RNA compositions for delivery of incretin agents

Abstract

The present disclosure provides compositions (e.g., pharmaceutical compositions) for delivery of incretin agents and related technologies (e.g., components thereof and / or methods relating thereto). Among other things, the present disclosure provides treatment methods using polyribonucleotides encoding incretin agents for various diseases.

Description

Attorney Docket No.: 2013237-0773 RNA COMPOSITIONS FOR DELIVERY OF INCRETIN AGENTS BACKGROUND

[0001] Obesity is the most prevalent chronic disease worldwide, affecting approximately 650 million adults today. Obesity is considered a starting point for and critical contributor to pre-diabetes, type 2 diabetes (T2D, with its complications), non- alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular and renal diseases, and premature mortality. It is estimated that the number of obese (BMI>30kg / m2) people will exceed one billion by 2030, about 10% of which will suffer from severe class III obesity (BMI>40kg / m2). Half of all men living with obesity live in just nine countries: USA, China, India, Brazil, Mexico, Russia, Egypt, Germany and Turkey. In addition, childhood obesity is sharply on the rise worldwide. T2D, NAFLD, NASH, cardiovascular and renal diseases are also prevalent independent of obesity. There exists a need to develop further therapies for treatment and / or prevention of obesity and other related diseases. SUMMARY

[0002] Treatment of obesity and T2D with incretins and incretin mimetics, in particular GLP1 and GIP receptor agonists or combinations thereof has shown tremendous benefits for people living with the diseases. Due to production and cost limitations, access to this new class of active agents is limited, depriving millions of people of the necessary treatment. The present disclosure recognizes these shortcomings and provides a new therapeutic modality to deliver incretin mimetics by using polyribonucleotide precursors of said incretins, obviating the need of producing the incretins themselves. The present disclosure provides, among other things, polyribonucleotide precursors of said incretins as molecular entities, their production, formulation, and administration to treat obesity and its sequelae, including T2D, early T1D, cardiovascular diseases, renal diseases, NASH and NAFLD. The present disclosure also provides methods where these agents are used to treat diseases including T2D, early T1D, cardiovascular diseases, renal diseases, NASH and NAFLD independent of obesity. The present disclosure also provides methods where these agents are used to treat sequelae of NASH, including liver fibrosis and cirrhosis.Attorney Docket No.: 2013237-0773

[0003] The present disclosure also recognizes that such a treatment approach, i.e., delivery of polyribonucleotide precursors of incretins presents additional benefits over current treatments, including but not limited to, broader accessibility for obese people who will otherwise have no access to current products due to limited supply, high price, lack of health insurance, formulations that require lower injection volume compared to marketed peptide-based products, a lower rate of treatment discontinuation by patients due to factors such as gastrointestinal side-effects, and improved properties such as an improved pharmacokinetic profile. In some embodiments, the improved pharmacokinetic profile has the advantage of lower administration frequency by virtue of a longer-acting therapeutic.

[0004] 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 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 triple receptor agonist. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NOs: 5-7. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NOs: 8-9. In some embodiments, the incretin agent comprises an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NOs: 11-15.

[0005] In some embodiments, the incretin agent comprises an incretin peptide fused to a signal peptide, optionally via the N-terminus of the incretin peptide, optionally via a linker. In some embodiments, the signal peptide has an amino acid sequence according to any one of SEQ ID NOs: 16-39. In some embodiments, the signal peptide has an amino acid sequence according to any one of SEQ ID NOs: 16-21. In some embodiments, the signal peptide has an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the incretin agent comprises an incretin peptide fused to a signal peptide that comprises an amino acid sequence according to any one of SEQ ID NOs: 41-45.

[0006] In some embodiments, the incretin agent comprises an incretin peptide fused to a half-life extending moiety, optionally via the C-terminus or N-terminus of the incretinAttorney Docket No.: 2013237-0773 peptide, optionally via a linker. In some embodiments, the half-life extending moiety comprises a peptide moiety that binds albumin. In some embodiments, the half-life extending moiety comprises an albumin binding domain (ABD) derived from protein G of Streptococcus strain GI48 and / or from protein PAB of Finegoldia magna, such as ABD035 and SA21. In some embodiments, the half-life extending moiety comprises an albumin binding domain (ABD) that 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 half-life extending moiety comprises ABDCon. In some embodiments, the half-life extending moiety comprises an albumin binding domain (ABD) derived from the bacterial protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus, such as M11.12 and M18.2.5. In some embodiments, the half-life extending moiety comprises a DARPin that binds albumin. In some embodiments, the half-life extending moiety comprises an immunoglobulin domain or fragment thereof. In some embodiments, the half-life extending moiety comprises a fully human domain antibody (dAb) that binds albumin, such as AlbudAb. In some embodiments, the half-life extending moiety comprises a Fab that binds albumin, such as dsFv CA645. In some embodiments, the half-life extending moiety comprises a 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 does not comprise a 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 a heavy chain only (VHH) antibody, such as a nanobody, that binds albumin. 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 incretin peptide is fused to the half-life extending moiety via a linker. In some embodiments, the linker is fused to the C-terminus of the incretin peptide and the N-terminus of the half-life extending moiety. In some embodiments, the linker is fused to the C-terminus of the half- life extending moiety and the N-terminus of the incretin peptide. In some embodiments, the linker has an amino acid sequence of any one of SEQ ID NOs: 1-5.

[0007] In some embodiments, the polyribonucleotide comprises at least one non- coding sequence element that enhances RNA stability and / or translation efficiency. In some embodiments, the at least one non-coding sequence element comprises a 5’ cap structure, a 5’ UTR, a 3’ UTR, and / or a polyA tail. In some embodiments, the polyribonucleotideAttorney Docket No.: 2013237-0773 comprises, in a 5’ to 3’ direction: a 5’ UTR; a signal peptide-coding sequence; an incretin peptide-coding sequence; a 3’ UTR; and a polyA tail. In some embodiments, the polyribonucleotide comprises, in a 5’ to 3’ direction: a 5’ UTR; a signal peptide-coding sequence; an incretin peptide-coding sequence; a linker-coding sequence; a half-life extending moiety-coding sequence; a 3’ UTR; and a polyA tail. In some embodiments, the polyribonucleotide comprises, in a 5’ to 3’ direction: a 5’ UTR; a signal peptide-coding sequence; a half-life extending moiety-coding sequence; a linker-coding sequence; an incretin peptide-coding sequence; a 3’ UTR; and a polyA tail.

[0008] In some embodiments, the incretin peptide is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence, wherein the codon-optimization and / or the increase in the G / C content does not change the sequence of the encoded amino acid sequence.

[0009] In some embodiments, the polyribonucleotide comprises at least one modified ribonucleotide. In some embodiments, the polyribonucleotide comprises a modified nucleoside in place of uridine. In some embodiments, the polyribonucleotide comprises a modified nucleoside in place of each uridine. In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside is N1-methyl- pseudouridine (m1ψ).

[0010] In some embodiments, the polyribonucleotide comprises a 5’ cap structure.

[0011] In some embodiments, polyribonucleotide comprises a 5' UTR.

[0012] In some embodiments, the polyribonucleotide comprises a 3' UTR.

[0013] In some embodiments, the polyribonucleotide comprises a polyA tail. In some embodiments, polyA tail comprises at least 100 nucleotides.

[0014] In some embodiments, the polyribonucleotide is mRNA.

[0015] In some embodiments, the polyribonucleotide is formulated as a liquid, formulated as a solid, or a combination thereof.Attorney Docket No.: 2013237-0773

[0016] In some embodiments, the polyribonucleotide is formulated for injection. In some embodiments, the polyribonucleotide is formulated for intraperitoneal or intravenous administration. In some embodiments, the polyribonucleotide is formulated or is to be formulated as lipid particles. In some embodiments, the polyribonucleotide is formulated or is to be formulated as lipid nanoparticles. In some embodiments, the polyribonucleotide is encapsulated within the lipid nanoparticles. In some embodiments, the lipid nanoparticles are pancreas-targeting and / or gut-targeting lipid nanoparticles. In some embodiments, the lipid nanoparticles are cationic lipid nanoparticles. In some embodiments, lipids that form the lipid nanoparticles comprise: a polymer-conjugated lipid; a cationic lipid; and a neutral lipid. In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid. In some embodiments, the cationic lipid is an ionizable lipid-like material (lipidoid). In some embodiments, the cationic lipid has one of the following structures:Attorney Docket No.: 2013237-0773

[0017] In some embodiments, the neutral lipid comprises a helper lipid such as 1,2- distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol.

[0018] In some embodiments, the cationic lipid is selected from cationic lipid 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-conjugated lipid is C14-PEG2000.

[0019] In some embodiments, the lipid nanoparticles comprise: i) about 30 mol% to about 50 mol% of a cationic lipid; ii) about 1 mol% to 5 mol% of a PEG-conjugated lipid; iii) about 30 mol% to about 50 mol% of a helper lipid; and iv) about 20 mol% to about 40 mol% of cholesterol.

[0020] In some embodiments, the lipid nanoparticles comprise about 35 mol% of a cationic lipid; about 40 mol% of a helper lipid, about 22.5 mol% of cholesterol, and about 2.5 mol% of a PEG-conjugated lipid.

[0021] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-2, X-3, or X-4, about 40 mol% of DOTAP, DOPE, or PS, about 22.5 mol% of cholesterol, and about 2.5 mol% of C14-PEG2000.

[0022] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-2; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol, and about 2.5 mol% of C14-PEG2000.

[0023] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-3; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.Attorney Docket No.: 2013237-0773

[0024] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-4; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

[0025] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-2; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

[0026] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-3; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

[0027] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-4; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

[0028] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-2; about 40 mol% of PS; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

[0029] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-3; about 40 mol% of PS; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

[0030] In some embodiments, the lipid nanoparticles comprise about 35 mol% of cationic lipid X-4; about 40 mol% of PS; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

[0031] In some embodiments, the lipid nanoparticles are formulated for intraperitoneal (i.p.) delivery.

[0032] In some embodiments, the lipid nanoparticles have an average size of about 50-150 nm.

[0033] In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.Attorney Docket No.: 2013237-0773

[0034] In some embodiments, the composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is sucrose.

[0035] In some embodiments, the composition further comprises an aqueous buffered solution. In some embodiments, the aqueous buffered solution includes sodium ions.

[0036] In one aspect, the present disclosure provides a method of treating a disease- state in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of any one of the aforementioned compositions. In one aspect, the present disclosure provides uses of any one of the aforementioned compositions for the treatment of a disease-state in a subject in need thereof.

[0037] 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 type 1 diabetes (T1D), non-alcoholic fatty liver disease (NAFLD), non- alcoholic steatohepatitis (NASH), a cardiovascular (CV) disease, a renal disease, or elevated risk of premature mortality. In some embodiments, 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). In some embodiments, the method improves weight management in said subject. In some embodiments, the method reduces weight gain or induces weight loss in said subject.

[0038] In some embodiments, the disease-state is diabetes. In some embodiments, the method improves glycemic control in said subject. In some embodiments, the method lowers HbA1c in said subject. In some embodiments, the diabetes is pre-diabetes, type 2 diabetes (T2D), or early type 1 diabetes (T1D).

[0039] In some embodiments, the disease-state is a cardiovascular (CV) disease. In some embodiments, the cardiovascular 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). In some embodiments, the method improves a subject’s blood pressure and / or blood lipids in said subject.

[0040] In some embodiments, the disease-state is a renal disease.Attorney Docket No.: 2013237-0773

[0041] In some embodiments, the disease-state is non-alcoholic fatty liver disease (NAFLD).

[0042] In some embodiments, the disease-state is non-alcoholic steatohepatitis (NASH) and optionally its sequelae, liver fibrosis and cirrhosis.

[0043] In some embodiments, administering the composition to the subject comprises administering one or more doses of the composition to the subject. In some embodiments, the one or more doses of the composition are administered to the subject daily, every other day or once a week. In some embodiments, the one or more doses of the composition are administered to the subject less frequently than once a week. In some embodiments, the one or more doses of the composition are administered to the subject once every 2, 3 or 4 weeks.

[0044] In some embodiments, the composition is administered via 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).

[0045] In some embodiments, administration of the composition results in expression of the incretin agent in the subject.

[0046] In some embodiments, the composition is administered in a volume that is less than 0.5 mL.

[0047] In one aspect, the present disclosure provides a method of producing an incretin agent comprising administering to cells any one of the aforementioned compositions so that the cells express and secrete the incretin agent.

[0048] In one aspect, the present disclosure provides an incretin agent that comprises an incretin peptide fused to a signal peptide. In some embodiments, the incretin agent is a GLP1 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 triple receptor agonist. In some embodiments, the incretin agent comprises an incretin peptide having anAttorney Docket No.: 2013237-0773 amino acid sequence according to any one of SEQ ID NOs: 5-7. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NOs: 8-9. In some embodiments, the incretin agent comprises an amino acid sequence according to SEQ ID NO: 10. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NOs: 11-15.

[0049] In some embodiments, the incretin agent comprises an incretin peptide fused to a signal peptide, optionally via the N-terminus of the incretin peptide, optionally via a linker. In some embodiments, the signal peptide has an amino acid sequence according to any one of SEQ ID NOs: 16-39. In some embodiments, the signal peptide has an amino acid sequence according to any one of SEQ ID NOs: 16-21. In some embodiments, the signal peptide has an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the incretin agent comprises an incretin peptide fused to a signal peptide that comprises an amino acid sequence according to any one of SEQ ID NOs: 41-45.

