of one or more polyribonucleotides (e.g., encoding an incretin agent) as described herein to the subject. In some embodiments, administering the pharmaceutical composition to the subject results in expression of an incretin agent encoded by the administered polyribonucleotide in the subject. In some embodiments, administering the pharmaceutical composition to the subject results in expression of an incretin agent encoded by the administered polyribonucleotide in the subject.

In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve administering a fixed number of doses.

In some embodiments, administration may involve administration of intermittent (e.g., multiple doses separated in time) and/or periodic (e.g., individual doses separated by a common time period) dosing. In some embodiments, administration may involve continuous administration (e.g., infusion) for at least a selected period of time.

In some embodiments, the dosing regimen comprises a plurality of doses, each of the plurality of doses being separated in time from the other doses. In some embodiments, the individual doses are separated from each other by a period of the same length, and in some embodiments, the dosing regimen comprises a plurality of doses and at least two different periods of time separating the individual doses. In some embodiments, all doses within a dosing regimen have the same unit dose amount. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, the dosing regimen includes a first dose of a first dose amount followed by one or more additional doses of a second dose amount different from the first dose amount. In some embodiments, the dosing regimen includes a first dose of a first dose amount followed by one or more additional doses of a second dose amount that is the same as the first dose amount. In some embodiments, the dosing regimen is associated with a desired or beneficial outcome (i.e., is a therapeutic dosing regimen) when administered in the relevant population.

Those skilled in the art will appreciate that the therapy may be administered in a dosing cycle. In some embodiments, the pharmaceutical compositions described herein are administered during one or more dosing cycles.

In some embodiments, one dosing cycle is at least 3 days or longer (including, for example, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, or at least 30 days). In some embodiments, one dosing cycle is at least 21 days.

In some embodiments, one dosing cycle may involve multiple doses, e.g., according to a pattern, such as, e.g., a dose may be administered daily within the dosing cycle, or a dose may be administered every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 2 weeks, every month, every 2 months within one cycle.

In some embodiments, multiple dosing cycles may be administered. For example, in some embodiments, at least 2 dosing cycles (including, for example, at least 3 dosing cycles, at least 4 dosing cycles, at least 5 dosing cycles, at least 6 dosing cycles, at least 7 dosing cycles, at least 8 dosing cycles, at least 9 dosing cycles, at least 10 dosing cycles, or more) may be administered. In some embodiments, the number of dosing cycles to be administered may vary with the type of treatment (e.g., monotherapy versus combination therapy). In some embodiments, at least 3-8 dosing cycles may be administered.

In some embodiments, there may be a "rest period" between dosing cycles, and in some embodiments, there may be no rest period between dosing cycles. In some embodiments, there may be periods of rest and sometimes no periods of rest between dosing cycles.

In some embodiments, the rest period may have a length in the range of days to months. For example, in some embodiments, the rest period may have a length of at least 3 days or more, including, for example, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, or more. In some embodiments, the rest period may have a length of at least 1 week or more, including, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, or more.

The dosage of the pharmaceutical compositions described herein may vary depending on a variety of factors including, for example, but not limited to, the weight of the individual to be treated, the type of disease and/or the stage of the disease and/or monotherapy or combination therapy. In some embodiments, the dosing cycle involves administering a set number and/or pattern of doses. For example, in some embodiments, at least one dose of the pharmaceutical compositions described herein is administered per administration cycle, including, for example, at least two doses per administration cycle, at least three doses per administration cycle, at least four doses per administration cycle, or more.

In some embodiments, the dosing cycle involves setting a cumulative dose, e.g., over a particular period of time and optionally by multiple dose administrations, which may be administered, e.g., at set intervals and/or according to a set pattern. In some embodiments, the set cumulative dose may be administered at set intervals through multiple doses such that there is at least some temporal overlap of biological and/or pharmacokinetic effects resulting from such multiple doses on the target cells or on the treated individual. In some embodiments, the set cumulative dose may be administered at set intervals through multiple doses, such that biological and/or pharmacokinetic effects resulting from such multiple doses on the target cells or on the treated individual may be additive. For example only, in some embodiments, a set cumulative dose of X mg may be administered by two doses, each of X/2 mg, wherein the two doses are administered close enough in time that the biological and/or pharmacokinetic effects produced by each of the X/2 mg doses on the target cells or on the individual being treated may be additive.

In some embodiments, dosing may be adjusted based on the response of the individual receiving the therapy. For example, in some embodiments, administration may involve administration of a higher dose followed by administration of a lower dose if one or more parameters for safety pharmacology assessment indicate that the previous dose may not meet medical safety requirements according to a physician. In some embodiments, dose escalation may be performed at one or more levels. Without wishing to be bound by any particular theory, the present disclosure provides, inter alia, insight that a pharmaceutically directed dose escalation (PGDE) method can be applied to determine the appropriate dose of the pharmaceutical composition described herein.

In some embodiments, the pharmaceutical compositions described herein can be administered to an individual as monotherapy.

In some embodiments, the pharmaceutical compositions provided herein may be administered as part of a combination therapy. In some embodiments, the pharmaceutical compositions provided herein may be administered as part of a combination therapy comprising the pharmaceutical composition and one or more incretins. In some embodiments, the one or more incretins may comprise any one of the incretins peptides as shown in tables 2-5, 8-9, and 11, or a combination thereof.

In some embodiments, the pharmaceutical compositions provided herein may be administered as part of a combination therapy comprising the pharmaceutical composition and another therapy, such as those described in tables 2-5, 8-9, and 11.

In some embodiments, the combination therapy may comprise administering a pharmaceutical composition comprising at least one polyribonucleotide encoding an incretin agent and administering a pharmaceutical composition comprising a dipeptidyl peptidase-4 (DPP-4) inhibitor. Without wishing to be bound by any theory, administration of a pharmaceutical composition comprising at least one polyribonucleotide encoding an incretin agent in combination with a pharmaceutical composition DPP-4 inhibitor may increase the efficacy of the pharmaceutical composition by prolonging the activity of the incretin agent. In some embodiments, a pharmaceutical composition comprising at least one polyribonucleotide encoding an incretin agent is administered with another pharmaceutical composition comprising one or more DPP-4 inhibitors.

Administration of the DPP-4 inhibitor or the pharmaceutical composition comprising the DPP-4 inhibitor may for example be by oral administration. In some embodiments, the DPP-4 inhibitor comprises sitagliptin, vildagliptin, saxagliptin, linagliptin, gemagliptin, alagliptin, tigliptin, alogliptin, trelagliptin, aogliptin, vildagliptin, dulgliptin, neogliptin, ragliptin, dulgliptin, colgliptin, fogliptin, pra Lu Gelie, small bane, or any combination thereof.

In some embodiments, a composition (e.g., a pharmaceutical composition) comprising at least one polyribonucleotide encoding an incretin agent is administered, e.g., by intramuscular administration, intraperitoneal administration, intravenous administration or subcutaneous administration, enteral administration, intraarterial administration, intradermal administration, intragastric administration, intramedullary administration, intranasal administration, intrathecal administration, intraventricular administration, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), and the like. In some embodiments, administration may be intramuscular, intraperitoneal, intravenous, or subcutaneous. In some embodiments, the pharmaceutical composition comprising at least one polyribonucleotide encoding an incretin agent is administered subcutaneously. In some embodiments, a pharmaceutical composition comprising one or more DPP-4 inhibitors and a pharmaceutical composition comprising at least one polyribonucleotide encoding an incretin agent are administered simultaneously. In some embodiments, a pharmaceutical composition comprising one or more DPP-4 inhibitors and a pharmaceutical composition comprising at least one polyribonucleotide encoding an incretin agent may be administered daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 2 weeks, monthly or every 2 months.

In some embodiments, a pharmaceutical composition comprising one or more DPP-4 inhibitors and a pharmaceutical composition comprising at least one polyribonucleotide encoding incretin are administered sequentially. In some embodiments, a pharmaceutical composition comprising one or more DPP-4 inhibitors is administered prior to a pharmaceutical composition comprising at least one polyribonucleotide encoding incretin. In some embodiments, the pharmaceutical composition comprising one or more DPP-4 inhibitors is administered after the pharmaceutical composition comprising at least one polyribonucleotide encoding incretin. In some embodiments, a pharmaceutical composition comprising one or more DPP-4 inhibitors and a pharmaceutical composition comprising at least one polyribonucleotide encoding incretin are administered on the same day. In some embodiments, a pharmaceutical composition comprising one or more DPP-4 inhibitors and a pharmaceutical composition comprising at least one polyribonucleotide encoding incretins are administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or 2 months apart. In some embodiments, a pharmaceutical composition comprising one or more DPP-4 inhibitors and a pharmaceutical composition comprising at least one polyribonucleotide encoding incretins are administered at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or at least 2 months before the pharmaceutical composition comprising at least one polyribonucleotide encoding incretins. In some embodiments, a pharmaceutical composition comprising at least one polyribonucleotide encoding incretins is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 1 month, or at least 2 months prior to a pharmaceutical composition comprising one or more DPP-4 inhibitors.

In some embodiments, one dosing cycle may involve, for example, simultaneous or sequential administration of a pharmaceutical composition comprising one or more DPP-4 inhibitors and a pharmaceutical composition comprising at least one polyribonucleotide encoding incretins. In some embodiments, multiple dosing cycles may be administered. For example, in some embodiments, at least 2 dosing cycles (including, for example, at least 3 dosing cycles, at least 4 dosing cycles, at least 5 dosing cycles, at least 6 dosing cycles, at least 7 dosing cycles, at least 8 dosing cycles, at least 9 dosing cycles, at least 10 dosing cycles, or more) may be administered. In some embodiments, at least 3-8 dosing cycles may be administered.

The pharmaceutical compositions comprising one or more DPP-4 inhibitors and the dosage of the pharmaceutical compositions described herein may vary depending on a variety of factors including, for example, but not limited to, the weight, age, weight or disease stage of the individual to be treated. In some embodiments, the dosing cycle involves administering a set number and/or pattern of doses. For example, in some embodiments, at least one dose of the pharmaceutical compositions described herein is administered per administration cycle, including, for example, at least two doses per administration cycle, at least three doses per administration cycle, at least four doses per administration cycle, or more.

In some embodiments, individuals receiving the compositions (e.g., pharmaceutical compositions) provided herein can be monitored periodically in a dosing regimen to assess the efficacy of the administered treatment. For example, in some embodiments, the efficacy of the administered treatment may be assessed periodically, e.g., weekly, biweekly, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer.

Preparation method

Individual polyribonucleotides can be produced by methods known in the art. For example, in some embodiments, the polyribonucleotides can be produced by in vitro transcription, e.g., using a DNA template. Plasmid DNA that is used as an in vitro transcription template to produce the polyribonucleotides described herein is also within the scope of the present disclosure.

In the presence of an appropriate RNA polymerase (e.g., recombinant RNA polymerase such as T7 RNA polymerase) having ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP), the DNA template can be used for in vitro RNA synthesis. In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide described herein) can be synthesized in the presence of a modified ribonucleotide triphosphate. For example only, in some embodiments, pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), or 5-methyl-uridine (m 5U) may be used in place of Uridine Triphosphate (UTP). In some embodiments, pseudouridine (ψ) can be used in place of Uridine Triphosphate (UTP). In some embodiments, N1-methyl-pseudouridine (m1ψ) may be used in place of Uridine Triphosphate (UTP). In some embodiments, 5-methyl-uridine (m 5U) can be used in place of Uridine Triphosphate (UTP).