[0050] In some embodiments, the incretin agent comprises an incretin peptide fused to a half-life extending moiety, optionally via the C-terminus or N-terminus of the incretin peptide, optionally via a linker. In some embodiments, the half-life extending moiety comprises a peptide moiety that binds albumin. In some embodiments, the half-life extending moiety comprises an albumin binding domain (ABD) derived from protein G of Streptococcus strain GI48 and / or from protein PAB of Finegoldia magna, such as ABD035 and SA21. In some embodiments, the half-life extending moiety comprises an albumin binding domain (ABD) that 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 half-life extending moiety comprises ABDCon. In some embodiments, the half-life extending moiety comprises an albumin binding domain (ABD) derived from the bacterial protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus, such as M11.12 and M18.2.5. In some embodiments, the half-life extending moiety comprises a DARPin that binds albumin. In some embodiments, the half-life extending moiety comprises an immunoglobulin domain or fragment thereof. In some embodiments, the half-life extending moiety comprises a fully human domain antibody (dAb) that binds albumin, such as AlbudAb. In some embodiments, the half-life extending moiety comprises a Fab that binds albumin, such as dsFv CA645. In some embodiments, the half-life extending moietyAttorney Docket No.: 2013237-0773 comprises a 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 does not comprise a 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 a heavy chain only (VHH) antibody, such as a nanobody, that binds albumin. 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 incretin peptide is fused to the half-life extending moiety via a linker. In some embodiments, the linker is fused to the C-terminus of the incretin peptide and the N-terminus of the half-life extending moiety. In some embodiments, the linker is fused to the C-terminus of the half- life extending moiety and the N-terminus of the incretin peptide. In some embodiments, the linker has an amino acid sequence of any one of SEQ ID NOs: 1-5. BRIEF DESCRIPTION OF THE DRAWING

[0051] Figure 1 shows an exemplary therapeutic strategy utilizing polyribonucleotides as described herein for delivery and in vivo expression of incretin agents.

[0052] Figure 2 shows a schematic of an exemplary polyribonucleotide that encodes an incretin agent. DEFINITIONS

[0053] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that 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 referred value.

[0054] Agent: As used herein, the term “agent,” may refer to a physical entity. In some embodiments, an agent may be characterized by a particular feature and / or effect. For example, as used herein, the term “therapeutic agent” refers to a physical entity has aAttorney Docket No.: 2013237-0773 therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.

[0055] Aliphatic: The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C1-6). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6 alkyl.

[0056] 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., C1-12, C1-10, C1-8, C1-6, C1-4, C1-3, or C1-2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0057] Alkylene: The term “alkylene” is refers to a bivalent alkyl group. In some embodiments, “alkylene” is a bivalent straight or branched alkyl group. In some embodiments, an “alkylene chain” is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, e.g., from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include thoseAttorney Docket No.: 2013237-0773 described in the specification herein. It will be appreciated that two substituents of the alkylene group may be taken together to form a ring system. In certain embodiments, two substituents can be taken together to form a 3- to 7-membered ring. The substituents can be on the same or different atoms. The suffix “-ene” or “-enyl” when appended to certain groups herein are intended to refer to a bifunctional moiety of said group. For example, “- ene” or “-enyl”, when appended to “cyclopropyl” becomes “cyclopropylene” or “cyclopropylenyl” and is intended to refer to a bifunctional cyclopropyl group, .

[0058] Alkenyl: The term “alkenyl”, used alone or as part of a largerto an optionally substituted straight or branched chain 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., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). 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 about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0059] Alkynyl: 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., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0060] Amino acid: In its broadest sense, as used herein, the term “amino acid” refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(R)–COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an 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. “Nonstandard amino acid” refers to any amino acid, other than the standard aminoAttorney Docket No.: 2013237-0773 acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0061] Aryl: The term “aryl” refers to monocyclic and bicyclic ring 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 between six and twelve total 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 which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons. In some embodiments, an “aryl” ring system is an aromatic ring (e.g., phenyl) that is fused to a non- aromatic ring (e.g., cycloalkyl). Examples of aryl rings include that are fused include .events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to,Attorney Docket No.: 2013237-0773 severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0063] Co-administration: As used herein, the term “co-administration” refers to use of a composition (e.g., a pharmaceutical composition) described herein and one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents comprises at least one polyribonucleotide encoding another therapeutic agent (e.g., an incretin agent). The combined use of a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order). In some embodiments, a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent may be combined in one pharmaceutically-acceptable excipient, or they may be placed in separate excipient and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of “co-administration” or “combination,” provided that a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.

[0064] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents (e.g., two or more incretin agents)). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) inAttorney Docket No.: 2013237-0773 the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition. In some embodiments, a combination therapy comprises polyribonucleotides encoding two or more incretin agents.

[0065] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0066] Corresponding to: As used herein, the term “corresponding to” 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 element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric 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, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinaryAttorney Docket No.: 2013237-0773 skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, 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 utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but 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, in some embodiments, that it plays an analogous role or performs an analogous function and / or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

[0067] Cycloaliphatic: As used herein, the term “cycloaliphatic” refers to a monocyclic C3-8 hydrocarbon or a bicyclic C6-10 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.

[0068] Cycloalkyl: As used herein, the term “cycloalkyl” refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0069] Derived: In the context of an amino acid sequence (peptide or polypeptide) “derived from” a designated amino acid sequence (peptide or polypeptide), refers to a structural analogue of a designated amino acid sequence. In some embodiments, an amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, incretinAttorney Docket No.: 2013237-0773 agents utilized according to the present disclosure may include amino acid sequences derived from two or more incretin agents, e.g., two or more naturally produced incretins.

[0070] Detecting: The term “detecting” is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest or any form of measurement of an entity of interest in a sample. Thus, “detecting” may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of an entity of interest. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term “quantifying” when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification. Absolute quantification may be accomplished by correlating a detected level of an entity of interest to known control standards (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different entities of interest to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.

[0071] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” (or “therapeutic regimen”) may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.

[0072] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to aAttorney Docket No.: 2013237-0773 coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0073] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.

[0074] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0075] Heteroaliphatic: The term “heteroaliphatic” or “heteroaliphatic group,” as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight–chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, 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 quaternized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1–10 carbon atoms wherein 1–3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1–4 carbon atoms, wherein 1–2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet otherAttorney Docket No.: 2013237-0773 embodiments, heteroaliphatic groups contain 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, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O- CH2-CH2-O-CH2-CH2-O-CH3, and the like.

[0076] Heteroaryl: The terms “heteroaryl” and “heteroar–”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups 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 π-electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, 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]pyridyl, imidazo[4,5-b]pyridyl, imidazo[4,5- c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar–”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, 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 benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

[0077] Heteroatom: The term “heteroatom” as used herein refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.Attorney Docket No.: 2013237-0773

[0078] Heterocycle: As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10-membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0–3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4- dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+(as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3- dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.

[0079] 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 to be “homologous” to one another if their sequences 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 to be “homologous” toAttorney Docket No.: 2013237-0773 one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “non- polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0080] Identity: As used herein, the term “identity” 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 between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence 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 a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. 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, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty ofAttorney Docket No.: 2013237-0773 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0081] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be “increased” relative to that obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or 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 subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by 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 higher, as compared to a comparable reference.

[0082] In order: As used herein with reference to a polynucleotide or polyribonucleotide, “in order” refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with reference to a polypeptide, “in order” refers to the order of features moving from the N-terminal-most of the features to the C-terminal-most of the features along the polypeptide. “In order” does not mean that no additional features can be present among the listed features. For example, if Features A, B,Attorney Docket No.: 2013237-0773 and C of a polynucleotide are described herein as being “in order, Feature A, Feature B, and Feature C,” this description does not exclude, e.g., Feature D being located between Features A and B.

[0083] Ionizable: The term “ionizable” refers to a compound or group or atom that is charged at a certain pH. In the context of an ionizable amino lipid, such a lipid or a function group or atom thereof bears a positive charge at a certain pH. In some embodiments, an ionizable amino lipid is positively charged at an acidic pH. In some embodiments, an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some embodiments about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, an ionizable amino lipid may have a pKa within a range of about 5 to about 7.

[0084] Isolated: The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0085] Lipid: As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.

[0086] RNA lipid nanoparticle: As used herein, the term “RNA lipid nanoparticle” refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size (e.g., Z-average) 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, RNA lipidAttorney Docket No.: 2013237-0773 nanoparticles can 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 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.

[0087] Neutralization: As used herein, the term “neutralization” refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term “neutralization” refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells.

[0088] Nucleic acid / Polynucleotide: As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-Attorney Docket No.: 2013237-0773 fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5- methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro), reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a 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 long.

[0089] Pharmaceutically effective amount: The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease (e.g., obesity), a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., obesity). In some embodiments, such inhibition may comprise slowing down the progress of a disease (e.g., obesity) and / or interrupting or reversing the progress of the disease (e.g., obesity). In some embodiments, a desired reaction in a treatment of a disease (e.g., obesity) may be or comprise delay or prevention of the onset of a disease (e.g., obesity) or a condition (e.g., a condition associated with obesity). An effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on disease (e.g., obesity) or a condition (e.g., a condition associated with obesity) to be treated, the severity of such a disease (e.g., obesity) or a condition (e.g., a condition associated with obesity), individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, theAttorney Docket No.: 2013237-0773 type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of a composition (e.g., a pharmaceutical composition) described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0090] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequenceAttorney Docket No.: 2013237-0773 motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch 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, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0091] Prevent: As used herein, the term “prevent” 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 to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0092] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.Attorney Docket No.: 2013237-0773

[0093] Ribonucleic acid (RNA) or Polyribonucleotide: As used herein, the term “ribonucleic acid,” “RNA,” or “polyribonucleotide” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is a mRNA. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 3' end a poly(A) region. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0094] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses 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 may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0095] Risk: As will be understood from context, “risk” of a disease, disorder, and / or condition refers to a 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, risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group ofAttorney Docket No.: 2013237-0773 reference samples have a known risk of a disease, disorder, condition and / or event. In some embodiments, a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and / or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes.

[0096] Specific: The term “specific,” when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.

[0097] Substituted or optionally substituted: As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g., refers groupAttorney Docket No.: 2013237-0773 may have a suitable substituent 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 a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes provided herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.

[0098] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; –(CH2)0–4R°; –(CH2)0–4OR°; - O(CH2)0-4Ro, –O–(CH2)0–4C(O)OR°; –(CH2)0–4CH(OR°)2; –(CH2)0–4SR°; –(CH2)0–4Ph, which may be substituted with R°; –(CH2)0–4O(CH2)0–1Ph which may be substituted with R°; –CH=CHPh, which may be substituted with R°; –(CH2)0–4O(CH2)0–1-pyridyl which may be substituted with R°; –NO2; –CN; –N3; -(CH2)0–4N(R°)2; –(CH2)0–4N(R°)C(O)R°; – N(R°)C(S)R°; –(CH2)0–4N(R°)C(O)NR°2; -N(R°)C(S)NR°2; –(CH2)0–4N(R°)C(O)OR°; - N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; –(CH2)0–4C(O)R°; C(S)R°; –(CH2)0–4C(O)OR°; –(CH2)0–4C(O)SR°; -(CH2)0–4C(O)OSiR°3; –(CH2)0–4OC(O)R°; –OC(O)(CH2)0–4SR°; –(CH2)0–4SC(O)R°; –(CH2)0–4C(O)NR°2; –C(S)NR°2; –C(S)SR°; – SC(S)SR°, -(CH2)0–4OC(O)NR°2; -C(O)N(OR°)R°; –C(O)C(O)R°; –C(O)CH2C(O)R°; – C(NOR°)R°; -(CH2)0–4SSR°; –(CH2)0–4S(O)2R°; –(CH2)0–4S(O)2OR°; –(CH2)0–4OS(O)2R°; – S(O)2NR°2; -(CH2)0–4S(O)R°; -N(R°)S(O)2NR°2; –N(R°)S(O)2R°; –N(OR°)R°; – C(NH)NR°2; –P(O)2R°; -P(O)R°2; -OP(O)R°2; –OP(O)(OR°)2; SiR°3; –(C1–4 straight or branched alkylene)O–N(R°)2; or –(C1–4 straight or branched alkylene)C(O)O–N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, C1– 6 aliphatic, –CH2Ph, –O(CH2)0–1Ph, -CH2-(5- to 6-membered heteroaryl ring), or a 3- to 6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3- to 12-membered saturated, partially unsaturated, or aryl mono– or bicyclic ringAttorney Docket No.: 2013237-0773 having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0099] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, –(CH2)0–2Rl, –(haloRl), –(CH2)0–2OH, –(CH2)0–2ORl, –(CH2)0–2CH 2, -O , –CN, –N3, – 0–2C Rl, – 0–2C OH, – 0–2C ORl, –or one or more and is independently selected from C1–4aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 3- to 6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =O and =S.