It will be apparent to those of skill in the art that during in vitro transcription, RNA polymerase (e.g., as described and/or utilized herein) typically passes through at least a portion of a single stranded DNA template in a 3'→5' direction to produce single stranded complementary RNA in the 5'→3' direction.

In some embodiments where the polyribonucleotide comprises a polyA tail, those skilled in the art will appreciate that such polyA tail may be encoded in the DNA template, for example by using a suitable tailed PCR primer, or the polyA tail may be post-transcriptionally added to the polyribonucleotide, for example by enzymatic treatment (for example using a poly (A) polymerase, such as E.coli poly (A) polymerase). Suitable poly (A) tails are described above. In some embodiments, the poly (a) tail comprises a plurality of a residues interrupted by a linker peptide. In some embodiments, the connecting peptide comprises nucleotide sequence GCATATGAC (SEQ ID NO: 40).

In some embodiments, one of skill in the art will appreciate that adding a 5' cap to RNA (e.g., mRNA) can facilitate RNA recognition and ligation to ribosomes to initiate translation and enhance translation efficiency. Those skilled in the art will also appreciate that the 5 'cap may also protect the RNA product from 5' exonuclease mediated degradation and thus increase half-life. Methods for capping are known in the art, and one of ordinary skill in the art will appreciate that in some embodiments, capping can be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system, such as a capping enzyme of a vaccinia virus). In some embodiments, the cap and the plurality of ribonucleotides triphosphate can be introduced during in vitro transcription such that the cap is incorporated into the polyribonucleotides during transcription (also referred to as co-transcription capping). In some embodiments, the RNA can be capped effectively by adding a GTP fed-batch procedure multiple times during the reaction to maintain a low concentration of GTP. Suitable 5' caps are described above. For example, in some embodiments, the 5' cap comprises m7 (3 ' ome g) (5 ') ppp (5 ') (2 ' ome a) pG.

After transcription of the RNA, the DNA template is broken down. In some embodiments, the decomposition may be achieved using dnase I under appropriate conditions.

In some embodiments, the in vitro transcribed polyribonucleotides may be generated in a buffer solution, e.g., in a buffer such as HEPES, phosphate buffer, citrate buffer, acetate buffer, in some embodiments, such a solution may be buffered to a pH in the range of, e.g., about 6.5 to about 7.5, in some embodiments, about 7.0. In some embodiments, the production of polyribonucleotides may further include one or more of purification, mixing, filtration, and/or packing.

In some embodiments, the polyribonucleotides (e.g., in some embodiments after an in vitro transcription reaction) can be purified, e.g., to remove components utilized or formed during production, such as proteins, DNA fragments, and/or nucleotides. As described in the present disclosure, various nucleic acid purification methods known in the art may be used. Some purification steps may be or include, for example, one or more of precipitation, column chromatography (including, for example, but not limited to, anion, cation, hydrophobic Interaction Chromatography (HIC)), solid substrate-based purification (e.g., magnetic bead-based purification). In some embodiments, the polynucleic acids may be purified using magnetic bead-based purification, which in some embodiments may be or comprise magnetic bead-based chromatography. In some embodiments, the polyribonucleotides may be purified using Hydrophobic Interaction Chromatography (HIC) and/or diafiltration. In some embodiments, the polyribonucleotides may be purified using diafiltration after HIC.

In some embodiments, the dsRNA may be obtained as a byproduct during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination. For example, in some embodiments, cellulosic material (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, e.g., in some embodiments in chromatographic form. In some embodiments, the cellulosic material (e.g., microcrystalline cellulose) may be pretreated to inactivate potential rnase contamination, for example by autoclaving in some embodiments, followed by incubation with an aqueous alkaline solution (e.g., naOH). In some embodiments, the cellulosic material may be used to purify polyribonucleotides according to the method described in WO2017/182524, the entire contents of which are incorporated herein by reference.

In some embodiments, a batch of polynucleic acids may be further processed by one or more filtration and/or concentration steps. For example, in some embodiments, the polyribonucleotides may be further diafiltered (e.g., by tangential flow filtration in some embodiments), e.g., after removal of dsRNA contamination, e.g., to adjust the concentration of polyribonucleotides to a desired RNA concentration and/or to exchange buffer for drug substance buffer.

In some embodiments, the polyribonucleotides can be processed by 0.2 μm filtration before filling them into an appropriate container.

In some embodiments, the polyribonucleotides and compositions thereof can be manufactured according to processes as described herein or as otherwise known in the art.

In some embodiments, the polyribonucleotides and compositions thereof can be manufactured in large scale. For example, in some embodiments, a supply of polyribonucleotides can be manufactured on a scale of greater than 1 g, greater than 2 g, greater than 3g, greater than 4 g, greater than 5 g, greater than 6 g, greater than 7 g, greater than 8 g, greater than 9 g, greater than 10 g, greater than 15 g, greater than 20 g, or greater.

In some embodiments, RNA quality control can be performed and/or monitored at any time during the production process of the polyribonucleotides and/or compositions comprising the same. For example, in some embodiments, RNA quality control parameters, including one or more of RNA identity (e.g., sequence, length, and/or RNA properties), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, can be assessed and/or monitored after each or some steps of the polyribonucleotide manufacturing process (e.g., after in vitro transcription) and/or after each purification step.

In some embodiments, the stability of a polyribonucleotide (e.g., produced by in vitro transcription) and/or a composition comprising one or more RNAs can be assessed over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or more) under various test storage conditions (e.g., at room temperature versus refrigerator or subzero temperature). In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide described herein) and/or a composition thereof can be stably stored at refrigerator temperatures (e.g., about 4 ℃ to about 10 ℃) for at least 1 month or more, including at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or more. In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide described herein) and/or a composition thereof can be stably stored at subzero temperatures (e.g., -20 ℃ or less) for at least 1 month or more, including at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or more. In some embodiments, a polyribonucleotide (e.g., a polyribonucleotide described herein) and/or a composition thereof can be stable for storage at room temperature (e.g., at about 25 ℃) for at least 1 month or more.

In some embodiments, one or more evaluations (e.g., as a release test) may be utilized during preparation, or other preparation or use of the polyribonucleotide.

In some embodiments, one or more quality control parameters may be evaluated to determine whether the polynucleic nucleotides described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for partitioning). In some embodiments, such quality control parameters may include, but are not limited to, RNA integrity, RNA concentration, residual DNA template, and/or residual dsRNA. Certain methods for assessing RNA quality are known in the art, for example, one skilled in the art will recognize that in some embodiments, one or more analytical tests may be used for RNA quality assessment. Examples of such certain analytical tests may include, but are not limited to, gel electrophoresis, UV absorption, and/or PCR assays.

In some embodiments, a batch of polynucleic acids may be evaluated for one or more features as described herein to determine a next action step. For example, if the RNA quality assessment indicates that a batch of polynucleotides meets or exceeds relevant acceptance criteria, the batch of polynucleotides may be designated for one or more further steps of manufacture and/or formulation and/or distribution. Otherwise, if a lot of polyribonucleotides does not meet or exceed the acceptance criteria, then an alternative action may be taken (e.g., discard the lot).

In some embodiments, a batch of polyribonucleotides that meet the evaluation result can be used for one or more further steps of manufacturing and/or formulation and/or distribution.

DNA constructs

The present disclosure provides, inter alia, DNA constructs, e.g., which may encode one or more incretins or components thereof as described herein. In some embodiments, DNA constructs provided by and/or as used by the present disclosure are contained in a vector.

Non-limiting examples of vectors include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors (e.g., retrovirus, adenovirus, or baculovirus vectors), or artificial chromosome vectors (e.g., bacterial Artificial Chromosome (BAC), yeast Artificial Chromosome (YAC), or P1 Artificial Chromosome (PAC)). In some embodiments, the vector is an expression vector. In some embodiments, the vector is a cloning vector. Generally, a vector is a nucleic acid construct (e.g., a construct that is or encodes a load, or imparts a particular functionality, etc.) that can receive or otherwise be linked to a nucleic acid component of interest.

The expression vector may be a plasmid or virus or other vector, which typically includes a target expressible sequence (e.g., coding sequence) functionally linked to one or more control components (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control components are selected for expression in the target system. In some embodiments, the system is ex vivo (e.g., an in vitro transcription system), in some embodiments, the system is in vivo (e.g., bacteria, yeast, plants, insects, fish, vertebrates, mammalian cells or tissues, etc.).

Cloning vectors are generally used for modification, engineering and/or replication (e.g., by replication in vivo, e.g., in a simple system such as bacteria or yeast, or in vitro, e.g., by amplification such as polymerase chain reaction or other amplification process). In some embodiments, the cloning vector may lack an expression signal.

In many embodiments, the vector may include replication components, such as a primer binding site and/or an origin of replication. In many embodiments, the vector may include insertion or modification sites, such as restriction endonuclease recognition sites and/or guide RNA binding sites, and the like.

In some embodiments, the vector is a viral vector (e.g., an AAV vector). In some embodiments, the vector is a non-viral vector. In some embodiments, the vector is a plasmid.

Those of skill in the art recognize that a variety of techniques may be used to produce a recombinant polynucleotide (e.g., DNA or RNA) as described herein. Such as, for example, restriction decomposition, reverse transcription, amplification (e.g., via polymerase chain reaction), gibson assembly, etc. Alternatively or additionally, certain nucleic acids may be prepared or assembled by chemical and/or enzymatic synthesis. In some embodiments, the recombinant polynucleotide is prepared using a combination of known methods.

In some embodiments, polynucleotides of the present disclosure are included in DNA constructs (e.g., vectors) suitable for transcription and/or translation.

In some embodiments, the expression vector comprises a polynucleotide encoding a protein and/or polypeptide of the disclosure operably linked to one or more sequences that control expression (e.g., a promoter, initiation signal, termination signal, polyadenylation signal, activating agent, repressor, etc.). In some embodiments, one or more sequences that control expression are selected to achieve a desired expression level. In some embodiments, more than one sequence (e.g., a promoter) is utilized that controls expression. In some embodiments, more than one sequence (e.g., a promoter) that controls expression is utilized to achieve a desired level of expression of a plurality of polynucleotides encoding a plurality of proteins and/or polypeptides. In some embodiments, multiple recombinant proteins and/or polypeptides are expressed from the same vector (e.g., a bicistronic vector, a tricistronic vector, a polycistronic vector). In some embodiments, multiple polypeptides are expressed, each of which is expressed from a separate vector.

In some embodiments, expression vectors comprising polynucleotides of the present disclosure are used to produce RNA and/or proteins and/or polypeptides in host cells. In some embodiments, the host cell may be in vitro (e.g., a cell line), such as a cell or cell line (e.g., a human embryonic kidney (HEK cell), chinese hamster ovary cell, etc.) suitable for producing the polynucleotides of the present disclosure and the proteins and/or polypeptides encoded by the polynucleotides.