[0100] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =O (“oxo”), =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))2–3S–, wherein each independent occurrence of R*is selected from hydrogen, C1–6aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: –O(CR*2)2–3O–, wherein each independent occurrence of R*is selected from hydrogen, C1–6aliphatic which may be substituted as defined below, or an unsubstituted 5–6–membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0101] Suitable substituents on the aliphatic group of R*include halogen, – Rl, -(haloRl), -OH, –ORl, –O(haloRl), –CN, –C(O)OH, –C(O)ORl, –NH2, –NHRl, –NRl2, or –NO2, wherein each Rlis unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1–4 aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.Attorney Docket No.: 2013237-0773

[0102] Suitable substituents on a 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, C1–6aliphatic which may be substituted as defined below, unsubstituted –OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0103] Suitable substituents on the aliphatic group of R†are independently halogen, –Rl, -(haloRl), –OH, –ORl, –O(haloRl), –CN, –C(O)OH, –C(O)ORl, –NH2, –NHRl, –NRl2, or -NO2, wherein each Rlis unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1–4aliphatic, –CH2Ph, –O(CH2)0–1Ph, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0104] Subject: As used herein, the term “subject” refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, a subject is aAttorney Docket No.: 2013237-0773 patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0105] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.) has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0106] Susceptible to: An individual who is “susceptible to” a disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.) is one who has a higher risk of developing the disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.) than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition (e.g., obesity, a condition associated with obesity, etc.) may not have been diagnosed with the disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.) may exhibit symptoms of the disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.) may not exhibit symptoms of the disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.) will develop the disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.). In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.) will not develop the disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.).

[0107] Therapy: The term “therapy” refers to an 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., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., obesity, a conditionAttorney Docket No.: 2013237-0773 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, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0108] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.). Treatment may be administered to a subject who does not exhibit 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 a subject who exhibits only early signs of the disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition (e.g., obesity, a condition associated with obesity, etc.).

[0109] Compounds of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0110] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. ForAttorney Docket No.: 2013237-0773 example, in some cases, provided compounds show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and / or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.

[0111] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS A. Incretins and Their Use in Treatment of Disease

[0112] Incretins are peptide hormones that are released in the gastrointestinal (GI) tract in response to glucose consumption, which stimulates insulin secretion by the pancreas and decreases glucagon production, lowering blood sugar levels. Incretins exert their effects by binding to their respective receptors on pancreatic beta cells, leading to insulin release. Glucagon-like peptide-1 (GLP1) and glucose-dependent insulinotropic polypeptide (GIP) are two incretins that have been identified for their role in postprandial insulin secretion. GIP is largely responsible for insulin release in response to glucose intake. GLP1 stimulates satiety, slows gastric emptying, lowers glucagon secretion, and decreases food intake, resulting in weight loss. Agonizing GIP and GLP1 receptors has been shown to produce an additive effect when insulin is secreted. See Chim, US Pharm.2022; 47(10):18-22, which is herein incorporated by reference in its entirety.

[0113] Because of their role in controlling blood glucose, satiety, etc., incretins and incretin mimetics have the potential to treat various diseases including obesity, pre-diabetes, type 2 diabetes (T2D, with its complications), early 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 a major cardiovascular event (MACE), including CV death, non-fatal myocardial infarction, non- fatal stroke, or heart failure with preserved ejection fraction (HFpEF)), renal disease, andAttorney Docket No.: 2013237-0773 elevated risk of premature mortality. Such chronic diseases are prevalent worldwide and often present as co-morbidities. 1. Obesity

[0114] Obesity is the most prevalent chronic disease worldwide, affecting approximately 650 million adults. Obesity is considered a starting point and critical contributor to pre-diabetes, type 2 diabetes (T2D, with its complications), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular and renal disease, and premature mortality. Obesity imposes a considerable economic burden with additional direct medical costs, productivity costs (absenteeism, presenteeism, disability support, premature mortality), transportation costs (including increased CO2 footprint), human capital accumulation costs (school absenteeism, highest educational degree achieved). It is estimated that the number of obese (BMI>30kg / m2) people will exceed one billion by 2030, about 10% of which will suffer from severe class III obesity (BMI>40kg / m2). Half of all men living with obesity live in just nine countries: USA, China, India, Brazil, Mexico, Russia, Egypt, Germany and Turkey. Childhood obesity is also sharply on the rise worldwide.

[0115] Obesity was declared a disease by the American Association of Clinical Endocrinologists (AACE) only in 2011, and is managed based on severity, starting with lifestyle / behavioral intervention, and increased physical activity, then pharmacotherapy, and finally bariatric surgery.

[0116] Based on short-term studies, weight loss is recommended for overweight or obese patients with T2D. The Look AHEAD study investigating long-term effects of weight loss on cardiovascular disease in more than 5100 people with T2D was halted for futility after almost 10 years in 2012, as it was shown that intensive lifestyle intervention focusing on weight loss did not reduce the rate of cardiovascular events in overweight or obese adults with T2D.

[0117] Overall, the success of pharmacotherapy for obesity has been limited so far, with only modest placebo-corrected weight loss, e.g., 3% for Xenical® (orlistat), 4-5% for Belviq® (lorcaserin) and Contrave® (naltrexone SR / bupropion SR), paired with sociallyAttorney Docket No.: 2013237-0773 constraining side-effects for Xenical®, and undesirable central nervous effects of the centrally acting agents. Acomplia® (rimonabant) was rejected by FDA due to concerns that using it could increase suicidal thoughts and depression.

[0118] The most effective intervention for obesity remains bariatric surgery, however, only 1% of eligible patients undergo the procedure due to perceived severe complications, including mortality. Bariatric surgery has shown to substantially alter the release of endocrine hormones, which sparked an interest in targeting the pathways of endogenous nutrient-stimulated hormones, essentially mimicking the effect of bariatric surgery with a chemical agent, coined “incretin mimetics”. 2. Existing treatments using Incretin Mimetics

[0119] Current therapies using incretin mimetics include Glucagon-like peptide-1 (GLP1) receptor agonists like Trulicity® (dulaglutide), Byetta® (exenatide), Ozempic® / Rybelsus® (semaglutide injectable / oral), Victoza® (liraglutide) and Suliqua® (lixisenatide, only in combination with insulin glargine), which are approved for lowering blood sugar in people living with T2D without the need to continuously check blood sugar levels. An added benefit was the loss of body weight (2-4%) and positive effects on cardiovascular and renal parameters. Recent research combined the activity of GLP1 receptor agonists with GIP receptor agonists and / or glucagon (GCG) receptor agonists (dual / triple agonists), aiming at even better control of blood sugar and greater weight loss. Blood sugar control and weight loss was demonstrated with the GLP1 / GCG receptor dual agonist SAR425899; however, the program was discontinued due to unacceptable gastrointestinal side effects in 2019. More recently, the GLP1 / GIP receptor dual agonist tirzepatide, now in the market as Mounjaro®, was approved as an injectable medicine for adults with T2D used along with diet and exercise to improve blood sugar. GIP’s function is to regulate energy balance through cell-surface receptor signaling in the brain and adipose tissue. The SURPASS-2 study demonstrated non-inferiority and superiority of tirzepatide against semaglutide in lowering blood sugar. Weight loss however was only a secondary endpoint.

[0120] Phase 3 trials have been performed with weight reduction as a primary endpoint in a population of non-diabetic, overweight / obese patients – see SCALEAttorney Docket No.: 2013237-0773 (liraglutide), STEP-1 (semaglutide) and SURMOUNT-1 (tirzepatide) trials. The SCALE trial demonstrated a 5.4% placebo-corrected weight loss and less progression to pre- diabetes, at a dose of 3 mg liraglutide once daily plus lifestyle intervention. The STEP-1 trial produced 12.4% weight loss in overweight or obese participants on 2.4 mg of semaglutide once weekly plus lifestyle intervention. The SURMOUNT-1 trial demonstrated 17.8% placebo-corrected weight loss on the highest dose of 15 mg tirzepatide once weekly. Cardiometabolic measures improved in all studies. Gastrointestinal side-effects were as expected, especially when starting the treatment, and were manageable. Approved obesity products now include Saxenda® (liraglutide 3 mg daily injection) and Wegovy® (semaglutide once-weekly injection). The FDA granted Fast Track designation for tirzepatide for the treatment of adults with obesity, or overweight with weight-related comorbidities in October 2022 which could lead to approval for this indication in early 2024, based on a rolling submission of further data from the SURMOUNT study series. Topline results were recently published from the SELECT trial, testing Wegovy® in patients with established cardiovascular disease and overweight / obesity but without T2D and found that semaglutide at 2.4 mg reduces the risk of major adverse cardiovascular events by 20% in overweight or obese adults. Another study using incretin mimetics is OASIS-1, a study in non-diabetic overweight / obese patients with oral semaglutide 25 or 50 mg. Oral semaglutide at a maintenance dose of 14 mg is already approved as Rybelsus® for T2D. Phase 2 results for retratutide, LY3437943, a triple receptor agonist (GIP / GLP1 / GCG receptors), were recently published showing 22.1% placebo-corrected weight-loss in people living with obesity after 48 weeks of treatment. Semaglutide was also recently shown to show a benefit in early T1D (within 3 months after diagnosis of T1D). In a small study, the need for prandial insulin was eliminated in all participants, and basal insulin in most.

[0121] Exemplary treatments of obesity / T2D using incretin mimetics are shown in Table 1 below. Table 1: Exemplary incretin mimetics for Treatment of Obesity / T2D Drug Name Targets Indication Trulicity® (dulaglutide) GLP1 receptor agonist T2D Byetta® (exenatide) GLP1 receptor agonist T2DAttorney Docket No.: 2013237-0773 Ozempic® (semaglutide) GLP1 receptor agonist T2D (injectable) Rybelsus® (semaglutide) GLP1 receptor agonist T2D (oral) Victoza (liraglutide) GLP1 receptor agonist T2D Saxenda® (liraglutide) GLP1 receptor agonist Obesity / weight loss Suliqua® (lixisenatide) GLP1 receptor agonist T2D Wegovy® (semaglutide) GLP1 receptor agonist Obesity Mounjaro® (tirzepatide) GLP1 / GIP dual receptor agonist T2D

[0122] The present disclosure recognizes, among other things, the current problems in the market of incretin mimetics for the treatment of obesity, pre-diabetes, T2D, early T1D, NAFLD, NASH, cardiovascular disease, renal disease, and elevated risk of premature mortality, including but not limited to, limited supply, high price, lack of health insurance covering such treatments, frequent injections (e.g., once weekly), high injection volumes, and gastrointestinal side effects.

[0123] The present disclosure provides, among other things, a more efficacious and cost effective way to use incretins and incretin mimetics for treatment of obesity, pre- diabetes, T2D, early T1D, NAFLD, NASH, cardiovascular disease, renal disease, and elevated risk of premature mortality through the delivery of incretins and incretin mimetics (collectively encompassed by the term “incretin agents”) encoded by one or more polyribonucleotides. In some embodiments, polyribonucleotides encoding incretin agents provide for a therapeutic treatment of obesity, and / or pre-diabetes, T2D, early T1D, NAFLD, NASH, cardiovascular disease, renal disease, and elevated risk of premature mortality (e.g., diseases related to obesity), which have improved properties compared to known incretin mimetic therapies, including the need for fewer injections (no more than once-weekly injection), lower injection volumes (e.g., no more than 0.5 ml), and fewer or less severe side effects. Additionally, polyribonucleotides for delivery of incretin agents provide for expression of incretin agents in a cell at therapeutically relevant levels, comparable to doses of current peptide-based therapies.Attorney Docket No.: 2013237-0773 B. Polyribonucleotides for Delivery of Incretin Agents

[0124] The present disclosure, among other things, utilizes RNA technologies as a modality to express incretin agents directly in a subject as a novel class of therapeutics that agonize GLP1, GIP, and / or GCG receptors to effectively treat a disease state such as obesity, pre-diabetes, T2D, early T1D, NAFLD, NASH, cardiovascular disease, renal disease, and / or elevated risk of premature mortality. In some embodiments, a polyribonucleotide as described herein encodes an incretin agent found in nature, or a fragment or variant thereof. In some embodiments, a polyribonucleotide as described herein encodes an incretin agent, or a fragment or variant thereof, that has been modified from its natural form.

[0125] As used herein, the term “incretin agents” refers to an agent that comprises an incretin or incretin mimetic. Exemplary incretins include GLP1, GIP, and GCG. Exemplary incretin mimetics are shown in, e.g., Table 1. In some embodiments, an incretin agent is a biologically active portion or fragment of an incretin peptide. In some embodiments, an incretin agent comprises an incretin peptide or incretin mimetic that is part of a fusion. For example, in some embodiments, an incretin agent is an incretin peptide or incretin mimetic that is fused to another peptide moiety (e.g., a half-life extending moiety).

[0126] In some embodiments, an incretin peptide or incretin mimetic is fused to another peptide (e.g., a half-life extending moiety) via a linker. In some embodiments, a linker contains at least one Gly (G) amino acid residue. Suitable linkers can be readily selected and can be of various lengths, such as from 1 amino acid (e.g., Gly) to 25 amino acids, from 2 amino acids to 15 amino acids, from 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 linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS: 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 linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly moreAttorney Docket No.: 2013237-0773 phi-psi space than even alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem.11:173-142 (1992)). In some embodiments, a linker comprises an amino acid sequence of SEQ ID NO: 3 (GGGGSGGGGSGGGGSGGGGS or “(G4S)4 linker) or SEQ ID NO: 4 (GGGGSGGGGSGGGGSGGGGSGGGGS or “(G4S)5 linker).

[0127] In some embodiments a half-life extending moiety binds albumin. Various albumin binding moieties (i.e., albumin binding protein domains) may be utilized as a half- life extending moiety for incretin agents described herein (see, e.g., Zorzi et al., MedChemComm, 2019, 10.7, 1068-1081, which is herein incorporated by reference in its entirety). In some embodiments, an albumin binding protein domain comprises an albumin- binding domain (ABD) derived from protein G of Streptococcus strain GI48 and / or from protein PAB of Finegoldia magna, such as ABD035 and SA21 (as described in Levy, et al., PLoS One, 2014, 9(2), e87704, which is herein incorporated by reference in its entirety) and ABD094 (NCT02690142) (as described in Frejd and Kim, Exp. Mol. Med., 2017, 49(3), e306, which is herein incorporated by reference in its entirety). In some embodiments, an ABD binds to domain II of human serum albumin and does not overlap or interfere with binding to the FcRn-binding site on albumin.

[0128] In some embodiments, an ABD comprises ABDCon, a three-helix bundle albumin-binding domain, as described in Jacobs et al., Protein Eng., Des. Sel., 2015, 28(10), 385–393, which is herein incorporated by reference in its entirety. In some embodiments, an ABD is derived from the bacterial protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus, e.g., 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 are herein incorporated by reference in their entirety). In some embodiments, an ABD comprises a DARPin, as described in Pluckthun, Annu. Rev. Pharmacol. Toxicol., 2015, 55, 489–511, which is herein incorporated by reference in its entirety.

[0129] In some embodiments, an ABD comprises an immunoglobulin domain or fragment thereof. In some embodiments, an ABD comprises a fully human domain antibody (dAb). For example, in some embodiments, an ABD comprises an AlbudAb, as described in Holt et al., Protein Eng., Des. Sel., 2008, 21(5), 283–288, which is herein incorporated by reference in its entirety. In some embodiments, an ABD comprises a Fab, e.g., dsFv CA645,Attorney Docket No.: 2013237-0773 as described in Adams et al., mAbs, 2016, 8(7), 1336–1346, which is herein incorporated by reference in its entirety. In some embodiments, am ABD is or comprises an Fc domain, e.g., of a human IgG (e.g., a human IgG1, IgG2, IgG3, or IgG4). In some embodiments, an ABD does not comprise an Fc domain, e.g., of a human IgG (e.g., a human IgG1, IgG2, IgG3, or IgG4).

[0130] In some embodiments, an ABD comprises a heavy chain only (VHH) antibody, i.e., nanobody, as described in Steeland et al., Drug Discovery Today, 2016, 21(7), 1076–1113, which is herein incorporated by reference in its entirety. In some embodiments, an ABD comprises a VNAR, as described in Muller et al., mAbs, 2012, 4(6), 673–685, which is herein incorporated by reference in its entirety.

[0131] In some embodiments, a half-life extending moiety is an XTEN sequence as described in Podust et al., Journal of Controlled Release, 2016, 240, 52-66, which is herein incorporated by reference in its entirety. Other albumin-binding domains are known in the art, see e.g., Zorzi et al., MedChemComm, 2019, 10.7, 1068-1081, which is herein incorporated by reference in its entirety.

[0132] In some embodiments, an incretin agent comprises a GLP1 receptor agonist such as GLP1. In some embodiments, an incretin agent comprises a GIP receptor agonist such as GIP. In some embodiments, an incretin agent comprises a dual GIP and GLP1 receptor agonist. In some embodiments, an incretin agent comprises a triple GIP, GLP1 and GCG receptor agonist.

[0133] In some embodiments, an incretin agent comprises a wild type (i.e., unmutated) incretin peptide sequence, or fragment thereof. For example, in some embodiments, an incretin agent comprises any one of the incretin peptides as represented in SEQ ID NOs: 5-15.

[0134] In some embodiments, an incretin agent comprises an incretin peptide, or fragment thereof and comprises at least one mutated amino acid residue in comparison to a wild type reference sequence. In some embodiments, a mutated amino acid residue comprises a substitution of a natural amino acid residue with another natural amino acid residue. In some embodiments, an incretin agent comprises any one of the incretin peptides as represented in SEQ ID NOs: 5-10. In some embodiments a mutated amino acid residueAttorney Docket No.: 2013237-0773 may confer dual or triple agonizing properties to an incretin agent. For example, in some embodiments, one or more amino acid substitutions are introduced into a GLP1, GIP or GCG peptide sequence (e.g., as shown in SEQ ID NOs: 12-15) in order to confer binding properties to two or more of the GLP1, GIP and / or GCG receptors.

[0135] In some embodiments, an incretin agent comprises a combination of incretin peptides. For example, in some embodiments, an integrin agent comprises a GLP1 receptor agonist (e.g., GLP1 peptide, or fragment or variant thereof) and a GIP receptor agonist (e.g., a GIP peptide, or fragment or variant thereof). In some embodiments, an incretin agent comprises one or more incretin peptides selected from SEQ ID NOs: 5-15 and one of more incretin peptides selected from SEQ ID NOs: 5-15. In some embodiments, an integrin agent comprises a GLP1 receptor agonist (e.g., GLP1 peptide, or fragment or variant thereof), a GIP receptor agonist (e.g., a GIP peptide, or fragment or variant thereof), and a GCG receptor agonist (e.g., a GCG peptide, or fragment of variant thereof).

[0136] In some embodiments, the present disclosure provides one or more polyribonucleotides encoding an incretin agent that comprises a combination of incretin peptides. In such embodiments, one or more polyribonucleotides may encode an incretin agent. In some embodiments, a first polyribonucleotide may encode a first incretin peptide of an incretin agent and a second polyribonucleotide may encode a second incretin peptide of an incretin agent.

[0137] Exemplary incretin agents include those that include the incretin peptide sequences presented below in Table 2 (which also identified the reference incretin or incretin mimetic). Table 2: Exemplary Integrin Agents Reference incretin or SEQ Sequence incretin mimetic ID NO GLP1 (7-37) 5 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG  GLP1 (7-37) (Arg34 mutant) 6 HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG  GLP1 (7-36) 7 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR  GIP (1-42) 8 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKK NDWKHNITQ  GIP (1-30) 9 YAEGTFISDYSIAMDKIHQQDFVNWLLAQK Attorney Docket No.: 2013237-0773 Dulaglutide (GLP1 (7-37)- 10 HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGG Fc fusion protein) GGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLG Exenatide (GLP1R agonist) 11 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSA PPPS Tirzepatide (GLP1R / GIPR 12 YAEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSG dual agonist) APPPS NNC0090-2746 13 YAEGTFTSDYSIYLDKQAAKEFVNWLLAGGPSS (GLP1R / GIPR dual agonist) GAPPPS MAR701 (GLP1R / GIPR 14 YAEGTFTSDYSIYLDKQAAKEFVCWLLAGGPSS dual agonist) GAPPPS Retratutide 15 YXQGTFTSDYSIYLDKKAQXAFIEYLLEGGPSSG (GLP1R / GIPR / GCGR triple APPPS agonist) where X at position 2 is A or S where X at position 20 is K or Q C. Exemplary Polyribonucleotide Features

[0138] Polyribonucleotides described herein encode an incretin agent as described herein. Additionally, polyribonucleotides described herein, in some embodiments, include encode other elements such as a signal peptide. In some embodiments, polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5’UTR and / or a 3’ UTR. In some embodiments, polynucleotides described herein can comprise a nucleotide sequence that encodes a polyA tail. In some embodiments, polyribonucleotides described herein may comprise a 5’ cap, which may be incorporated during transcription, or joined to a polyribonucleotide post-transcription. 1. Signal peptide

[0139] According to certain embodiments, a signal peptide is fused, either directly or through a linker, to an encoded incretin agent described herein.Attorney Docket No.: 2013237-0773

[0140] In some embodiments, an open reading frame of the RNA described herein encodes an incretin agent described herein with a signal peptide, e.g., that is functional in mammalian cells.

[0141] In some embodiments, signal peptides are sequences, which are typically characterized by a length of about 15 to 30 amino acids.

[0142] In many embodiments, signal peptides are positioned at the N-terminus of an incretin agent, without being limited thereto. In some embodiments, signal peptides preferably allow the transport of an incretin agent encoded by RNAs of the present disclosure with which they are associated into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.

[0143] In some embodiments, a ribonucleic acid sequence encoding a signal peptide allows an incretin agent encoded by the polyribonucleotide to be secreted upon translation by cells, e.g., present in a subject, thus yielding a plasma concentration of a biologically active incretin agent.

[0144] In some embodiments, a ribonucleic acid sequence encoding a signal peptide included in a polyribonucleotide consists of or comprises a nucleotide sequence that encodes a human signal peptide. In some embodiments, a ribonucleic acid sequence encoding a secretion signal included in a polyribonucleotide consists of or comprises a nucleotide sequence that encodes a non-human secretion signal. For example, in some embodiments, such a signal peptide may be or comprises the amino acid sequence of MRVLVLLACLAAASNA (SP1-2; SEQ ID NO: 17).

[0145] In some embodiments, a signal peptide sequence is selected from those included in the Table 3 below, or a fragment or variant thereof: Table 3: Exemplary Signal Peptide Sequences Signal peptide SEQ Sequence (Amino Acid) ID NO: SP1 16 MRVLVLLACLAAASA SP1-2 17 MRVLVLLACLAAASNA SP1-3 18 MRRVLVLLACLAAASAAttorney Docket No.: 2013237-0773 SP1-4 19 MRRVLVLLACLAAASNA SP1-5 20 MRRRVLVLLACLAAASA SP1-6 21 MRRRVLVLLACLAAASNA HSV-1 gD SP 22 MGGAAARLGAVILFVVIVGLHGVRSKY HSV-2 gD SP 23 MGRLTSGVGTAALLVVAVGLRVVCA HSV-2 24 MGRLTSGVGTAALLVVAVGLRVVCAKYA SARS-CoV-2-S 25 MFVFLVLLPLVSSQCVNLT Human Ig heavy chain 26 MDWIWRILFLVGAATGAHSQM signal peptide Human Ig heavy chain 27 MDWTWRVFCLLAVAPGAHS signal peptide HuIgGk signal peptide 28 METPAQLLFLLLLWLPDTTG IgE heavy chain epsilon- 29 MDWTWILFLVAAATRVHS 1signal peptide Japanese encephalitis 30 MLGSNSGQRVVFTILLLLVAPAYS PRM signal peptide VSVg protein signal 31 MKCLLYLAFLFIGVNCA peptide Exemplary signal peptide 32 MDWTWILFLVAAATRVHS Exemplary signal peptide 33 ETPAQLLFLLLLWLPDTTG Exemplary signal peptide 34 MLGSNSGQRVVFTILLLLVAPAYS Exemplary signal peptide 35 MKCLLYLAFLFIGVNCA Exemplary signal peptide 36 MWLVSLAIVTACAGA Exemplary signal peptide 37 MFVFLVLLPLVSSQC husec signal peptide 38 MRVMAPRTLILLLSGALALTETWAGS husec3 signal peptide 39 MEFGLSWLFLVAILKGVQC

[0146] In some embodiments, a polyribonucleotide comprises, in a 5’ to 3’ direction a signal peptide-coding sequence and an incretin peptide-coding sequence. In some embodiments, a polyribonucleotide comprises, in a 5’ to 3’ direction a signal peptide-coding sequence; an incretin peptide-coding sequence; a linker-coding sequence; and a half-life extending moiety-coding sequence. In some embodiments, a polyribonucleotide comprises, in a 5’ to 3’ direction a signal peptide-coding sequence; a half-life extending moiety-coding sequence; a linker-coding sequence; and an incretin peptide-coding sequence.Attorney Docket No.: 2013237-0773 2. 5’ Cap

[0147] A structural feature of mRNAs is a cap structure at the 5’-end. Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5´ to 5´- triphosphate bridge resulting in cap0 structure (m7GpppN). In most eukaryotic mRNA and some viral mRNA, further modifications can occur at the 2’-hydroxy-group (2’-OH) (e.g., the 2’-hydroxyl group may be methylated to form 2’-O-Me) of the first and subsequent nucleotides producing “cap1” and “cap2” five-prime ends, respectively. Diamond et al., (2014) Cytokine & growth Factor Reviews, 25:543–550 reported that cap0-mRNA cannot be translated as efficiently as cap1-mRNA in which the role of 2’-O-Me in the penultimate position at the mRNA 5’ end is determinant. Lack of the 2’-O-Me has been shown to trigger innate immunity and activate IFN response. Daffis et al., (2010) Nature, 468:452-456; and Züst et al. (2011) Nature Immunology, 12:137-143.

[0148] RNA capping is well researched and is described, e.g., in Decroly et al., (2012) Nature Reviews 10: 51-65; and in Ramanathan et al., (2016) Nucleic Acids Res; 44(16): 7511–7526, the entire contents of each of which is hereby incorporated by reference. For example, in some embodiments, a 5’ cap structure which may be suitable in the context of the present invention is a cap0 (methylation of the first nucleobase, e.g. m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2ndnucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rdnucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), 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.