In some embodiments, the expression vector is an RNA expression vector. In some embodiments, the RNA expression vector comprises a polynucleotide template for producing RNA in a cell-free enzyme mixture. In some embodiments, the RNA expression vector comprising the polynucleotide template is enzymatically linearized prior to in vitro transcription. In some embodiments, the polynucleotide template is generated as a linearized polynucleotide template by PCR. In some embodiments, the linearized polynucleotide is mixed with enzymes suitable for RNA synthesis, RNA capping, and/or purification. In some embodiments, the resulting RNA is suitable for producing a protein encoded by the RNA.

Various methods of introducing expression vectors into host cells are known in the art. In some embodiments, the vector may be introduced into the host cell using transfection. In some embodiments, transfection is accomplished, for example, using calcium phosphate transfection, lipofection, or polyethyleneimine mediated transfection. In some embodiments, the vector may be introduced into the host cell using transduction.

In some embodiments, after introducing the vector into the host cell, the transformed host cell is cultured to allow expression of the recombinant polynucleotide. In some embodiments, the transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, or more. The transformed host cells are cultured in growth conditions (e.g., temperature, carbon dioxide level, growth medium) according to the requirements of the host cell selected. Those skilled in the art will recognize that the culture conditions of the selected host cells are well known in the art.

Examples

Example 1: generation of encoded exemplary intestinal promotions polyribonucleotides of insulin agents

This example describes a method for producing a polyribonucleotide sequence encoding an incretin agent. This example further describes the design of polyribonucleotides that can achieve transient in vivo incretin agent production after i.p./i.v./i.m./s.c. delivery.

The method of the embodiment comprises the following steps:

(1) DNA fragments encoding an incretin agent (e.g., GLP1, GIP, or mutants thereof) are cloned into DNA plasmids suitable for RNA expression. Suitable DNA plasmids may encode RNA features including, for example, a 5 'untranslated region (5' UTR), a Kozak sequence, a 3 'untranslated region (3' UTR), and/or a polyA tail sequence. DNA plasmids also typically include restriction sites that enable cloning of DNA fragments encoding incretins downstream of the 5 'UTR and Kozak sequence coding regions and upstream of the 3' UTR and polyA tail sequence coding regions. Examples of suitable DNA plasmids are also found in WO2021/214204, which is hereby incorporated by reference in its entirety.

(2) Selected clones were verified by restriction analysis and optionally sequencing.

(3) Linearization of DNA plasmids encoding incretins.

(4) Encoding incretins synthesis of polyribonucleotides.

(5) Biochemical characterization of polyribonucleotides encoding incretins.

(6) The polyribonucleotides encoding the incretins are transfected into HEK cells and the incretins levels are quantified.

Codon optimization

For optimal expression of exemplary incretins, DNA sequences were generated based on the amino acid sequences of GLP1 (7-37), GIP (1-42) and truncated or mutant mutants thereof fused to exemplary Signal Peptides (SPs) including the sequences (SP 1-2, MRVLVLLACLAAASNA) and SPs shown in SEQ ID NOS: 65 and 66, as shown in Table 11, above.

The amino acid sequence is translated into a DNA nucleotide sequence. If either Eam1104I (GAAGAG)、BamHI (GGATCC)、PstI (CTGCAG)、SbfI (CCTGCAGG)、XhoI (CTCGAG)、SpeI (ACTAGT)、BspEI (TCCGGA)、SacI (GAGCTC)、Ear1 (CTCTTCN^NNN) and NheI (GCTAGC) are used for linearization or cloning of the plasmid, the restriction sites for these enzymes can be selectively removed after the optimization process. The sequence was also examined to see if there was a region of high homology to the T7 RNA polymerase termination signal sequence "ATCTGTT" followed by multiple "T" residues.

The optimization was performed using the GeneOptimezer software supplied by Life Technologies GmbH GeneArt cube. This software adjusts codon usage by using the most frequent codons and adjusts the GC content of the upload sequence for the chosen expression system (homo sapiens in this case). At the same time, the GeneOptimaizer removes sequence repeats, introns, cryptic splice sites, internal ribosome entry sites and RNA destabilizing sequence components (e.g., upA-dinucleotides), adds RNA stabilizing sequence components (e.g., cpG-dinucleotides) and avoids stabilizing RNA secondary structures as well as unwanted sequences such as restriction sites. The output sequence is then used to order the DNA fragment strings. Those skilled in the art will appreciate that alternative methods for codon optimization are available. Furthermore, additional information about codon optimization methods is provided herein.

Cloning

Each incretin sequence was cloned into a DNA plasmid (e.g., pST 5). This may be accomplished, for example, by in vivo assembly, volume .Garcia-Nafria, "IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly," Scientific Reports , article number 27459 (2016), which is incorporated herein by reference in its entirety.

Plasmid DNA preparation

Plasmid DNA was prepared by selecting clones for inoculation into the medium. The selected clones are optionally verified by restriction analysis and optionally sequencing. Cultures were grown and after cell harvest, purified according to manufacturer's instructions, for example using QIAGEN PLASMID Plus Maxi kit. DNA concentration can be determined by UV spectroscopy. DNA was stored in certified rnase-free and dnase-free reaction tubes.

Linearization and DNA purification

Linearization of plasmid DNA was performed using appropriate restriction enzymes, followed by purification of the linearized DNA template using magnetic beads (e.g., dynabeadsTM MyOneTM Carboxylic Acid) according to the manufacturer's protocol. DNA concentration was measured by UV spectroscopy, restriction analysis and optionally sequencing.

In vitro transcription

RNA, optionally capped RNA, is then produced, for example, following the procedures disclosed in Kreiter et al, cancer immunol. Immunother. 2007, 56, 1577-87 and WO2021/214204, each of which is incorporated herein by reference in its entirety. Methyl pseudouridine can be used in vitro transcription reactions and incorporated into the produced RNA. The resulting RNA was subjected to cellulose purification to isolate single-stranded RNA, followed by concentration measurement by UV spectrometry. RNA integrity was determined by microfluidic-based electrophoresis. The resulting RNA is optionally further biochemically characterized.

Transfection and expression

RNA encoding the incretins is transfected into HEK cells, e.g., by electroporation, and the resulting incretins levels are quantified. HEK cells, such as HEK293T cells, were washed with cooled medium. Electroporation was performed in pre-cooled cuvettes. The cells and RNA in each sample are at typical concentrations for RNA electroporation. Following electroporation, the cells were incubated on ice.

Cells are then transferred to expression medium (e.g., expi293 medium) and counted. Cells are seeded at typical concentrations for expression and incubated at 37 ℃ for example 48 hours. The supernatant was then harvested by centrifugation, followed by careful aspiration so as not to interfere with cell sedimentation, and then stored at 4 ℃.

Expression of the incretins is quantified, for example, by ELISA or Wester blot analysis of cell culture supernatants.

EXAMPLE 2 Generation of reporter cell lines to monitor incretin Activity

This example describes a method for generating a reporter cell line to monitor incretin activity.

The method of the embodiment comprises the following steps:

(1) The DNA fragment encoding the incretin receptor (e.g., GLP1R and/or GIPR) is cloned into a DNA plasmid (e.g., pT 2).

(2) HEK293 cells were stably transfected with DNA plasmids encoding the incretin receptor and mRNA transposase.

(3) Cells are sorted by FACS for high, medium and low intestinal insulinotropic receptor expression (e.g., GLP1R and/or GIPR expression), optionally with bulk sorting followed by single cell sorting.

(4) Stable expression of incretin receptors (e.g., GLP1R and/or GIPR) was confirmed.

(5) A master cell bank is generated.

Cloning

Exemplary DNA sequences encoding incretin receptor sequences are shown in table 15. Such DNA sequences are cloned into DNA plasmids (e.g., pT 2). The mutants of GLP1R_ mutR and GLP1R_mutL encoding GLP1R are slightly different (GLP1R_ mutR encodes GLP1R having an L260F mutation relative to the GLP1R encoded by GLP1R_mutL). This may be accomplished, for example, by in vivo assembly, volume .Garcia-Nafria, "IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly," Scientific Reports , article number 27459 (2016), which is incorporated herein by reference in its entirety.

TABLE 17 sequences encoding exemplary incretin receptors

Plasmid DNA preparation

Transfection

Plasmid DNA encoding the incretin receptor and RNA encoding a transposase (e.g., SB100X transposase) are transfected into HEK cells, e.g., by electroporation. HEK cells, such as HEK293 cells, are washed with cooled medium. Electroporation was performed in pre-cooled cuvettes. The cells, plasmid DNA and RNA in each sample were at typical concentrations for electroporation. Following electroporation, the cells were incubated on ice.

Cell sorting

Cells are then sorted by FACS (e.g., using BD FACSMelodyTM cell sorter) for high, medium and low intestinal insulinotropic receptor expression (e.g., GLP1R and/or GIPR expression), optionally with extensive sorting followed by single cell sorting. Stable expression of the selected cloned incretin receptor was confirmed and a master cell pool was generated. The resulting cell lines may include one or more of HEK293_GLP1R high, HEK293_GLP1R medium, HEK293_GLP1R low, HEK293_GIPR high, HEK293_GIPR medium, HEK293_GIPR low, HEK293_GLP1R high/GIPR high, HEK293_GLP1R medium/GIPR medium, and HEK293_GLP1R low/GIPR low.

Example 3: assessing the functionality of polyribonucleotide encoded incretins

This example describes a method for assessing the functionality of polyribonucleotide encoded incretins.

The method of the embodiment comprises the following steps:

(1) HEK 293-GLP 1R/GIPR reporter cells of example 2 were transfected with GLP1 or GIP RNA of example 1. The reporter cells may be incubated, for example, for 24 hours after transfection, prior to collection of the supernatant.

(2) Downstream signal induction is quantified by measuring, for example, cAMP release in the supernatant, for example, using cAMP-GloTM detection or ELISA.

The method of the embodiment also includes:

(1) Wild-type HEK293 cells were transfected with GLP1 or GIP RNA from example 1.

(2) The supernatant containing GLP1 and GIP was collected.

(3) HEK 293-GLP 1R/GIPR reporter cells of example 2 were incubated with the supernatant. The reporter cells may be incubated, for example, for 24 hours, prior to collection of the supernatant.

(4) Downstream signal induction is quantified by measuring, for example, cAMP release in the supernatant, for example, using cAMP-GloTM detection or ELISA.

Example 4 in vitro functionality of polyribonucleotides encoding incretins

This example demonstrates that polyribonucleotides encoding an incretin agent as described herein can induce the production of an incretin agent.

Method in this example, 6X 10 ⁴ HEK293t17 cells per well were seeded in three different 48-well plates and grown overnight in a 37℃5% CO ₂ incubator. Cells were transfected with 0.6 μg of the polyribonucleotide candidate (polyribonucleotides encoding GLP1 (7-37), GLP1 (7-37) - (K34R), GIP (1-30) and GIP (1-42) using Lipofectamine Messenger MAX kit (ThermoFisher Scientific, catalog No. LMRNA 003). The cells were further incubated. After 3 hours, 6 hours, 24 hours, 48 hours and 72 hours following transfection. For time points of 3 hours and 6 hours post-transfection, supernatants were collected and frozen at-80 ℃. At the 24 hour time point after transfection, supernatants were collected and fresh medium in the well plates was replaced. The well plate was further incubated until 48 hours and 72 hours post-transfection. Supernatants collected at 24, 48 and 72 hour time points post-transfection were stored at-80 ℃ until further analysis.