[0149] The term “5’-cap” as used herein refers to a structure found on the 5’-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5’- to 5’-triphosphate linkage (also referred to as Gppp or G(5’)ppp(5’)). In some embodiments, a guanosine nucleoside included in a 5’ cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and / or by methylation at one or more positions of a ribose. In some embodiments,Attorney Docket No.: 2013237-0773 a guanosine nucleoside included in a 5’ cap comprises a 3’O methylation at a ribose (3’OmeG). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and a 3’ O methylation at a ribose (m7(3’OmeG)). It will be understood that the notation used in the above paragraph, e.g., “(m27,3’-O)G” or “m7(3’OmeG)”, applies to other structures described herein.

[0150] In some embodiments, providing an RNA with a 5’-cap disclosed herein may be achieved by in vitro transcription, in which a 5’-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed herein improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase translation efficiency and / or translation rate of an RNA, and / or increase expression of an encoded polypeptide. In some embodiments, alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent decapping and increase RNA half-life.

[0151] In some embodiments, a utilized 5’ cap is a cap0, a cap1, or cap2 structure. See, e.g., Fig.1 of Ramanathan et al., and Fig.1 of Decroly et al., each of which is incorporated herein by reference in its entirety. In some embodiments, an RNA described herein comprises a cap1 structure. In some embodiments, an RNA described herein comprises a cap2 structure.

[0152] In some embodiments, an RNA described herein comprises a cap0 structure. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G). In some embodiments, such a cap0 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as (m7)Gppp. In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 2’- position of the ribose of guanosine In some embodiments, a cap0 structure comprises a guanosine nucleoside methylated at the 3’-position of the ribose of guanosine. In some embodiments, a guanosine nucleoside included in a 5’ cap comprises methylation at the 7- position of guanine and at the 2’-position of the ribose ((m27,2’-O)G). In some embodiments, aAttorney Docket No.: 2013237-0773 guanosine nucleoside included in a 5’ cap comprises methylation at the 7-position of guanine and at the 2’-position of the ribose ((m27,3’-O)G).

[0153] In some embodiments, a cap1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and optionally methylated at the 2’ or 3’ position of the ribose, and a 2’O methylated first nucleotide in an RNA ((m2’-O)N1). In some embodiments, a cap1 structure comprises a guanosine nucleoside methylated at the 7- position of guanine ((m7)G) and the 3’ position of the ribose, and a 2’O methylated first nucleotide in an RNA ((m2’-O)N1). In some embodiments, a cap1 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as, e.g., ((m7)Gppp(2'-O)N1) or (m27,3’-O)Gppp(2'-O)N1), wherein N1is as defined and described herein. In some embodiments, a cap1 structure comprises a second nucleotide, N2, which is at position 2 and is chosen from A, G, C, or U, e.g., (m7)Gppp(2'-O)N1pN2or (m27,3’-O)Gppp(2'-O)N1pN2, wherein each of N1 and N2 is as defined and described herein.

[0154] In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and optionally methylated at the 2’ or 3’ position of the ribose, and a 2’O methylated first and second nucleotides in an RNA ((m2’-O)N1p(m2’-O)N2). In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and the 3’ position of the ribose, and a 2’O methylated first and second nucleotide in an RNA. In some embodiments, a cap2 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as, e.g., ((m7)Gppp(2'-O)N1p(2'-O)N2) or (m27,3’-O)Gppp(2'-O)N1p(2'-O)N2), wherein each of N1 and N2is as defined and described herein.

[0155] In some embodiments, the 5’ cap is a dinucleotide cap structure. In some embodiments, the 5’ cap is a dinucleotide cap structure comprising N1, wherein N1 is as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap G*N1, wherein N1 is as defined above and herein, and G* comprises a structure of Formula (I):Attorney Docket No.: 2013237-0773 or a salt thereof,wherein each R2and R3is -OH or -OCH3; and X is O or S.

[0156] In some embodiments, R2is -OH. In some embodiments, R2is -OCH3. In some embodiments, R3is -OH. In some embodiments, R3is -OCH3. In some embodiments, R2is -OH and R3is -OH. In some embodiments, R2is -OH and R3is -CH3. In some embodiments, R2is -CH3 and R3is -OH. In some embodiments, R2is -CH3 and R3is -CH3.

[0157] In some embodiments, X is O. In some embodiments, X is S.

[0158] In some embodiments, the 5’ cap is a dinucleotide cap0 structure (e.g., (m7)GpppN1, (m27,2’-O)GpppN1, (m27,3’-O)GpppN1, (m7)GppSpN1, (m27,2’-O)GppSpN1, or (m27,3’-O)GppSpN1), wherein N1is as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap0 structure (e.g., (m7)GpppN1, (m27,2’-O)GpppN1, (m27,3’-O)GpppN1, (m7)GppSpN1, (m27,2’-O)GppSpN1, or (m27,3’-O)GppSpN1), wherein N1is G. In some embodiments, the 5’ cap is a dinucleotide cap0 structure (e.g., (m7)GpppN1, (m27,2’-O)GpppN1, (m27,3’-O)GpppN1, (m7)GppSpN1, (m27,2’-O)GppSpN1, or (m27,3’-O)GppSpN1), wherein N1 is A, U, or C. In some embodiments, the 5’ cap is a dinucleotide cap1 structure (e.g., (m7)Gppp(m2’-O)N1, (m27,2’-O)Gppp(m2’-O)N1, (m27,3’-O)Gppp(m2’-O)N1, (m7)GppSp(m2’-O)N1, (m27,2’-O)GppSp(m2’-O)N1, or (m27,3’-O)GppSp(m2’-O)N1), wherein N1 is as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m7)GpppG (“Ecap0”), (m7)Gppp(m2’-O)G (“Ecap1”), (m27,3’-O)GpppG (“ARCA”), and (m27,2’-O)GppSpG (“beta-S-ARCA”). In some embodiments, the 5’ cap is (m7)GpppG (“Ecap0”), having a structure:Attorney Docket No.: 2013237-0773 or a salt

[0159] In some embodiments, the 5’ cap is (m7)Gppp(m2’-O)G (“Ecap1”), having a structure: or a salt

[0160] In some embodiments, the 5’ cap is (m27,3’-O)GpppG (“ARCA”), having a structure: or a salt

[0161] In some embodiments, the 5’ cap is (m27,2’-O)GppSpG (“beta-S-ARCA”), having a structure:Attorney Docket No.: 2013237-0773 or a salt

[0162] In some embodiments, the 5’ cap is a trinucleotide cap structure. In some embodiments, the 5’ cap is a trinucleotide cap structure comprising N1pN2, wherein N1 and N2are as defined and described herein. In some embodiments, the 5’ cap is a dinucleotide cap G*N1pN2, wherein N1 and N2 are as defined above and herein, and G* comprises a structure of Formula (I): or a salt thereof,herein.

[0163] In some embodiments, the 5’ cap is a trinucleotide cap0 structure (e.g. (m7)GpppN1pN2, (m27,2’-O)GpppN1pN2, or (m27,3’-O)GpppN1pN2), wherein N1and N2are as defined and described herein). In some embodiments, the 5’ cap is a trinucleotide cap1 structure (e.g., (m7)Gppp(m2’-O)N1pN2, (m27,2’-O)Gppp(m2’-O)N1pN2, (m27,3’-O)Gppp(m2’-O)N1pN2), wherein N1 and N2 are as defined and described herein. In some embodiments, the 5’ cap is a trinucleotide cap2 structure (e.g., (m7)Gppp(m2’-O)N1p(m2’-O)N2, (m27,2’-O)Gppp(m2’-O)N1p(m2’-O)N2, (m27,3’-O)Gppp(m2’-O)N1p(m2’-O)N2), wherein N1 and N2 are as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m27,3’-O)Gppp(m2’-O)ApG (“CleanCap AG”, “CC413”), (m27,3’-O)Gppp(m2’-O)GpG (“CleanCap GG”), (m7)Gppp(m2’-O)ApG, (m7)Gppp(m2’-O)GpG, (m27,3’-O)Gppp(m26,2’-O)ApG, and (m7)Gppp(m2’-O)ApU.Attorney Docket No.: 2013237-0773

[0164] In some embodiments, the 5’ cap is (m27,3’-O)Gppp(m2’-O)ApG (“CleanCap AG”, “CC413”), having a structure: or a salt

[0165] In some embodiments, the 5’ cap is (m27,3’-O)Gppp(m2’-O)GpG (“CleanCap GG”), having a structure: or a salt

[0166] In some embodiments, the 5’ cap is (m7)Gppp(m2’-O)ApG, having a structure:Attorney Docket No.: 2013237-0773 or a salt

[0167] In some embodiments, the 5’ cap is (m7)Gppp(m2’-O)GpG, having a structure: or a

[0168] In some embodiments, the 5’ cap is (m27,3’-O)Gppp(m26,2’-O)ApG, having a structure:Attorney Docket No.: 2013237-0773 or a salt

[0169] In some embodiments, the 5’ cap is (m7)Gppp(m2’-O)ApU, having a structure: or a salt

[0170] In some embodiments, the 5’ cap is a tetranucleotide cap structure. In some embodiments, the 5’ cap is a tetranucleotide cap structure comprising N1pN2pN3, wherein N1, N2, and N3are as defined and described herein. In some embodiments, the 5’ cap is a tetranucleotide cap G*N1pN2pN3, wherein N1, N2, and N3 are as defined above and herein, and G* comprises a structure of Formula (I):Attorney Docket No.: 2013237-0773 herein.

[0171] In some embodiments, the 5’ cap is a tetranucleotide cap0 structure (e.g. (m7)GpppN1pN2pN3, (m27,2’-O)GpppN1pN2pN3, or (m27,3’-O)GpppN1N2pN3), wherein N1, N2, and N3 are as defined and described herein). In some embodiments, the 5’ cap is a tetranucleotide Cap1 structure (e.g., (m7)Gppp(m2’-O)N1pN2pN3, (m27,2’-O)Gppp(m2’-O)N1pN2pN3, (m27,3’-O)Gppp(m2’-O)N1pN2N3), wherein N1, N2, and N3 are as defined and described herein. In some embodiments, the 5’ cap is a tetranucleotide Cap2 structure (e.g., (m7)Gppp(m2’-O)N1p(m2’-O)N2pN3, (m27,2’-O)Gppp(m2’-O)N1p(m2’-O)N2pN3, (m27,3’-O)Gppp(m2’-O)N1p(m2’-O)N2pN3), wherein N1, N2, and N3are as defined and described herein. In some embodiments, the 5’ cap is selected from the group consisting of (m27,3’-O)Gppp(m2’-O)Ap(m2’-O)GpG, (m27,3’-O)Gppp(m2’-O)Gp(m2’-O)GpC, (m7)Gppp(m2’-O)Ap(m2’-O)UpA, and (m7)Gppp(m2’-O)Ap(m2’-O)GpG.

[0172] In some embodiments, the 5’ cap is (m27,3’-O)Gppp(m2’-O)Ap(m2’-O)GpG, having a structure:Attorney Docket No.: 2013237-0773 or a salt

[0173] In some embodiments, the 5’ cap is (m27,3’-O)Gppp(m2’-O)Gp(m2’-O)GpC, having a structure: or a salt

[0174] In some embodiments, the 5’ cap is (m7)Gppp(m2’-O)Ap(m2’-O)UpA, having a structure:Attorney Docket No.: 2013237-0773 or a salt

[0175] In some embodiments, the 5’ cap is (m7)Gppp(m2’-O)Ap(m2’-O)GpG, having a structure: or a saltAttorney Docket No.: 2013237-0773 3. Cap Proximal Sequences

[0176] In some embodiments, a 5’ UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5’ cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0177] In some embodiments, a cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +1 (N1) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1 and +2 (N1 and N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7Guanosine cap and nucleotides +1, +2, and +3 (N1, N2, and N3) of an RNA polynucleotide.

[0178] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and / or +5) may be included in an RNA by virtue of having been included in a cap entity (e.g., a cap1 or cap2 structure, etc.); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m27,3’-OGppp(m12’-O)ApG cap is utilized, +1 (i.e., N1) and +2 (i.e. N2) are the (m12’-O)A and G residues of the cap, and +3, +4, and +5 are added by polymerase (e.g., T7 polymerase).

[0179] In some embodiments, the 5’ cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises N1 of the 5’ cap, where N1 is any nucleotide, e.g., A, C, G or U. In some embodiments, the 5’ cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises N1 and N2 of the 5’ cap, wherein N1 and N2 are independently any nucleotide, e.g., A, C, G or U. In some embodiments, the 5’ cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequenceAttorney Docket No.: 2013237-0773 comprises N1, N2, and N3 of the 5’ cap, wherein N1, N2, and N3 are any nucleotide, e.g., A, C, G or U.

[0180] In some embodiments, e.g., where the 5’ cap is a dinucleotide cap structure, a cap proximal sequence comprises N1 of a the 5’ cap, and N2, N3, N4 and N5, wherein N1 to N5correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5’ cap is a trinucleotide cap structure, a cap proximal sequence comprises N1 and N2 of a the 5’ cap, and N3, N4 and N5, wherein N1 to N5 correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide. In some embodiments, e.g., where the 5’ cap is a tetranucleotide cap structure, a cap proximal sequence comprises N1, N2, and N3of a the 5’ cap, and N4and N5, wherein N1to N5correspond to positions +1, +2, +3, +4, and / or +5 of an RNA polynucleotide.