The concentrations of GIP and GLP1 in the supernatants were then quantified using ELISA. (human GIP (total) ELISA kit and GLP1 (7-36) active ELISA kit, merck Millipore). Statistical analysis was performed by single factor ANOVA followed by a post hoc Tukey test.

Results from ELISA showed that the polyribonucleotides encoding the incretins GLP1 (7-37), GLP1 (7-37) - (K34R) and GIP (1-42) were translated into proteins to reach maximum concentrations after 24 hours (see FIGS. 10, 11 and 12, respectively). Surprisingly, the results show that GLP1 (7-37) - (K34R) translates more efficiently than GLP1 (7-37), as indicated by a significant increase in GLP1 (7-37) - (K34R) concentration within 6 hours.

Example 5: generation of encoded exemplary intestinal promotions polyribonucleotides of insulin agents

This example describes the production of polyribonucleotides encoding various incretins. This example further describes the design of polyribonucleotides that can achieve transient in vivo incretin agent production after i.p./i.v./i.m./s.c. delivery.

The method of the embodiment comprises the following steps:

(1) DNA fragments encoding an incretin agent (e.g., GLP1, GIP, or mutants thereof) are cloned into DNA plasmids suitable for RNA expression. Suitable DNA plasmids may encode RNA features including, for example, a 5 'untranslated region (5' UTR), a Kozak sequence, a 3 'untranslated region (3' UTR), and/or a polyA tail sequence. DNA plasmids also typically include restriction sites that enable cloning of DNA fragments encoding incretins downstream of the coding region of the 5 'UTR and Kozak sequences and upstream of the coding region of the 3' UTR and polyA tail sequences. Examples of suitable DNA plasmids are also found in WO2021/214204, which is hereby incorporated by reference in its entirety.

(3) Linearization of DNA plasmids encoding incretins.

(4) Encoding incretins synthesis of polyribonucleotides.

(5) Biochemical characterization of polyribonucleotides encoding incretins.

Design of exemplary incretins for optimal expression of exemplary incretins DNA sequences were generated based on the amino acid sequences of GLP1 (7-37), GIP (1-42) and truncated or mutant mutants thereof fused to exemplary Signal Peptides (SPs) including viral signal peptide SP1-2 ("viral SP") of SEQ ID NO: 17 and husec (δGS) signal peptide ("husec") of SEQ ID NO: 65, as shown in tables 10 and 11 above. For an incretin designed to include husec signal peptides, two different codon optimization methods were tested to determine if the codon optimization method affected the translation efficiency and functionality of the final incretin peptide. In addition, certain incretins are designed to see if a linking peptide fused to an incretin peptide affects the expression and functionality of the incretin. Exemplary incretins produced in this example are shown in table 18 below.

TABLE 18 exemplary incretin agents

Codon optimization

And carrying out optimization by using Life Technologies GmbH GeneArt cube provided GeneOptimaizer cube software according to two strategies. Both strategies generally optimize codon usage by using the most frequent codons, and adjust the GC content of the uploading sequence for the chosen expression system (homo sapiens in this case). At the same time, codon optimization removes sequence repeats, introns, cryptic splice sites, internal ribosome entry sites and RNA destabilizing sequence components (e.g., upA-dinucleotides), adds RNA stabilizing sequence components (e.g., cpG-dinucleotides) and avoids stabilizing RNA secondary structures as well as unwanted sequences such as restriction sites. The output sequence is then used to order the DNA fragment strings. Those skilled in the art will appreciate that alternative methods for codon optimization are available. Furthermore, additional information about codon optimization methods is provided herein.

Cloning

Each incretin sequence was cloned into a DNA plasmid (e.g., pST 5). This may be accomplished, for example, by in vivo assembly, volume .Garcia-Nafria, "IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly," Scientific Reports , article number 27459 (2016).

The nucleotide sequences of plasmids encoding the incretins produced in this example are shown in Table 19 below.

Table 19: encoding incretins exemplary polyribonucleotides

Plasmid DNA preparation

Plasmid DNA is prepared, for example, by selecting clones for inoculation into the medium. The selected clones are optionally verified by restriction analysis and optionally sequencing. Cultures were grown and after cell harvest, purified according to manufacturer's instructions, for example using QIAGEN PLASMID Plus Maxi kit. DNA concentration was determined by UV spectroscopy. DNA was stored in certified rnase-free and dnase-free reaction tubes.

Linearization and DNA purification

In vitro transcription

RNA, optionally capped RNA, is then produced, for example, following the procedures disclosed in Kreiter et al, cancer immunol. Immunother. 2007, 56, 1577-87 and WO2021/214204, each of which is incorporated herein by reference in its entirety. Methyl pseudouridine is used in vitro transcription reactions and is incorporated into the produced RNA. The resulting RNA was subjected to cellulose purification to isolate single-stranded RNA, followed by concentration measurement by UV spectrometry. RNA integrity was determined by microfluidic-based electrophoresis. The resulting RNA is optionally further biochemically characterized.

Transfection and expression

Example 6 in vitro functionality of additional Polyribonucleotides encoding an incretin agent

This example examines the in vitro functionality of the polyribonucleotides encoding incretins produced and described in example 5 and compares the levels of peptide secretion. This example demonstrates that each of the polyribonucleotides produced in example 5 can induce the production of different levels of an incretin agent.

Method in this example, 6X 10 ⁴ HEK293t17 cells per well were seeded in three different 48-well plates and grown overnight in a 37℃5% CO ₂ incubator. Cells were transfected with 0.6 μg of polyribonucleotide candidate using Lipofectamine Messenger MAX kit (ThermoFisher Scientific, cat. No. LMRNA 003).

The cells were further incubated. Supernatants were collected after 3 hours, 6 hours, 24 hours, 48 hours and 72 hours period following transfection. For time points of 3 hours and 6 hours post-transfection, supernatants were collected and frozen at-80 ℃. At the 24 hour time point after transfection, supernatants were collected and fresh medium in the well plates was replaced. The well plate was further incubated until 48 hours and 72 hours post-transfection. Supernatants collected at 24, 48 and 72 hour time points post-transfection were stored at-80 ℃ until further analysis.

The concentrations of GIP and GLP1 in the supernatants were then quantified using ELISA (human GIP (total) ELISA kit and GLP1 (7-36) active ELISA kit, merck Millipore). Statistical analysis was performed by single factor ANOVA followed by a post hoc Tukey test.

Results FIG. 18 shows the concentration (pg/ml) of an exemplary GLP1 incretin agent in HEK29t17 cell supernatant transfected with polyribonucleotides encoding an exemplary GLP1 incretin agent containing a viral signal peptide ("viral SP") or husec signal peptide ("husec"). The polyribonucleotides encoding the incretins (1) husec GLP (7-37) with the A8G mutation and (2) husec GLP (7-37) with the A8G mutation, the linker peptide, showed much higher concentrations 24 hours after transfection. These results indicate that the incretins comprising husec signal peptide and A8G mutation show superior translational efficiency. In addition, the codon optimized mutant 1 ("opt 1") showed better translation than the codon optimized mutant 2 ("optp") for both (1) husec GLP1 (7-37) with the A8G mutation and (2) husec GLP (7-37) with the A8G mutation, the linker peptide, the incretins.

FIG. 19 shows exemplary concentrations (ng/ml) of GIP incretins in HEK29t17 cell supernatants transfected with polyribonucleotides encoding incretins containing viral signal peptide or husec signal peptide. The polyribonucleotides encoding viral SP-GIP (1-42) showed the highest concentration, especially 24 hours and 48 hours after transfection. These results indicate that inclusion of the viral signal peptide results in superior translational efficiency of the incretins. The codon-optimized mutants with husec signal peptide showed similar translational efficiencies.

Example 7 biological Activity of incretins translated from polyribonucleotides

This example demonstrates the biological activity of the incretins described herein delivered to cells with polyribonucleotides and subsequently translated. Various incretins encoded by polyribonucleotides as described in example 5 were tested.

The purpose of this experiment was to determine the biological activity of translated GIP and GLP1 incretin in GLP1R-CRE and GIPR-CRE luciferase reporter gene-HEK 293 cell lines (see FIG. 22).

Day 0 plating of HEK293 CRE reporter cells expressing GLP1R and GIPR

GLP1R-CRE and GIPR-CRE luciferase reporter HEK293 cells were each seeded at a density of approximately 38,000 cells/well in a white clear bottom 96-well plate containing 100. Mu.l of their specific medium per well. Cells were incubated in a CO ₂ incubator at 37 ℃ for 2 days.

Day 1 cell stimulation and luciferase assay

100 Μl of medium was gently removed from the wells 24 hours after transfection, and the cells were incubated at 5% CO ₂ ℃ for 6 hours. Mu.l ONE-StepTM luciferase assay reagent was added to each well. The cells were gently shaken 15min at room temperature and then luminescence was measured. Results are expressed as fold induction relative to control samples.

The biological activity of GIP and GLP1 candidates was tested in 100 μl of DMEM at concentrations of 1.75nM and 50pM, respectively. GLP1, semaglutin and telipopeptide were used as controls for GLP1 detection. GIP and telipopeptide were used as controls for GIP detection.

Results:

The results from the bioactivity assay of GLP1 incretins are shown in fig. 23. The results show that GLP1 incretins with husec signal peptide and A8G mutation show better biological activity than GLP1 incretins with viral signal peptide. In addition, the results show that codon optimization of GLP1 incretins does not affect biological activity. The specific biological activity of the GLP1 mutant encoded by the mRNA with the viral signal peptide was comparable to the controls (GLP 1, semaglutin and telipopeptide). Surprisingly, the specific biological activity of GLP1 mutants encoded by mRNA with husec signal peptide was superior to controls (GLP 1, semaglutin and telipopeptide).

The results from the bioactivity assay of GIP incretins are shown in fig. 24. The results showed that the GIP incretins with husec signal peptide and A2G mutation showed better biological activity than the GIP incretins with viral signal peptide. In addition, the results show that the codon optimization strategy does not affect the biological activity of the GIP incretins. The specific biological activity of the mRNA-encoded GIP incretins (using husec or viral signal peptide) was lower than the control (GIP and telipopeptide).

In summary, for the GLP1 incretins tested, changing the Signal Peptide (SP) from the viral signal peptide to husec signal peptide, and introducing the A8G mutation improved both translation and biological activity of the incretins. For GIP candidates, changing the signal peptide from a viral signal peptide to husec signal peptide, and introducing an A2G mutation reduced the translation rate of the mRNA, but surprisingly improved the specific biological activity of the translated peptide.

Without wishing to be bound by any theory, the choice of signal peptide in the context of the polyribonucleotides described herein that encode an incretin agent may affect the manner in which the N-terminus of the incretin peptide is cleaved. Certain signal peptides may result in alternative processing or cleavage sites, ultimately altering the final amino acid sequence of the mature incretin peptide. In such relatively small peptides, variations in amino acid sequence can greatly affect biological activity. Such results support the insight that in designing a polyribonucleotide construct encoding an incretin peptide (or other similar peptide), the signal peptide should be selected to affect proper cleavage of the N-terminus of the incretin peptide, or in other words, to produce a scar-free N-terminus to maintain the biological activity of the incretin peptide. FIGS. 20 and 21 show schematic representations of the locations of the theoretical cleavage sites of various signal peptides. FIG. 20 indicates that the A8G mutation promotes correct N-terminal processing of GLP1 incretins with husec signal peptide. Figure 21 indicates that the A2G mutation promotes correct N-terminal processing in GIP incretins containing husec signal peptide. The results in this example show that signal peptide selection affects translation and biological activity, probably because of the way the signal peptide is cleaved from the incretin peptide.

Example 8: generation of encoded exemplary intestinal promotions polyribonucleotides of insulin agents

This example describes the production of polyribonucleotides encoding various incretins. This example further describes the design of polyribonucleotides that can achieve transient in vivo incretin agent production after i.p./i.v./i.m./s.c. delivery. The exemplary incretins produced in this example utilize the various strategies described herein for improving the activity and half-life of incretins.

The method of the embodiment comprises the following steps:

(1) DNA fragments encoding an incretin agent (e.g., GLP1, GIP, or mutants thereof) are cloned into DNA plasmids suitable for RNA expression. Suitable DNA plasmids may encode RNA features including, for example, a 5 'untranslated region (5' UTR), a Kozak sequence, a 3 'untranslated region (3' UTR), and/or a polyA tail sequence. DNA plasmids also typically include restriction sites that enable cloning of DNA fragments encoding incretins downstream of the region encoding the 5 'UTR and Kozak sequences and upstream of the region encoding the 3' UTR and polyA tail sequences. Examples of suitable DNA plasmids are also found in WO2021/214204, which is hereby incorporated by reference in its entirety.

(3) Linearization of DNA plasmids encoding incretins.

(4) Encoding incretins synthesis of polyribonucleotides.

(5) Biochemical characterization of polyribonucleotides encoding incretins.

Design of exemplary incretins for optimal expression of exemplary incretins, DNA sequences were generated based on the amino acid sequences of GLP1 (7-37), GIP (1-42) and truncated or mutant mutants thereof fused to exemplary Signal Peptides (SPs) including the viral signal peptide gD1 of SEQ ID NO: 66 and the husec (δGS) signal peptide of SEQ ID NO: 65 ("husec"), as shown in tables 10 and 11 above. The incretins produced in this example were also examined for combinations of different half-life extending moieties and multiple incretins peptides. Exemplary incretins produced in this example are shown in table 20 below.

TABLE 20 exemplary incretins wherein the x2 and x4 examples include a linker peptide between each repeat unit and a furin cleavage site

Codon optimization

Cloning

The nucleotide sequences of plasmids encoding the incretins produced in this example are shown in Table 21 below.

TABLE 21 exemplary polyribonucleotides encoding incretins wherein the x2 and x4 examples include a linker peptide between each repeat unit and a furin cleavage site

Plasmid DNA preparation

Plasmid DNA was prepared by selecting clones for inoculation into the medium. The selected clones are optionally verified by restriction analysis and optionally sequencing. Cultures were grown and after cell harvest, purified according to manufacturer's instructions, for example using QIAGEN PLASMID Plus Maxi kit. DNA concentration was determined by UV spectroscopy. DNA was stored in certified rnase-free and dnase-free reaction tubes.

Linearization and DNA purification

In vitro transcription

Transfection and expression

Example 9 in vitro functionality and biological Activity of additional Polyribonucleotides encoding incretins

This example examines the in vitro functionality and biological activity of the polyribonucleotides encoding incretins produced as described in example 8. This example compares the levels of peptide secretion and demonstrates that each of the polyribonucleotides produced in example 8 can induce the production of different levels of incretin. In addition, this example also demonstrates the biological activity of the incretins described herein as a polyribonucleotide delivered to cells and subsequently translated. The biological activity of various incretins encoded by polyribonucleotides as described in example 8 was tested. This example also examines various design strategies to improve properties of expressed incretins, such as half-life, N-terminal cleavage, stability, translational efficiency, and bioactivity.

The method comprises the following steps:

In vitro expression in this example, 6×10 ⁴ HEK293t17 cells per well were seeded in three different 48-well plates and grown overnight in a 37 ℃ 5% CO ₂ incubator. Cells were transfected with 0.6 μg of polyribonucleotide candidate using Lipofectamine Messenger MAX kit (ThermoFisher Scientific, cat. No. LMRNA 003).

Biological Activity the purpose of this experiment was to determine the biological activity of translated GIP and GLP1 incretin in GLP1R-CRE and GIPR-CRE luciferase reporter gene-HEK 293 cell lines (see FIG. 22).

Day 0 plating of HEK293 CRE reporter cells expressing GLP1R and GIPR

Day 1 cell stimulation and luciferase assay

The biological activity of GIP and GLP1 candidates was tested in 100 μl of DMEM at concentrations of 1.75nM and 50pM, respectively. GLP1 (7-37) with the A8G mutation was used as a control for GLP1 detection. GIP (1-42) having the A2G mutation was used as a control for GIP detection. The assay was repeated in triplicate.

Results:

Figure 25 shows GIP expression for all incretins tested. Constructs comprising two or more incretin peptides (i.e., 4081 and 4082) and those fused to Dula _igg4 (i.e., 4092 and 4093) showed the highest expression, including at the later time point of 72 hours.

Figure 26 shows GIP bioactivity (in each of the three replicates) of all GIP-containing incretins tested. The same volume of supernatant (with different concentrations of incretin as shown in fig. 25) was used for each construct, and therefore different amounts of expressed protein were present in assessing biological activity in this experiment. For example, constructs 4081 and 4082 show high GIP expression in fig. 25, which may result in high GIP bioactivity in fig. 26.

Figure 27 shows GLP1 expression of all incretins tested.

FIG. 28 shows GLP1 bioactivity of all GLP1 containing incretins tested. The same volume of supernatant (with different concentrations of incretin as shown in fig. 27) was used for each construct, and therefore different amounts of expressed protein were present in assessing biological activity in this experiment.

FIG. 29 shows a comparison of GIP expression (A) and GIP bioactivity (B) in candidates with different signal peptides (husec vs gD 1). The incretins with the gD1 signaling peptide showed increased GIP expression (about 2X) and increased GIP bioactivity (about 5X) compared to the same incretins with husec signaling peptide. As described herein, the present disclosure recognizes that the selection of the signaling peptide used in the polyribonucleotide construct encoding the incretin agent is important to promote proper expression and cleavage of the signaling peptide such that the N-terminus of the incretin agent is "scar-free" and retains functionality to dock to its cognate receptor (e.g., GIPR).

FIG. 30 shows a comparison of GLP1 expression (A) with GLP1 bioactivity (B) in candidates with different signal peptides (husec vs gD 1). The incretin agent with the gD1 signaling peptide showed increased GLP1 expression compared to the incretin agent comprising husec signaling peptide (see figure 30A). However, incretins utilizing gD1 signaling peptides do not necessarily show better GLP1 bioactivity. The incretins (4071, 3815 and 4073) using husec signaling peptides showed lower GLP1 expression and thus less GLP1 peptide was used in the bioactivity assay, but 3815 showed comparable bioactivity to the gD1 containing incretins 4074, 4075 and 4076, samples of which contained higher levels of GLP1 peptide in the bioactivity assay, even if less GLP1 peptide was present. Thus, although an incretin agent containing gD1 appears to have increased GLP1 expression compared to an incretin agent containing husec signaling peptide, GLP1 expression does not necessarily result in increased GLP1 bioactivity.

Figure 31 shows a comparison of GIP expression (a) of GIP with and without various half-life extending (HLE) moieties with GIP biological activity (B). The results showed that the incretins (4086 and 4087) containing Dula _igg4 expressed well (a). Without wishing to be bound by any theory, high expression may be due to the overall size of the polyribonucleotides. However, the results showed that higher expression did not necessarily result in improved GIP bioactivity, which was relatively low (B). The albumin-containing incretins (4096) showed consistent levels of GIP expression and GIP bioactivity compared to other incretins. In addition, incretins (4097) containing an albumin-bound VHH (a-HSA VHH) have relatively low GIP expression levels (a), but show higher GIP bioactivity (B) even though the assayed proteins are less. The incretin agent (4089) containing the Fc fusion with KIH mutations (FcKIH-b) also showed consistent GIP expression and GIP bioactivity.

FIG. 32 shows a comparison of GLP1 expression (A) with and without various half-life extending (HLE) moieties to GLP1 bioactivity (B). The results showed that the incretin agent (4088) with the KIH mutant Fc domain fusion (FcKIH-a) showed consistent GLP1 expression (a) and GLP1 bioactivity (B), similar to the GIP expression/bioactivity (4089) of FcKIH-B in fig. 31 (1:1 translation/bioactivity), indicating that HLE moiety (FcKIH) did not interfere with bioactivity. The incretins containing Dula _igg4 (4084 and 4085) expressed well (a), possibly due to the overall size of the polyribonucleotides, but had relatively low GLP1 bioactivity (B), similar to the results of GIP expression/bioactivity in fig. 31. In addition, the results show that the incretin agent (4091) containing the albumin-bound VHH (a-HSA VHH) has a relatively low expression level (32A), but shows higher bioactivity (32B) even with fewer GLP1 peptides assayed, consistent with the results in fig. 31.

In summary, the results in fig. 31 and 32 surprisingly show that the HLE moiety described herein does not reduce the biological activity of the incretin agent, even though the size of the HLE moiety is larger compared to the incretin peptide.

FIGS. 33 and 34 show comparison of GIP and GLP1 expression (FIGS. 33A and 34A) and bioactivity (FIGS. 33B and 34B), respectively, in exemplary incretins containing both GIP and GLP1, wherein the sequence of the GIP and GLP1 peptide chains encoded by a single polyribonucleotide is altered. For example, incretin 4093 contains from N-terminus to C-terminus a GLP1 peptide (with H7Y, A8G, R G mutation), a furin cleavage site, a GIP peptide (with A2G mutation), fused to Dula _IgG4 (with LS mutation), whereas incretin 4094 contains from N-terminus to C-terminus a GIP peptide (with A2G mutation), a furin cleavage site, a GLP1 peptide (with H7Y, A8G, R G mutation), fused to Dula _IgG4 (with LS mutation).

Fig. 33 shows GIP expression in both 4093 and 4094 (a), however, 4094 (where GIP is at the N-terminus) has slightly better GIP bioactivity (B) considering the amount of GIP peptide detected. In fig. 34, GLP1 is expressed higher in the 4094 construct (wherein GLP1 is after GIP and adjacent to the furin cleavage site) than in the 4093 construct (a), however GLP1 shows higher bioactivity in the 4093 construct, wherein GL1 is adjacent to the husec signal peptide at the N-terminus (B). This class of results indicates that there is a position dependent effect at the N-terminus that includes GLP1 versus GIP. In particular, GIP appears to show better biological activity under furin cleavage, and GLP1 appears to show better biological activity when processed with husec signaling peptides. As described herein, to allow the incretin peptide to function and interact with its cognate receptors GIPR and GLP1R, it is preferably cleaved appropriately (i.e., with a signal peptide or furin) and has a scar-free N-terminus (i.e., cleavage/processing at the N-terminus does not alter the peptide structure).