[0181] In some embodiments, N1 is A. In some embodiments, N1 is C. In some embodiments, N1is G. In some embodiments, N1is U. In some embodiments, N2is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, N3is A. In some embodiments, N3is C. In some embodiments, N3is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N4 is C. In some embodiments, N4is G. In some embodiments, N4is U. In some embodiments, N5 is A. In some embodiments, N5 is C. In some embodiments, N5 is G. In some embodiments, N5 is U. It will be understood that, each of the embodiments described above and herein (e.g., for N1through N5) may be taken singly or in combination and / or may be combined with other embodiments of variables described above and herein (e.g., 5’ caps). 4. 5’ UTR

[0182] In some embodiments, a nucleic acid (e.g., DNA, RNA) utilized in accordance with the present disclosure comprises a 5' UTR. In some embodiments, 5’ UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element). In some embodiments a 5’ UTR comprises multiple different sequence elements.Attorney Docket No.: 2013237-0773

[0183] The term “untranslated region” or “UTR” is commonly used in the art to reference a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5' UTR) and / or 3' (downstream) of an open reading frame (3' UTR). As used herein, the terms “five prime untranslated region” or “5' UTR” refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide. In some embodiments, “5' UTR” refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context. In some embodiments, a 5' UTR comprises a Kozak sequence. A 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, a cap proximal sequence comprises a sequence adjacent to a 5’ cap.

[0184] Exemplary 5’ UTRs include a human alpha globin (hAg) 5’ UTR or a fragment thereof, a TEV 5’ UTR or a fragment thereof, a HSP705’ UTR or a fragment thereof, or a c-Jun 5’ UTR or a fragment thereof.

[0185] In some embodiments, an RNA disclosed herein comprises a hAg 5’ UTR or a fragment thereof.

[0186] In some embodiments, an RNA disclosed herein comprises a 5’ UTR 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 with the sequence according to AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 49). In some embodiments, an RNA disclosed herein comprises a 5’ UTR provided in SEQ ID NO: 49. 5. PolyA Tail

[0187] In some embodiments, a polynucleotide (e.g., DNA, RNA) disclosed herein comprises a polyadenylate or poly(A) sequence, e.g., as described herein. In someAttorney Docket No.: 2013237-0773 embodiments, a poly(A) sequence is situated downstream of a 3' UTR, e.g., adjacent to a 3' UTR.

[0188] As used herein, the term “poly(A) sequence” or “polyA tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3’ UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. In some embodiments, polynucleotides disclosed herein comprise an uninterrupted poly(A) sequence. In some embodiments, polynucleotides disclosed herein comprise interrupted poly(A) sequence. In some embodiments, RNAs disclosed herein can have a poly(A) sequence attached 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.

[0189] It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol.108, pp.4009-4017, which is herein incorporated by reference).

[0190] In some embodiments, a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A) sequence comprises, essentially consists 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, “essentially consists of” means that most 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 nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, “consists 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 adenylate.Attorney Docket No.: 2013237-0773

[0191] In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.

[0192] In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO2016 / 005324, hereby incorporated by reference. Any poly(A) cassette disclosed in WO2016 / 005324 may be used in accordance with the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

[0193] In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3'-end, i.e., the poly(A) sequence is not masked or followed at its 3'- end by a nucleotide other than A.

[0194] In some embodiments, the poly(A) sequence may 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 may essentially 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 may 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)Attorney Docket No.: 2013237-0773 sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

[0195] In some embodiments, a polyA tail comprises a specific number of Adenosines, 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 a polyA tail of a string construct may comprise 200 A residues or less. In some embodiments, a polyA tail of a string construct may comprise about 200 A residues. In some embodiments, a polyA tail of a string construct may comprise 180 A residues or less. In some embodiments, a polyA tail of a string construct may comprise about 180 A residues. In some embodiments, a polyA tail may comprise 150 residues or less.

[0196] In some embodiments, RNA comprises a poly(A) sequence comprising the nucleotide sequence of AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAA (SEQ ID NO: 50), 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, a poly(A) tail comprises a nucleotide sequence according to SEQ ID NO: 50.

[0197] In some embodiments, a polyA tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCAUAUGACU (SEQ ID NO: 40). 6. 3’ UTR

[0198] In some embodiments, an RNA utilized in accordance with the present disclosure comprises a 3' UTR. As used herein, the terms “three prime untranslated region,” “3' untranslated region,” or “3' UTR” refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context. The term “3' UTR” does preferably not include theAttorney Docket No.: 2013237-0773 poly(A) sequence. Thus, the 3' UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0199] In some embodiments, an RNA disclosed herein comprises a 3’ UTR comprising an F element and / or an I element. In some embodiments, a 3’ UTR or a proximal sequence thereto comprises a restriction site. In some embodiments, a restriction site is a BamHI site. In some embodiments, a restriction site is a XhoI site.

[0200] In some embodiments, an RNA construct comprises an F element. In some embodiments, a F element sequence is a 3’ UTR of amino-terminal enhancer of split (AES).

[0201] In some embodiments, an RNA disclosed herein comprises a 3’ UTR 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 3’ UTR with the sequence according to CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCC GAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCAC UCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCA AAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUA GCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUU CGUGCCAGCCACACC (SEQ ID NO: 51). In some embodiments, an RNA disclosed herein comprises a 3’ UTR provided in SEQ ID NO: 51.

[0202] In some embodiments, a 3’UTR is an FI element as described in WO2017 / 060314, which is herein incorporated by reference in its entirety. 7. RNA Formats

[0203] At least three distinct formats useful for RNA compositions (e.g., pharmaceutical compositions) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplifying mRNA (saRNA). Each of these platforms displays unique features. In general, in all three formats, RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR), and have a polyA-tail at the 3' end. An ORF of an uRNA and modRNA vectors encode an incretin agent. An saRNA has multiple ORFs.Attorney Docket No.: 2013237-0773

[0204] In some embodiments, the RNA described herein may have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.

[0205] The term “uracil,” as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is: .The term “uridine,” as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:structure: .Attorney Docket No.: 2013237-0773

[0209] “Pseudouridine” is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0210] Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1Ψ), which has the structure:uridine (m5U), which has the structure: .

[0213] In some embodiments, one or more uridine in the RNA described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.Attorney Docket No.: 2013237-0773

[0214] In some embodiments, RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, RNA comprises a modified nucleoside in place of each uridine.

[0215] In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). 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 (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl- pseudouridine (m1ψ), and 5-methyl-uridine (m5U). 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 (m5U). In some embodiments, the modified nucleosides comprise N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0216] In some embodiments, the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy- uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5- oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5- carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl- uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5- methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5- methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio- uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl- uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-Attorney Docket No.: 2013237-0773 pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 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 (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1- methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ψ), 5- (isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′- O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyl- uridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um), 3,2′- O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0217] In some embodiments, the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in some embodiments, in the RNA 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. In some embodiments, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5- methyl-uridine (m5U). 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.

[0218] In some embodiments of the present disclosure, the RNA is “replicon RNA” or simply a “replicon,” in particular “self-replicating RNA” or “self-amplifying RNA.” In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or comprises elements derived from a single-stranded (ss) RNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see José et al., Future Microbiol., 2009, vol.4, pp.837–856, which is incorporated herein by reference in its entirety). The totalAttorney Docket No.: 2013237-0773 genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5’ cap, and a 3’ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1–nsP4) are typically encoded together by a first ORF beginning near the 5′ terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3’ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol.87 pp.111–124). Following infection, i.e., at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234).

[0219] Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, a first ORF encodes an alphavirus-derived RNA-dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA. A second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest, e.g., an incretin agent. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

[0220] Features of a non-modified uridine platform may include, for example, one or more of intrinsic adjuvant effect, as well as good tolerability and safety. Features of modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effect,Attorney Docket No.: 2013237-0773 blunted immune innate immune sensor activating capacity and thus good tolerability and safety. Features of self-amplifying platform may include, for example, long duration of protein expression, good tolerability and safety, higher likelihood for efficacy with very low vaccine dose.

[0221] The present disclosure provides particular RNA constructs optimized, for example, for improved manufacturability, encapsulation, expression level (and / or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein. 8. Codon Optimization and GC Enrichment

[0222] As used herein, the term “codon-optimized” refers to alteration of codons in a coding region of a nucleic acid molecule (e.g., a polyribonucleotide) to reflect the typical codon usage of a host organism (e.g., a subject receiving a polyribonucleotide) without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments, coding regions are codon- optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons.” In some embodiments, codon-optimization may include increasing guanosine / cytosine (G / C) content of a coding region of RNA described herein as compared to the G / C content of the corresponding coding sequence of a wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.

[0223] In some embodiments, a coding sequence (also referred to as a “coding region”) is codon optimized for expression in the subject to whom a composition (e.g., a pharmaceutical composition) is to be administered (e.g., a human). Thus, in some embodiments, sequences in such a polynucleotide (e.g., a polyribonucleotide) may differ from wild type sequences encoding the relevant incretin agent, even when the amino acid sequence of the incretin agent is wild type.Attorney Docket No.: 2013237-0773

[0224] In some embodiments, strategies for codon optimization for expression in a relevant subject (e.g., a human), and even, in some cases, for expression in a particular cell or tissue.

[0225] Various species exhibit particular bias for certain codons of a particular amino acid. Without wishing to be bound by any one theory, codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell may generally be a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are available, for example, at the “Codon Usage Database” available at www.kazusa.orjp / codon / and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.

[0226] In some embodiments, a polynucleotide (e.g., a polyribonucleotide) of the present disclosure is codon optimized, wherein the codons in the polynucleotide (e.g., the polyribonucleotide) are adapted to human codon usage (herein referred to as “human codon optimized polynucleotide”). Codons encoding the same amino acid occur at different frequencies in a subject, e.g., a human. Accordingly, in some embodiments, the coding sequence of a polynucleotide of the present disclosure is modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage, e.g., as shown in Table 8. For example, in the case of the amino acid Ala, the wild type coding sequence is preferably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with 30 a frequency of 0.10 etc. (see Table 4). Accordingly, in some embodiments, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of a polynucleotide to obtain sequences adapted to human codon usage. Table 4: Human codon usage table with frequencies indicated for each amino acid. Amino acid codon frequency Amino acid codon frequencyAttorney Docket No.: 2013237-0773 Ala GCG 0.10 Pro CCG 0.11 Ala GCA 0.22 Pro CCA 0.27 Ala GCT 0.28 Pro CCT 0.29 Ala GCC* 0.40 Pro CCC* 0.33 Cys TGT 0.42 Gin CAG* 0.73 Cys TGC* 0.58 Gin CAA 0.27 Asp GAT 0.44 Arg AGG 0.22 Asp GAC* 0.56 Arg AGA* 0.21 Glu GAG* 0.59 Arg CGG 0.19 Glu GAA 0.41 Arg CGA 0.10 Phe TTT 0.43 Arg CGT 0.09 Phe TTC* 0.57 Arg CGC 0.19 Gly GGG 0.23 Ser AGT 0.14 Gly GGA 0.26 Ser AGC* 0.25 Gly GGT 0.18 Ser TCG 0.06 Gly GGC* 0.33 Ser TCA 0.15 His CAT 0.41 Ser TCT 0.18 His CAC* 0.59 Ser TCC 0.23 lle ATA 0.14 Thr ACG 0.12 lle ATT 0.35 Thr ACA 0.27 lle ATC* 0.52 Thr ACT 0.23 Lys AAG* 0.60 Tor ACC* 0.38 Lys AAA 0.40 Val GTG* 0.48 Leu TTG 0.12 Val GTA 0.10 Leu TTA 0.06 Val GTT 0.17 Leu CTG* 0.43 Val GTC 0.25 Leu CTA 0.07 Trp TGG* 1 Leu CTT 0.12 Tyr TAT 042 Lou CTC 0.20 Tyr TAC* 0.58 Met ATG* 1 Stop TGA* 061 Asn AAT 0.44 Stop TAG 0.17 Asn AAC* 0.56 Stop TAA 0.22Attorney Docket No.: 2013237-0773

[0227] Certain strategies for codon optimization and / or G / C enrichment for human expression are described in WO2002 / 098443, which is incorporated by reference herein in its entirety. In some embodiments, a coding sequence may be optimized using a multiparametric optimization strategy. In some embodiments, optimization parameters may include parameters that influence protein expression, which can be, for example, impacted on a transcription level, an mRNA level, and / or a translational level. In some embodiments, exemplary optimization parameters include, but are not limited to transcription-level parameters (including, e.g., GC content, consensus splice sites, cryptic splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); mRNA-level parameters (including, e.g., RNA instability motifs, ribosomal entry sites, repetitive sequences, and combinations thereof); translation-level parameters (including, e.g., codon usage, premature poly(A) sites, ribosomal entry sites, secondary structures, and combinations thereof); or combinations thereof. In some embodiments, a coding sequence may be optimized by a GeneOptimizer 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, the entire content of each of which is incorporated herein for the purposes described herein. In some embodiments, a coding sequence may be optimized by Eurofins’ adaption and optimization algorithm “GENEius” as described in Eurofins’ Application Notes: Eurofins’ adaption and optimization software “GENEius” in comparison to other optimization algorithms, the entire content of which is incorporated by reference for the purposes described herein.