Equivalent(s)

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but rather is set forth in the appended claims.

Claims

1. A composition comprising a polyribonucleotide encoding an incretin agent.

2. The composition of claim 1, wherein the incretin agent is a GLP1 receptor agonist.

3. The composition of claim 1, wherein the incretin agent is a GIP receptor agonist.

4. The composition of claim 1, wherein the incretin agent is a GLP1/GIP dual receptor agonist.

5. The composition of claim 1, wherein the incretin agent is a GLP1/GCG dual receptor agonist.

6. The composition of claim 1, wherein the incretin agent is a GLP1/GIP/GCG triple receptor agonist.

7. The composition of claim 2, wherein the incretin agent comprises an incretin peptide having an amino acid sequence as set forth in any one of SEQ ID NOs 5-7, 63-64, 69-70 and 74-75.

8. The composition of claim 3, wherein the incretin agent comprises an incretin peptide having an amino acid sequence set forth in any one of SEQ ID NOs 8-9, 62 and 72.

9. The composition of claim 2, wherein the incretin agent comprises an incretin peptide having the amino acid sequence set forth in SEQ ID No. 11.

10. The composition of claim 4, wherein the incretin agent comprises an incretin peptide having an amino acid sequence set forth in any one of SEQ ID NOs 12-14.

11. The composition of claim 6, wherein the incretin agent comprises an incretin peptide having the amino acid sequence set forth in SEQ ID No. 15.

12. The composition of any one of claims 7 to 11, wherein the incretin peptide is optionally fused to the signal peptide through the N-terminus of the incretin peptide, optionally through a linker peptide.

13. The composition of claim 12, wherein the signal peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 16-39 and 65-67.

14. The composition of claim 12, wherein the signal peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 16-21 and 65-67.

15. The composition of claim 12, wherein the signal peptide has an amino acid sequence as set forth in SEQ ID No. 17.

16. The composition of claim 12, wherein the signal peptide has the amino acid sequence set forth in SEQ ID No. 65.

17. The composition of claim 12, wherein the signal peptide has the amino acid sequence set forth in SEQ ID No. 66.

18. The composition of claim 1, wherein the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 41-45, 52-61, and 108-152.

19. The composition of any one of claims 1 to 18, wherein the incretin agent comprises an incretin peptide fused to one or more additional incretin peptides, optionally through one or more linking peptides.

20. The composition of claim 19, wherein the one or more connecting peptides comprise an amino acid sequence set forth in any one of SEQ ID NOs 1-5, 68 or 156.

21. The composition of claim 19 or 20, wherein the incretin agent comprises an incretin peptide fused to two or more incretin peptides.

22. The composition of any one of claims 19 to 21, wherein the incretin agent comprises at least one GLP1 receptor agonist and at least one GIP receptor agonist.

23. The composition of any one of claims 19 to 22, wherein the incretin agent comprises at least two GLP1 receptor agonists.

24. The composition of any one of claims 19-23, wherein the incretin agent comprises at least two GIP receptor agonists.

25. The composition of any one of claims 19 to 24, wherein the incretin agent comprises one or more furin cleavage sites (furin CLEAVAGE SITE).

26. The composition of claim 25, wherein the one or more furin cleavage sites are located between adjacent incretin peptides.

27. The composition of claim 27 or 28, wherein the one or more furin cleavage sites comprises the amino acid sequence set forth in SEQ ID No. 153.

28. The composition of any one of claims 19 to 27, wherein the incretin agent comprises one or more units each comprising, from N-terminus to C-terminus, a GLP1 receptor agonist-connecting peptide-furin cleavage site-GIP receptor agonist, e.g., wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 76, 77, 78, 79, 80, 81), two units (e.g., SEQ ID NO: 82), or four units (e.g., SEQ ID NO: 83).

29. The composition of any one of claims 19 to 30, wherein the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 76-83, 94-97, 102-107.

30. The composition of any one of claims 1 to 29, wherein the incretin agent comprises a half-life extending moiety.

31. The composition of claim 30, wherein the half-life extending moiety comprises albumin (e.g., human serum albumin).

32. The composition of claim 31, wherein the human serum albumin comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO 159.

33. The composition of claim 31 or 32, wherein the human serum albumin comprises the amino acid sequence set forth in SEQ ID No. 159.

34. The composition of any one of claims 31-33, wherein the incretin agent comprises albumin (e.g., human serum albumin) fused to one or more units each comprising from N-terminus to C-terminus:

(i) GLP1 receptor agonist-connecting peptide (e.g., SEQ ID NO: 98);

(ii) GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 100);

(iii) GLP1 receptor agonist-connecting peptide-furin cleavage site (e.g., SEQ ID NO: 102), or

(Iv) GLP1 receptor agonist-connecting peptide-furin cleavage site-GIP receptor agonist, for example, wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 104), two units (e.g., SEQ ID NO: 106), or four units (e.g., SEQ ID NO: 107).

35. The composition of any one of claims 31 to 34, wherein the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 98, 100, 102, 104, 106, 107, or any combination thereof.

36. The composition of claim 30, wherein the half-life extending moiety comprises an Albumin Binding Domain (ABD).

37. The composition of claim 36, wherein said ABD is derived from protein G of Streptococcus (Streptococcus) strain GI48 and/or protein PAB of pegoldens major (Finegoldia magna), such as ABD035 and SA21.

38. The composition of claim 36, wherein the half-life extending moiety comprises ABD that binds to domain II of human serum albumin and does not overlap or interfere with binding to an FcRn binding site on albumin.

39. The composition of claim 36, wherein the half-life extending moiety comprises ABDCon.

40. The composition of claim 36, wherein the half-life extending moiety comprises an Albumin Binding Domain (ABD) derived from bacterial protein Sso7d, such as M11.12 and M18.2.5, from sulfolobus sulphur (Sulfolobus solfataricus) from hyperthermophilic archaea (hyperthermophilic archaeon).

41. The composition of claim 36, wherein the half-life extending moiety comprises albumin-binding DARPin.

42. The composition of claim 36, wherein the ABD comprises an albumin-binding immunoglobulin domain or fragment thereof.

43. The composition of claim 36 or 42, wherein the ABD comprises a fully human domain antibody (dAb), such as an AlbudAb, that binds albumin.

44. The composition of claim 36 or 42 to 43, wherein said ABD comprises an albumin binding Fab, such as dsFv CA645.

45. The composition of any one of claims 36 or 42 to 44, wherein said ABD comprises a heavy chain only (VHH) antibody, such as a nanobody, that binds albumin.

46. The composition of claim 45, wherein the VHH antibody comprises a VHH domain having Complementarity Determining Region (CDR) sequences HCDR1, HCDR2 and/or HCDR3 as shown in SEQ ID NO: 191 (GFTLDYYA), SEQ ID NO: 192 (IASSGGST) and/or SEQ ID NO: 193 (AAAVLECRTVVRGYDY), respectively.

47. The composition of claim 46, wherein the VHH antibody comprises an amino acid sequence that is at least 90%, 95% or 99% identical to SEQ ID No. 154.

48. The composition of claim 46 or 47, wherein the VHH antibody comprises an amino acid sequence that is set forth in SEQ ID No. 154.

49. The composition of any one of claims 45 to 47, wherein the incretin agent comprises a VHH antibody that binds to albumin fused to a unit comprising from N-terminus to C-terminus:

(i) GLP 1-linked peptide (e.g., SEQ ID NO: 99);

(ii) GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 101), or

(Iii) GLP1 receptor agonist-connecting peptide-furin-GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 103 or 105).

50. The composition of any one of claims 45 to 49, wherein the incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 99, 101, 103, 105.

51. The composition of claim 30, wherein the half-life extending moiety does not comprise an Fc domain, such as from a human IgG, optionally from a human IgG1, igG2, igG3, or IgG4.

52. The composition of claim 30, wherein the half-life extending moiety comprises an Fc domain, such as from a human IgG, optionally from a human IgG1, igG2, igG3 or IgG4.

53. The composition of claim 52, wherein the human IgG is human IgG4.

54. The composition of claim 52 or 53, wherein the incretin agent comprises an IgG4 Fc domain fused to a unit comprising from N-terminus to C-terminus:

(i) GLP1 receptor agonist-connecting peptides (e.g., SEQ ID NOs: 10, 89, 90, 91);

(ii) GIP receptor agonist-connecting peptides (e.g., SEQ ID NOS: 92, 93), or

(Iii) GLP1 receptor agonist-connecting peptide-furin-GIP receptor agonist-connecting peptide (e.g., SEQ ID NOs: 94, 95, 96, 97).

55. The composition of claim 53 or 54, wherein said IgG4 Fc domain comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID No. 155.

56. The composition of claim 55, wherein said IgG4 Fc domain comprises the amino acid sequence of SEQ ID NO 155.

57. The composition of any one of claims 53 to 56, wherein said incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 10 and 89-97.

58. The composition of any one of claims 52 to 57, wherein the Fc domain comprises one or more mutations in one or both Fc constant domains that increase half-life and/or induce dimerization of the incretin agent.

59. The composition of claim 58, wherein the one or more mutations comprises one or more mutations in a CH3 domain.

60. The composition of claim 58 or 59, wherein said one or more mutations that induce dimerization comprise:

(i) Y349C, T366S, L368A and/or Y407V (according to EU numbering) or

(Ii) S354C and/or T366W (according to EU numbering).

61. The composition of any one of claims 58 to 60, wherein the one or more mutations comprises Y349C, T366S, L a and Y407V ("FcKIH-b", according to EU numbering), or S354C and T366W ("FcKIH-a", according to EU numbering).

62. The composition of any one of claims 58 to 61, wherein the incretin agent comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an incretin peptide fused to a first Fc domain, wherein the first Fc domain comprises mutations Y349C, T366S, L a and Y407V ("FcKIH-b", according to EU numbering), and wherein the second polypeptide chain comprises an incretin peptide fused to a second Fc domain, wherein the second Fc domain comprises mutations S354C and T366W ("FcKIH-a", according to EU numbering).

63. The composition of any one of claims 58 to 62, wherein the one or more mutations that increase half-life of the incretin agent comprises M428L and N434S ("LS", numbering according to EU).

64. The composition of any one of claims 58 to 63, wherein the incretin agent comprises an Fc domain having a FcKIH-a mutation in a first polypeptide chain and an Fc domain having a FcKIH-b mutation in a second polypeptide chain, wherein the Fc domains on each polypeptide chain are each independently fused to one or more units comprising from N-terminus to C-terminus:

(i) GLP1 receptor agonist-connecting peptides (e.g., SEQ ID NOs: 84, 85, 86, 87), or

(Ii) GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 88).

65. The composition of any one of claims 58 to 64, wherein said incretin agent comprises an amino acid sequence as set forth in any one of SEQ ID NOs 84-88.

66. The composition of any one of claims 58 to 64, wherein said Fc domain comprises one or more mutations that abrogate the effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q).

67. The composition of claim 66, wherein one or more mutations that abrogate effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q) comprises the following mutations L234S, L T and G236R ("STR", numbering according to EU).

68. The composition of claim 66, wherein the one or more mutations that abrogate effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q) comprise mutations L234A and L235A ("LALA", numbering according to EU).

69. The composition of claim 66, wherein the one or more mutations that abrogate effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q) comprises a mutation of L234A/L235A/P329G ("LALAPG", numbering according to EU).

70. The composition of claim 30, wherein the half-life extending moiety comprises an albumin-binding VNAR.

71. The composition of claim 30, wherein the half-life extending moiety comprises an XTEN sequence.

72. The composition of any one of claims 1-71, wherein the polyribonucleotide has a ribonucleic acid sequence that is at least 90% identical to any one of SEQ ID NOs 177-185 and 224-256.

73. The composition of claim 72, wherein the polyribonucleotide has a ribonucleic acid sequence as set forth in any of SEQ ID Nos. 177-185 and 224-256.

74. The composition of any one of claims 1 to 73, wherein the polyribonucleotide comprises at least one non-coding sequence component that enhances RNA stability and/or translation efficiency.

75. The composition of claim 74, wherein said at least one non-coding sequence component comprises a 5' cap structure, a 5' UTR, a 3' UTR, and/or a polyA tail.

76. The composition of claim 75, wherein the polyribonucleotide comprises in the 5 'to 3' direction:

a. 5' UTR;

b. a signal peptide coding sequence;

c. An incretin peptide coding sequence;

d. 3' UTR, and

E. polyA tail.

77. The composition of claim 75, wherein the polyribonucleotide comprises in the 5 'to 3' direction:

(1)

a. 5' UTR;

b. a signal peptide coding sequence;

c. An incretin peptide coding sequence;

d. a linker peptide coding sequence;

e. half-life extending moiety coding sequences;

f. 3' UTR, and

G. polyA tail, or

(2)

a. 5' UTR;

B. a signal peptide coding sequence;

c. Half-life extending moiety coding sequences;

d. a linker peptide coding sequence;

e. an incretin peptide coding sequence;

f. 3' UTR, and

G. polyA tail.

78. The composition of any one of claims 1 to 77, wherein said incretin peptide is encoded by a coding sequence that is codon optimized and/or has an increased G/C content as compared to a wild-type coding sequence, wherein said codon optimization and/or increase in the G/C content does not alter the sequence of the encoded amino acid sequence.

79. The composition of any one of claims 1 to 78, wherein the polyribonucleotide comprises at least one modified ribonucleotide.

80. The composition of claim 79, wherein the polyribonucleotide comprises a modified nucleoside that replaces uridine.

81. The composition of claim 80, wherein the polyribonucleotides comprise modified nucleosides that replace each uridine.

82. The composition of claim 81, wherein the modified nucleoside is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), and 5-methyl-uridine (m 5U).

83. The composition of any one of claims 80 to 83, wherein said modified nucleoside is N1-methyl-pseudouridine (m1ψ).

84. The composition of any one of claims 1 to 83, wherein the polyribonucleotide comprises a 5' cap structure.

85. The composition of any one of claims 1 to 84, wherein the polyribonucleotide comprises a 5' UTR.

86. The composition of any one of claims 1 to 85, wherein the polyribonucleotide comprises a 3' UTR.

87. The composition of any one of claims 1 to 86, wherein the polyribonucleotide comprises a polyA tail.

88. The composition of claim 87, wherein the polyA tail comprises at least 100 nucleotides.

89. The composition of any one of claims 1 to 88, wherein the polyribonucleotide is mRNA.

90. The composition of any one of claims 1 to 89, wherein the polyribonucleotide is formulated as a liquid, formulated as a solid, or a combination thereof.

91. The composition of any one of claims 1 to 90, wherein the polyribonucleotide is formulated for injection.

92. The composition of any one of claims 1-91, wherein the polyribonucleotide is formulated for intraperitoneal or intravenous administration.

93. The composition of any one of claims 1 to 92, wherein the polyribonucleotide is formulated or to be formulated as a lipid particle.

94. The composition of claim 93, wherein the polyribonucleotide is formulated or to be formulated as a lipid nanoparticle.

95. The composition of claim 94, wherein the polyribonucleotides are encapsulated within isopolysaccharide nanoparticles.

96. The composition of claim 94 or 95, wherein the isopolysaccharide nanoparticle is a pancreas-and/or intestine-targeted lipid nanoparticle.

97. The composition of any one of claims 94-96, wherein said isopolysaccharide nanoparticle is a cationic lipid nanoparticle.

98. The composition of claim 97, wherein the lipid forming the isopolysaccharide nanoparticle comprises:

a. Polymer coupled lipids;

b. cationic lipid, and

C. neutral lipids.

99. The composition of claim 98, wherein the polymer-coupled lipid is a PEG-coupled lipid.

100. The composition of claim 98 or 99, wherein the cationic lipid is an ionizable lipid-like material (lipid).

101. The composition of claim 100, wherein the cationic lipid has one of the following structures:

X-1

X-2

X-3

X-4。

102. The composition of any one of claims 99 to 101, wherein the neutral lipid comprises a helper lipid, such as 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DPSC) and/or cholesterol.

103. The composition of any one of claims 99-102, wherein the cationic lipid is selected from cationic lipids X-2, X-3 or X-4 and the neutral lipid comprises a helper lipid such as DOTAP, DOPE or PS and cholesterol.

104. The composition of claim 103, wherein the polymer-coupled lipid is C14-PEG2000.

105. The composition of any one of claims 99 to 104, wherein the isopolysaccharide nanoparticle comprises i) about 30 mol% to about 50 mol% cationic lipid, ii) about 1 mol% to 5 mol% PEG conjugated lipid, iii) about 30 mol% to about 50 mol% helper lipid, and iv) about 20 mol% to about 40 mol% cholesterol.

106. The composition of any one of claims 99 to 104, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid, about 40 mol% helper lipid, about 22.5 mol% cholesterol, and about 2.5 mol% PEG conjugated lipid.

107. The composition of claim 106, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-2, X-3, or X-4, about 40 mol% DOTAP, DOPE, or PS, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000.

108. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-2, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000.

109. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-3, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000.

110. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-4, about 40. 40 mol% DOTAP, about 22.5. 22.5 mol% cholesterol, and about 2.5. 2.5 mol% C14-PEG2000.

111. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-2, about 40 mol% DOPE, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000.

112. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-3, about 40 mol% DOPE, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000.

113. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-4, about 40 mol% DOPE, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000.

114. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-2, about 40 mol% PS, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000.

115. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-3, about 40 mol% PS, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000.

116. The composition of claim 107, wherein the isopolysaccharide nanoparticle comprises about 35 mol% cationic lipid X-4, about 40 mol% PS, about 22.5 mol% cholesterol, and about 2.5 mol% C14-PEG2000.

117. The composition of any one of claims 94-116, wherein the isopolysaccharide nanoparticle is formulated for intraperitoneal (i.p.) delivery.

118. The composition of any one of claims 94-117, wherein said isopipidemic nanoparticle has an average size of about 50-150 nm.

119. The composition of any one of claims 1 to 118, further comprising one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

120. The composition of claim 119, further comprising a cryoprotectant.

121. The composition of claim 120, wherein the cryoprotectant is sucrose.

122. The composition of any one of claims 119-121, further comprising an aqueous buffer solution.

123. The composition of claim 122, wherein said aqueous buffer solution comprises sodium ions.

124. A method of treating a disease state in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the composition of any one of claims 1-123.

125. The method of claim 124, further comprising administering one or more DPP-4 inhibitors.

126. The method of claim 125, wherein the one or more DPP-4 inhibitors and the composition are administered simultaneously.

127. The method of claim 125, wherein the one or more DPP-4 inhibitors and the composition are administered sequentially.

128. The method of claim 127, wherein the one or more DPP-4 inhibitors are administered prior to the composition.

129. The method of claim 127, wherein the one or more DPP-4 inhibitors are administered after the composition.

130. The method of any one of claims 125-129, wherein the one or more DPP-4 inhibitors comprise sitagliptin (sitagliptin), vildagliptin (vildagliptin), saxagliptin (saxagliptin), linagliptin (linagliptin), gemagliptin (gemigliptin), alagliptin (anagliptin), tigliptin (TENELIGLIPTIN), alogliptin (alogliptin), trelagliptin (TRELAGLIPTIN), australitin (omarigliptin), ebagliptin (evogliptin), agogliptin (gosogliptin), duloxetine (dutogliptin), neogliptin (neogliptin), regagliptin (retagliptin), dulgliptin (denagliptin), colagliptin (cofroglipin), fogliptin (fotagliptin), pragliptin (prusogliptin), ponine (berberine), or any combination thereof.

131. The method of any one of claims 125-130, wherein the one or more DPP-4 inhibitors are administered orally.

132. The method of claim 124, wherein the disease state is obesity or an obesity-related disorder.

133. The method of claim 132, wherein the obesity-related disorder is pre-diabetes, type 2 diabetes (T2D), early stage type 1 diabetes (T1D), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease, kidney disease, or an increased risk of premature death.

134. The method of claim 133, wherein the Cardiovascular (CV) disease comprises a major cardiovascular event (MACE), including CV death, non-fatal myocardial infarction, non-fatal stroke, and/or heart failure with preserved ejection fraction (HFpEF).

135. The method of claim 132, wherein the method improves weight management in the subject.

136. The method of claim 132, wherein the method reduces weight gain or induces weight loss in the subject.

137. The method of claim 124, wherein the disease state is diabetes.

138. The method of claim 137, wherein the method improves glycemic control of the individual.

139. The method of claim 137, wherein the method reduces HbA1c in the individual.

140. The method of claim 137, wherein the diabetes is pre-diabetes, type 2 diabetes (T2D), or early stage type 1 diabetes (T1D).

141. The method of claim 124, wherein the disease state is a Cardiovascular (CV) disease.

142. The method of claim 141, wherein the cardiovascular disease comprises major cardiovascular events (MACEs) including CV death, non-fatal myocardial infarction, non-fatal stroke, and/or heart failure with retention of ejection fraction (HfpEF).

143. The method of claim 141, wherein the method improves blood pressure and/or blood lipid in the individual of the individual.

144. The method of claim 124, wherein the disease state is kidney disease.

145. The method of claim 124, wherein the disease state is non-alcoholic fatty liver disease (NAFLD).

146. The method of claim 124, wherein the disease state is non-alcoholic steatohepatitis (NASH) and optionally sequelae liver fibrosis and cirrhosis.

147. The method of any one of claims 124-146, wherein administering the composition to the subject comprises administering one or more doses of the composition to the subject.

148. The method of claim 147, wherein the one or more doses of the composition are administered to the individual daily, every other day, or weekly.

149. The method of claim 147, wherein the one or more doses of the composition are administered to the individual less frequently than once a week.

150. The method of claim 147, wherein the one or more doses of the composition are administered to the individual once every 2, 3, or 4 weeks.

151. The method of any one of claims 124-150, wherein the composition is administered by injection.

152. The method of claim 151, wherein the composition is administered subcutaneously, intravenously, intramuscularly, or intraperitoneally.

153. The method of claim 152, wherein the composition is administered intraperitoneally.

154. The method of any one of claims 124-150, wherein the composition is administered non-invasively (e.g., orally or nasally).

155. The method of any one of claims 124-154, wherein administration of the composition results in expression of the incretin agent in the individual.