[0228] In some embodiments, a coding sequence utilized in accordance with the present disclosure has G / C content which is increased compared to a wild type coding sequence for a relevant incretin agent. In some embodiments, guanosine / cytidine (G / C) content of a coding region is modified relative to a wild type coding sequence for a relevantAttorney Docket No.: 2013237-0773 incretin agent, but the amino acid sequence encoded by the polyribonucleotide is not modified.

[0229] Without wishing to be bound by any particular theory, it is proposed that GC enrichment may improve translation of a payload sequence. Typically, sequences having an increased G (guanosine) / C (cytidine) content are more stable than sequences having an increased A (adenosine) / U (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by a polyribonucleotide, there are various possibilities for modification of the ribonucleic acid sequence, compared to its wild type sequence. In particular, codons which contain A and / or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and / or U or contain a lower content of A and / or U nucleosides.

[0230] In some embodiments, G / C content of a coding region of a polyribonucleotide 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 prior to codon optimization, e.g., of the wild type RNA. In some embodiments, G / C content of a coding region of a polyribonucleotide described herein is decreased 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 prior to codon optimization, e.g., of the wild type RNA.

[0231] In some embodiments, stability and translation efficiency of an polyribonucleotide may incorporate one or more elements established to contribute to stability and / or translation efficiency of the polyribonucleotide; exemplary such elements are described, for example, in WO2007 / 036366 incorporated herein by reference. In some embodiments, to increase expression of a polyribonucleotide used according to the present disclosure, a 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, for example so as to increase the GC-content to increase mRNA stability and / or to perform a codon optimization and, thus, enhance translation in cells.Attorney Docket No.: 2013237-0773 D. RNA Delivery Technologies

[0232] Provided polyribonucleotides may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNAs, or delivery mediated by viral and / or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid- polymer hybrid nanoparticles, etc.), and / or peptide-based vectors. See, e.g., Wadhwa et al. “Opportunities and Challenges in the Delivery of mRNA-Based Vaccines” Pharmaceutics (2020) 102 (27 pages), the content of which is incorporated herein by reference, for information on various approaches that may be useful for delivery polyribonucleotides described herein.

[0233] In some embodiments, one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery (e.g., administration).

[0234] In some embodiments, lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and / or engineered for systemic delivery of the RNA to target cells (e.g., liver, gut or pancreas cells). In some embodiments, such lipid nanoparticles may be particularly useful to deliver polyribonucleotides when polyribonucleotides are intraperitoneally, intravenously or intramuscularly administered to a subject. 1. Particles for Delivery of at Least One Polyribonucleotide

[0235] Polyribonucleotides provided herein can be delivered by particles. In the context of the present disclosure, the term “particle” relates to a structured entity formed by molecules or molecule complexes. In some embodiments, the term “particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure dispersed in a medium. In some embodiments, a particle is a nucleic acid containing particle such as a particle comprising a polyribonucleotide.

[0236] Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid (e.g., a polyribonucleotide) are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles (e.g., ribonucleic acid particles). In some embodiments, a nucleic acid particle (e.g., ribonucleic acid particle) is a nanoparticle.Attorney Docket No.: 2013237-0773

[0237] A “nucleic acid particle” (e.g., a ribonucleic acid particle) are particles that encompass or contain a nucleic acid, and are used to deliver nucleic acid (e.g., a polyribonucleotide) to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle (e.g., a ribonucleic acid particle) may be formed from (i) at least one cationic or cationically ionizable lipid or lipid-like material, (ii) at least one cationic polymer such as protamine, or a mixture of (i) and (ii), and (iii) nucleic acid (e.g., a polyribonucleotide). Nucleic acid particles (e.g., a ribonucleic acid particle) include lipid nanoparticles (lipid nanoparticle) and lipoplexes (LPX).

[0238] In some embodiments, nucleic acid particles (e.g., ribonucleic acid particles) comprise more than one type of nucleic acid molecules (e.g., polyribonucleotides), where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.

[0239] In some embodiments, provided nucleic acid particles (e.g., ribonucleic acid particles) can comprise lipid nanoparticles. As used in the present disclosure, “nanoparticle” refers to a particle having an average diameter suitable for parenteral administration. In various embodiments, lipid nanoparticles can have an average size (e.g., mean 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 in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 60 nm to about 120 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean 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.

[0240] Nucleic acid particles (e.g., ribonucleic acid particles) described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, orAttorney Docket No.: 2013237-0773 about 0.2 or less. By way of example, the nucleic acid particles (e.g., ribonucleic acid particles) can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

[0241] 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 (the “N” in N / P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N / P) in RNA. It is understood that a cationic group is one that is either in cationic form (e.g., N+), or one that is ionizable to become cationic. Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 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., a ribonucleic acid particle) described herein has an N / P ratio greater than or equal to 5. In some embodiments, a nucleic acid particle (e.g., a ribonucleic acid particle) described herein has an N / P ratio that is about 5, 6, 7, 8, 9, or 10. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., a ribonucleic acid particle) described herein is from about 10 to about 50. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., a ribonucleic acid particle) described herein is from about 10 to about 70. In some embodiments, an N / P ratio for a nucleic acid particle (e.g., a ribonucleic acid particle) described herein is from about 10 to about 120.

[0242] Nucleic acid particles (e.g., ribonucleic acid particles) described herein can be prepared using a wide range of methods that may 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 nucleic acid to obtain nucleic acid particles.

[0243] The term “colloid” as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture can be microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term “colloid” only refers to the particles in the mixture and not the entire suspension.

[0244] The term “average diameter” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-Attorney Docket No.: 2013237-0773 called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys.57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference). Herein “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0245] The “polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

[0246] Different types of nucleic acid particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g., Kaczmarek et al., 2017, Genome Medicine 9, 60, which is herein incorporated by reference). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

[0247] The present disclosure describes particles comprising nucleic acid (e.g., a polyribonucleotide), at least one cationic or cationically ionizable lipid or lipid-like material, and / or at least one cationic polymer which associate with the nucleic acid (e.g., a polyribonucleotide) to form nucleic acid particles (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) and compositions comprising such particles. The nucleic acid particles (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) may comprise nucleic acid (e.g., a polyribonucleotide) which is complexed in different forms by non- covalent interactions to the particle. The particles described herein are not viral particles, in particular, they are not infectious viral particles, i.e., they are not able to virally infect cells.

[0248] 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).

[0249] In a nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation, it is possible that each nucleic acid species (e.g., polyribonucleotide species) is separately formulated as an individual nucleic acid particleAttorney Docket No.: 2013237-0773 (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation. In that case, each individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation will comprise one nucleic acid species (e.g., polyribonucleotide species). The individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations may be present as separate entities, e.g., in separate containers. Such formulations are obtainable by providing each nucleic acid species (e.g., polyribonucleotide species) separately (typically each in the form of a nucleic acid- containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific nucleic acid species (e.g., polyribonucleotide species) that is being provided when the particles are formed (individual particulate formulations).

[0250] In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulation. Respective pharmaceutical compositions are referred to as “mixed particulate formulations.” Mixed particulate formulations according to the invention are obtainable by forming, separately, individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations, as described above, followed by a step of mixing of the individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing particles is obtainable. Individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) populations may be together in one container, comprising a mixed population of individual nucleic acid particle (e.g., ribonucleic acid particle, e.g., ribonucleic acid nanoparticle) formulations.

[0251] Alternatively, it is possible that different nucleic acid species (e.g., polyribonucleotide species) are formulated together as a “combined particulate formulation.” Such formulations are obtainable by providing a combined formulation (typically combined solution) of different nucleic acid species (e.g., polyribonucleotide species) species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a “mixed particulate formulation,” a “combined particulate formulation” will typically comprise particles that comprise more than one nucleic acid species (e.g., polyribonucleotide species) species. In a combined particulate compositionAttorney Docket No.: 2013237-0773 different nucleic acid species (e.g., polyribonucleotide species) are typically present together in a single particle.

[0252] In certain embodiments, nucleic acids (e.g., polyribonucleotides), when present in provided nucleic acid particles (e.g., ribonucleic acid particles, e.g., lipid nanoparticles) are resistant in aqueous solution to degradation with a nuclease.

[0253] In some embodiments, nucleic acid particles (e.g., ribonucleic acid particles) are lipid nanoparticles. In some embodiments, lipid nanoparticles are liver-targeting lipid nanoparticles. In some embodiments, lipid nanoparticles are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., ones described herein). In some embodiments, cationic lipid nanoparticles may comprise at least one cationic lipid, at least one polymer- conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid). E. Cationic Polymeric Materials

[0254] Cationic polymers have been recognized as useful for developing particle delivery vehicles, as reported in PCT App. Pub. No. WO2021 / 001417, the entirety of which is incorporated herein by reference. As used herein, the term “polymer” refers to a composition comprising one or more molecules that comprise repeating units of one or more monomers. As used herein, “polymer,” “polymeric material,” and “polymer composition” are used interchangeably, and unless otherwise specified, refer to a composition of polymer molecules. A person of skill in the art will appreciate that a polymer composition comprises polymer molecules having molecules of different lengths (e.g., comprising varying amounts of monomers). Polymer compositions described herein are characterized by one or more of a normalized molecular weight (Mn), a weight average molecular weight (Mw), and / or a polydispersity index (PDI). In some embodiments, such repeat units can all be identical (a “homopolymer”); alternatively, in some cases, there can be more than one type of repeat unit present within the polymeric material (a “heteropolymer” or a “copolymer”). In some cases, a polymer is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymeric material, for example targeting moieties such as those described herein.

[0255] In some embodiments, a polymer utilized in accordance with the present disclosure may be a copolymer. Repeat units forming the copolymer can be arranged in anyAttorney Docket No.: 2013237-0773 fashion. For example, in some embodiments, repeat units can be arranged in a random order; alternatively or additionally, in some embodiments, repeat units may be arranged in an alternating order, or as a “block” copolymer, e.g., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

[0256] In certain embodiments, a polymeric material for use in accordance with the present disclosure is biocompatible. In certain embodiments, a biocompatible material is biodegradable, e.g., is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body.

[0257] In certain embodiments, a polymeric material may be or comprise protamine or polyalkyleneimine.

[0258] As those skilled in the art are aware term “protamine” is often used to refer to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term “protamine” is often used to refer to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

[0259] In some embodiments, the term “protamine” as used herein is refers to a protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and / or multimeric forms of said amino acid sequence or fragment thereof, as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

[0260] In some embodiments, a polyalkyleneimine comprises polyethylenimine (PEI) and / or polypropylenimine. In some embodiments, a preferred polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the average molecular weight of PEI is preferably 0.75∙102 to 107 Da, preferably 1000 to 105 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.Attorney Docket No.: 2013237-0773

[0261] Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those which are able to electrostatically bind nucleic acid. In some embodiments, cationic polymeric materials contemplated for use herein include any cationic polymeric materials with which nucleic acid can be associated, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0262] In some embodiments, particles described herein may comprise polymers other than cationic polymers, e.g., non-cationic polymeric materials and / or anionic polymeric materials. Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials. F. Lipid Compositions 1. Lipids and Lipid-Like Materials

[0263] Lipids and lipid-like materials have also been recognized as useful for developing particle delivery vehicles. The terms “lipid” and “lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and / or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

[0264] Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non- polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have bothAttorney Docket No.: 2013237-0773 a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

[0265] A “lipid-like material” is a substance that is structurally and / or functionally related to a lipid but may not be considered a lipid in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar / unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.

[0266] Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.

[0267] Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterols and prenol lipids (derived from condensation of isoprene subunits). Although the term “lipid” is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0268] Fatty acids are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule’s configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more doubleAttorney Docket No.: 2013237-0773 bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.

[0269] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word “triacylglycerol” is sometimes used synonymously with “triglyceride”. In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.

[0270] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid- derived “tails” by ester linkages and to one “head” group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

[0271] Sphingolipids are members of a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0272] Sterols, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with the glycerophospholipids and sphingomyelins.Attorney Docket No.: 2013237-0773

[0273] Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram- negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0274] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0275] 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.

[0276] 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 each of which are incorporated herein by reference for the purposes described herein. 2. Cationic or Cationically Ionizable Lipids or Lipid-like Materials

[0277] In some embodiments cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In one embodiment, cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.Attorney Docket No.: 2013237-0773

[0278] Cationic lipids or lipid-like materials are characterized in that they have a net positive charge (e.g., at a relevant pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

[0279] In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

[0280] In some embodiments, a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.

[0281] Examples of cationic lipids include, but are not limited to 1,2-dioleoyl-3- trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N—(N′,N′- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3- dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3- aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3- trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]- N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en- 3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5′- (cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′- octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamineAttorney Docket No.: 2013237-0773 (DMOBA), 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3- Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N′-Dilinoleylcarbamyl-3- dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2- dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4- (2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3- aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1- propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N- dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4- carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (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-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4- di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl) 8,8’- ((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,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- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2- dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)- [2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1 ,2- dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1 -Attorney Docket No.: 2013237-0773 (2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing. Further suitable cationic lipids for use in the present disclosure include those described in WO2020 / 128031 and US2020 / 0163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein. Further suitable cationic lipids for use in the present disclosure include those described in WO2010 / 053572 (including C12-200 described at paragraph

[0225] ) and WO2012 / 170930, both of which 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).