156. The method of any one of claims 124-155, wherein the composition is administered in a volume of less than 0.5 mL.

157. Use of the composition of any one of claims 1 to 123 for treating a disease state in a subject in need thereof.

158. A method of producing an incretin agent comprising administering the composition of any one of claims 1-123 to a cell such that the cell expresses and secretes the incretin agent.

159. An incretin agent comprising an incretin peptide fused to a signal peptide.

160. The incretin agent of claim 159, wherein the incretin peptide is fused to the signal peptide through the N-terminus of the incretin peptide, optionally through a linker peptide.

161. The incretin agent of claim 159 or 160, wherein the signal peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 16-39 and 65-67.

162. The incretin agent of claim 161, wherein the signal peptide has an amino acid sequence as set forth in any one of SEQ ID NOs 16-21 and 65-67.

163. The incretin agent of claim 161, wherein the signal peptide has an amino acid sequence as set forth in SEQ ID No. 17.

164. The incretin agent of claim 161, wherein the signal peptide has an amino acid sequence as set forth in SEQ ID No. 65.

165. The incretin agent of claim 161, wherein the signal peptide has an amino acid sequence as set forth in SEQ ID No. 66.

166. The incretin agent of any one of claims 159-165, wherein the incretin agent comprises an incretin peptide fused to a signal peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs 41-45, 52-61, and 108-152.

167. The incretin agent of any one of claims 159-166, wherein the incretin agent comprises an incretin peptide fused to one or more additional incretin peptides, optionally through one or more linking peptides.

168. The incretin agent of claim 167, wherein the one or more connecting peptides comprise an amino acid sequence set forth in any one of SEQ ID NOs 1-5, 68, or 156.

169. The incretin agent of claim 167 or 168, wherein the incretin agent comprises an incretin peptide fused to two or more incretin peptides.

170. The incretin agent of any one of claims 167-169, wherein the incretin agent comprises at least one GLP1 receptor agonist and at least one GIP receptor agonist.

171. The incretin agent of any one of claims 167 to 170, wherein the incretin agent comprises at least two GLP1 receptor agonists.

172. The incretin agent of any one of claims 167-171, wherein the incretin agent comprises at least two GIP receptor agonists.

173. The composition of any one of claims 167-172, wherein said incretin agent comprises one or more furin cleavage sites.

174. The incretin agent of claim 173, wherein the one or more furin cleavage sites are located between adjacent incretin peptides.

175. The incretin agent of claim 173 or 174, wherein the one or more furin cleavage sites comprises the amino acid sequence set forth in SEQ ID No. 153.

176. The incretin agent of any one of claims 167-175, wherein the incretin agent comprises one or more units each comprising, from N-terminus to C-terminus, a GLP1 receptor agonist-connecting peptide-furin cleavage site-GIP receptor agonist, e.g., wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 76, 77, 78, 79, 80, 81), two units (e.g., SEQ ID NO: 82), or four units (e.g., SEQ ID NO: 83).

177. The incretin agent of any one of claims 167-176, wherein the incretin agent comprises an amino acid sequence set forth in any one of SEQ ID NOs 76-83, 94-97, 102-107.

178. The incretin agent of any one of claims 159-177, wherein the incretin agent comprises a half-life extending moiety.

179. The incretin agent of claim 178, wherein the half-life extending moiety comprises albumin (e.g., human serum albumin).

180. The incretin agent of claim 180, wherein the human serum albumin comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO 159.

181. The incretin agent of claim 179 or 180, wherein the human serum albumin comprises the amino acid sequence set forth in SEQ ID NO 159.

182. The incretin agent of any one of claims 172-174, wherein the incretin agent comprises albumin (e.g., human serum albumin) fused to one or more units each comprising from N-terminus to C-terminus:

(i) GLP1 receptor agonist-connecting peptide (e.g., SEQ ID NO: 98);

(ii) GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 100);

183. The incretin agent of any one of claims 179-182, wherein the incretin agent comprises an amino acid sequence set forth in any one of SEQ ID NOs 98, 100, 102, 104, 106, 107, or any combination thereof.

184. The incretin agent of claim 178, wherein the half-life extending moiety comprises an Albumin Binding Domain (ABD).

185. The incretin agent of claim 184, wherein the ABD is derived from protein G of streptococcus strain GI48 and/or protein PAB of megagoldens, such as ABD035 and SA21.

186. The incretin agent of claim 184, wherein the half-life extending moiety comprises an ABD that binds to domain II of human serum albumin and does not overlap or interfere with binding to an FcRn binding site on albumin.

187. The incretin agent of claim 184, wherein the half-life extending moiety comprises ABDCon.

188. The incretin agent of claim 184, wherein the half-life extending moiety comprises an ABD derived from a bacterial protein Sso7d, such as M11.12 and M18.2.5, from the hyperthermophilic archaea sulfolobus.

189. The incretin agent of claim 178, wherein the half-life extending moiety comprises albumin-binding DARPin.

190. The incretin agent of claim 184, wherein the ABD comprises an albumin-binding immunoglobulin domain or fragment thereof.

191. The incretin agent of claim 184 or 190, wherein said ABD comprises a fully human domain antibody (dAb), such as an AlbudAb, that binds albumin.

192. The incretin agent of any one of claims 184 or 190-191, wherein the ABD comprises an albumin-binding Fab, such as dsFv CA645.

193. The incretin agent of claims 184 or 190-192, wherein said ABD comprises a heavy chain only (VHH) antibody, such as a nanobody, that binds albumin.

194. The incretin agent of claim 193, wherein the VHH antibody comprises a VHH domain having Complementarity Determining Region (CDR) sequences HCDR1, HCDR2 and/or HCDR3 as shown in SEQ ID No. 191 (GFTLDYYA), SEQ ID No. 192 (IASSGGST) and/or SEQ ID No. 193 (AAAVLECRTVVRGYDY), respectively.

195. The incretin agent of claim 193 or 194, wherein the VHH antibody comprises an amino acid sequence that is at least 90%, 95% or 99% identical to SEQ ID No. 154.

196. The incretin agent of claim 195, wherein the VHH antibody comprises an amino acid sequence that is set forth in SEQ ID No. 154.

197. The incretin agent of any one of claims 193-196, wherein the incretin agent comprises a VHH antibody that binds to albumin fused to a unit comprising from N-terminus to C-terminus:

(i) GLP 1-linked peptide (e.g., SEQ ID NO: 99);

(ii) GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 101), or

198. The incretin agent of any one of claims 193-197, wherein the incretin agent comprises an amino acid sequence set forth in any one of SEQ ID NOs 99, 101, 103, 105.

199. The incretin agent of claim, wherein the half-life extending moiety does not comprise an Fc domain, such as from a human IgG, optionally from a human IgG1, igG2, igG3, or IgG4.

200. The incretin agent of claim 178, wherein the half-life extending moiety comprises an Fc domain, such as from a human IgG, optionally from a human IgG1, igG2, igG3, or IgG4.

201. The incretin agent of claim 200, wherein the human IgG is human IgG4.

202. The incretin agent of claim 200 or 201, wherein the incretin agent comprises an IgG4 Fc domain fused to a unit comprising from N-terminus to C-terminus:

(ii) GIP receptor agonist-connecting peptides (e.g., SEQ ID NOS: 92, 93), or

203. The incretin agent of any one of claims 200-202, wherein the IgG4 Fc domain comprises an amino acid sequence having at least 90%, 95% or 99% identity to SEQ ID NO 155.

204. The incretin agent of claim 203, wherein the IgG4 Fc domain comprises an amino acid sequence as set forth in SEQ ID No. 155.

205. The incretin agent of any one of claims 200-204, wherein the incretin agent comprises an amino acid sequence set forth in any one of SEQ ID NOs 10, 89-97.

206. The incretin agent of any one of claims 200-205, wherein the Fc domain comprises one or more mutations in one or both Fc constant domains that increase half-life and/or induce dimerization of the incretin agent.

207. The incretin agent of claim 206, wherein the one or more mutations comprise one or more mutations in a CH3 domain.

208. The incretin agent of claim 206 or 207, wherein the one or more mutations that induce dimerization comprise:

(i) Y349C, T366S, L368A and/or Y407V (according to EU numbering) or

(Ii) S354C and/or T366W (according to EU numbering).

209. The incretin agent of any of claims 206-208, wherein the one or more mutations comprise Y349C, T366S, L a and Y407V ("FcKIH-b", according to EU numbering), or S354C and T366W ("FcKIH-a", according to EU numbering).

210. The incretin agent of any one of claims 206-209, wherein the incretin agent comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an incretin peptide fused to a first Fc domain, wherein the first Fc domain comprises mutations Y349C, T a S, L368A and Y407V ("FcKIH-b", according to EU numbering), and wherein the second polypeptide chain comprises an incretin peptide fused to a second Fc domain, wherein the second Fc domain comprises mutations S354C and T366W ("FcKIH-a", according to EU numbering).

211. The incretin agent of any one of claims 206-210, wherein the one or more mutations that increase half-life of the incretin agent comprise M428L and N434S ("LS", numbering according to EU).

212. The incretin agent of any one of claims 206-211, wherein the incretin agent comprises an Fc domain having a FcKIH-a mutation on a first polypeptide chain and an Fc domain having a FcKIH-b mutation on a second polypeptide chain, wherein the Fc domain on each polypeptide chain is independently fused to one or more units comprising from N-terminus to C-terminus:

(Ii) GIP receptor agonist-connecting peptide (e.g., SEQ ID NO: 88).

213. The incretin agent of any one of claims 206-212, wherein the incretin agent comprises an amino acid sequence set forth in any one of SEQ ID NOs 84-88.

214. The incretin agent of any one of claims 206-213, wherein the Fc domain comprises one or more mutations that abrogate the effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q).

215. The composition of claim 214, wherein one or more mutations that abrogate effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q) comprises the following mutations L234S, L T and G236R ("STR", numbering according to EU).

216. The composition of claim 215, wherein the one or more mutations that abrogate effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q) comprise mutations L234A and L235A ("LALA", numbering according to EU).

217. The composition of claim 216, wherein the one or more mutations that abrogate effector activity of the Fc domain (e.g., binding to fcγ receptor or C1 q) comprises a mutation of L234A/L235A/P329G ("LALAPG", numbering according to EU).

218. The incretin agent of claim 178, wherein the half-life extending moiety comprises a VNAR that binds albumin.

219. The incretin agent of claim 178, wherein the half-life extending moiety comprises an XTEN sequence.

220. An incretin agent comprising:

husec signal peptide;

an incretin peptide comprising a GLP1 incretin peptide or a fragment or mutant thereof;

Wherein the GLP1 incretin peptide comprises an amino acid sequence having an A8G substitution mutation compared to the wild-type GLP1 amino acid sequence.

221. A polynucleic acid (polynucleic acid), encoding an incretin agent according to claim 220.

222. An incretin agent comprising:

husec signal peptide;

an incretin peptide comprising a GIP incretin peptide or a fragment or mutant thereof;

wherein the GIP incretin peptide comprises an amino acid sequence having an A2G substitution mutation as compared to the wild-type GIP amino acid sequence.

223. A polynucleic acid (polynucleic acid), encoding an incretin agent according to claim 222.