[0282] In some embodiments, formulations that are useful for pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) compositions as described herein can comprise at least one cationic lipid. Representative cationic lipids include, but are not limited to, 1 ,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2- dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2- linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3- trimethylaminopropane chloride salt (DLin-TMA.CI), 1 ,2-dilinoleoyl-3- trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-1 ,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1 ,2-propanediol (DOAP), 1 ,2-dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1 ,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US2010 / 0324120, which is incorporated herein by reference in its entirety).

[0283] In some embodiments, amino or cationic lipids useful in accordance with the present disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at aAttorney Docket No.: 2013237-0773 second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise suitable in the context of the present invention.

[0284] In some embodiments, a protonatable lipid has 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.

[0285] In some embodiments, a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of total lipid present in a lipid composition utilized in accordance with the present disclosure. 3. Additional Lipids or Lipid-like materials

[0286] In some embodiments, formulations utilized in accordance with 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-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. In some embodiments, optimizing a formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable / cationic lipid or lipid-like material may, for example, enhance particle stability and efficacy of nucleic acid delivery.

[0287] In some embodiments, a lipid or lipid-like material may be incorporated which may or may not affect the overall charge of particles. In certain embodiments, such lipid or lipid-like material is a non-cationic lipid or lipid-like material.

[0288] In some embodiments, a non-cationic lipid may comprise, e.g., one or more anionic lipids and / or neutral lipids. An “anionic lipid” is negatively charged (e.g., at a selected pH).Attorney Docket No.: 2013237-0773

[0289] A “helper lipid” exists either in an uncharged, neutral zwitterionic form (e.g., at a selected pH), or, in some embodiments, as having a cationic or positive charge at physiological pH. In some embodiments, a formulation comprises one of the following helper lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2’-hydroxyethyl ether, cholesteryl-4’-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0290] Specific exemplary phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl- phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.

[0291] In certain embodiments, a formulation utilized in accordance with the present disclosure includes DSPC or DSPC and cholesterol.

[0292] In some embodiments, a formulation utilized in accordance with the present disclosure comprises a helper lipid that is ionizable or cationic.Attorney Docket No.: 2013237-0773

[0293] In certain embodiments, formulations utilized in accordance with the present disclosure include both a cationic lipid and an additional (non-cationic) lipid.

[0294] In some embodiments, formulations herein include a polymer conjugated lipid such as a pegylated lipid. “Pegylated lipids” or “PEG-conjugated lipids” comprise both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

[0295] Without wishing to be bound by theory, the amount of (total) cationic lipid compared to the amount of other lipid(s) in formulation may affect important characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. 4. Lipoplex Particles

[0296] In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipoplex particles.

[0297] An “RNA lipoplex particle” contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.

[0298] In certain embodiments, RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0299] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific 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 an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.Attorney Docket No.: 2013237-0773

[0300] In some embodiments, RNA lipoplex particles have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex 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 an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0301] RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration. The RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM. Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex 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-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and / or 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplexAttorney Docket No.: 2013237-0773 particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2- di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

[0302] Spleen targeting RNA lipoplex particles are described in WO2013 / 143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression in the lung and / or liver occurs. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and / or macrophages. 5. Lipid Nanoparticles (LNPs)

[0303] In some embodiments, nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs). In some embodiments, LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0304] In some embodiments, an LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids, helper lipids, and pegylated lipids.

[0305] In some embodiments, an LNP comprises a cationic lipid, a helper lipid, a sterol, a polymer conjugated lipid; and an RNA, encapsulated within or associated with the lipid nanoparticle.

[0306] In some embodiments, a helper lipid is a phosphotidylcholine such as 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-Attorney Docket No.: 2013237-0773 palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), phophatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), sphingomyelins (SM). In some embodiments, a helper lipid is a 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1-α-phosphatidylserine (PS), or DOPE. In some embodiments, a 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.

[0307] In some embodiments, a sterol is cholesterol.

[0308] In some embodiments, a polymer conjugated lipid is a pegylated lipid (a PEG lipid). In some embodiments, a PEG lipid is selected from pegylated diacylglycerol (PEG- DAG) such as l-(monomethoxy-polyethyleneglycol)- 2,3-dimyristoylglycerol (PEG-DMG) (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG)), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S- DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω-m ethoxy (polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate, and 2,3-di(tetradecanoxy)propy 1-Ν-(ω methoxy(polyethoxy)ethyl)carbamate. In some embodiments, a pegylated lipid is 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG2000). In some embodiments, a pegylated lipid has the following structure:   or a pharmaceuticallythereof, wherein: R12and R13are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionallyAttorney Docket No.: 2013237-0773 interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some embodiments, R12and R13are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, w has a mean value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R12and R13are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.

[0309] In some embodiments, a pegylated lipid is DMG-PEG 2000, e.g., having the following structure: .

[0310] glycol)-2000]-N,N-ditetradecylacetamide with a chemical structure as shown below: O N or a45 to 50

[0311] In some embodiments, a cationic lipid component of LNPs has the structure of Formula (III): R3G3or a pharmaceuticallystereoisomer thereof, wherein: one of L1or L2is –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 L1or L2is –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; G1and G2are each independently unsubstituted C1-C12alkylene or C1-C12alkenylene;Attorney Docket No.: 2013237-0773 G3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Rais H or C1-C12alkyl; R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl; R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or –NR5C(=O)R4; R4is C1-C12 alkyl; R5is H or C1-C6alkyl; and x is 0, 1 or 2.

[0312] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB): wherein:A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R6is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.

[0313] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

[0314] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID):wherein y and z are each independently integers ranging from 1 to 12.

[0315] In any of the foregoing embodiments of Formula (III), one of L1or L2is -O(C=O)-. For example, in some embodiments each of L1and L2are -O(C=O)-. In some different embodiments of any of the foregoing, L1and L2are eachAttorney Docket No.: 2013237-0773 independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L1and L2is -(C=O)O-.

[0316] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF): .

[0317] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ): ; .

[0318] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 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.

[0319] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.Attorney Docket No.: 2013237-0773

[0320] In some of the foregoing embodiments of Formula (III), R6is H. In other of the foregoing embodiments, R6is C1-C24alkyl. In other embodiments, R6is OH.

[0321] In some embodiments of Formula (III), G3is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3is linear C1-C24 alkylene or linear C1-C24alkenylene.

[0322] In some other foregoing embodiments of Formula (III), R1or R2, or both, is C6-C24alkenyl. For example, in some embodiments, R1and R2each, independently have the following structure: , wherein:R7aand R7bare, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7band a are each selected such that R1and R2each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0323] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7ais H. For example, in some embodiments, R7ais H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7bis C1-C8 alkyl. For example, in some embodiments, C1-C8alkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0324] In different embodiments of Formula (III), R1or R2, or both, has one of the following structures:Attorney Docket No.: 2013237-0773 ;, CN, -C(=O)OR4, -OC(=O)R4or –NHC(=O)R4. In some embodiments, R4is methyl or ethyl.

[0326] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in in Table 5 below. Table 5: Exemplary Cationic Lipid Structures of Formula (III) No. Structure III-1   O H O N O III-2 O O   III-3  Attorney Docket No.: 2013237-0773 No. Structure III-4   III-5   III-6   III-7   III-8   III-9   III-10  Attorney Docket No.: 2013237-0773 No. Structure III-11   III-12   III-13   III-14   III-15   III-16   III-17  Attorney Docket No.: 2013237-0773 No. Structure III-18   III-19   III-20   III-21   III-22   III-23   III-24  Attorney Docket No.: 2013237-0773 No. Structure III-25   III-26   III-27   III-28   III-29   H O N O OH O III-30 O O   III-31  Attorney Docket No.: 2013237-0773 No. Structure III-32   III-33   III-34   III-35   III-36

[0327] In various different embodiments, a cationic lipid has one of the structures set forth in Table 6 below. Table 6: Exemplary Cationic Lipid Structures of Formula A-F No. Structure O ANO Attorney Docket No.: 2013237-0773 No. Structure H O O N O B O O   O H O N O C O O   H O O N O D O O   O O H O E N O O   O H O N F O

[0328] In some embodiments, an LNP comprises a cationic lipid that is an ionizable lipid-like material (lipidoid). In some embodiments, a 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):Attorney Docket No.: 2013237-0773

[0329] In some embodiments, lipid nanoparticles can have an average size (e.g., mean 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 in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 60 nm to about 120 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean 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” or “mean diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as resultsAttorney Docket No.: 2013237-0773 the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, Chem. Phys.57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference). Here “average diameter,” “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.

[0330] In some embodiments, lipid nanoparticles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, lipid nanoparticles can exhibit a polydispersity index in a 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 the so-called cumulant analysis as mentioned in the definition of the “average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

[0331] Lipid nanoparticles described herein can be characterized by an “N / P ratio,” which is the molar ratio of cationic (nitrogen) groups (the “N” in N / P) in the cationic polymer to the anionic (phosphate) groups (the “P” in N / P) in RNA. It is understood that a cationic group is one that is either in cationic form (e.g., N+), or one that is ionizable to become cationic. Use of a single number in an N / P ratio (e.g., an N / P ratio of about 5) is intended to refer to that number over 1, e.g., an N / P ratio of about 5 is intended to mean 5:1. In some embodiments, a lipid nanoparticle described herein has an N / P ratio greater than or equal to 5. In some embodiments, a lipid nanoparticle described herein has an N / P ratio that is about 5, 6, 7, 8, 9, or 10. In some embodiments, an N / P ratio for a lipid nanoparticle described herein is from about 10 to about 50. In some embodiments, an N / P ratio for a lipid nanoparticle described herein is from about 10 to about 70. In some embodiments, an N / P ratio for a lipid nanoparticle described herein is from about 10 to about 120.

[0332] In some embodiments, a formulation described herein comprising lipid nanoparticles is formulated for intramuscular (i.m.) or intravenous (i.v.) delivery, and a lipid nanoparticle comprises i) about 30 to about 50 mol% of the cationic lipid; ii) about 1 to about 5 mol% of a PEG-conjugated lipid; iii) about 5 to about 15 mol% of a helper lipid; and iv) about 30 to about 50 mol% of a steroid.Attorney Docket No.: 2013237-0773

[0333] In some embodiments, a formulation described herein comprising lipid nanoparticles is formulated for intraperitoneal (i.p.) delivery, and a lipid nanoparticle comprises: i) about 30 mol% to about 50 mol% of a cationic lipid; ii) about 1 mol% to 5 mol% of a PEG-conjugated lipid; iii) about 30 mol% to about 50 mol% of a helper lipid; and iv) about 20 mol% to about 40 mol% of cholesterol. Without wishing to be limited to any theory, intraperitoneal (i.p.) delivery of these formulations is expected to lead to enhanced delivery of the lipid nanoparticles to pancreatic beta cells and enhanced in vivo expression of encoded incretin agents in pancreatic beta cells.

[0334] In some embodiments, a formulation for i.p. delivery comprises lipid nanoparticles, wherein a lipid nanoparticle comprises about 35 mol% of a cationic lipid; about 40 mol% of a helper lipid, about 22.5 mol% of cholesterol, and about 2.5 mol% of a PEG-conjugated lipid. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-2, X-3, or X-4, about 40 mol% of DOTAP, DOPE, or PS, about 22.5 mol% of cholesterol, and about 2.5 mol% of C14-PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-2; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol, and about 2.5 mol% of C14-PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-3; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-4; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14- PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-2; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-3; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-4; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-2; about 40 mol% of PS; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-3; about 40 mol% of PS; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000. In some embodiments, a lipid nanoparticle comprises about 35 mol% of cationic lipid X-4; about 40 mol% of PS; about 22.5 mol% of cholesterol;Attorney Docket No.: 2013237-0773 and about 2.5 mol% of C14-PEG2000. Without wishing to be limited to any theory, these formulations are expected to lead to enhanced delivery of the lipid nanoparticles to pancreatic beta cells and in vivo expression of encoded incretin agents in pancreatic beta cells, particularly when administered via intraperitoneal (i.p.) delivery. G. Exemplary Methods of Making Lipid Nanoparticles

[0335] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Nos.8,569,256, 5,965,542 and U.S. Patent Publication Nos.2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 086373, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 017054, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, 1999 / 009076 and PCT Pub. Nos. WO99 / 39741, WO2018 / 081480, WO2017 / 004143, WO2017 / 075531, WO2015 / 199952, WO2014 / 008334, WO2013 / 086373, WO2013 / 086322, WO2013 / 016058, WO2013 / 086373, W02011 / 141705, and WO2001 / 07548, the full disclosures each of which are herein incorporated by reference in their entirety for the purposes described herein.

[0336] For example, in some embodiments, cationic lipids, helper lipids, (e.g., DSPC, and / or cholesterol) and polymer-conjugated lipids can be solubilized in ethanol at a pre-determined molar ratio (e.g., ones described herein). In some embodiments, lipid nanoparticles (lipid nanoparticle) are prepared at a total lipid to polyribonucleotides weight ratio of approximately 10: 1 to 30: 1. In some embodiments, such polyribonucleotides can be diluted to 0.2 mg / mL in acetate buffer.

[0337] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising polyribonucleotides can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, helper lipids, and polymer- conjugated lipids, is injected into an aqueous solution comprising polyribonucleotides (e.g., ones described herein).Attorney Docket No.: 2013237-0773

[0338] In some embodiments, lipid and polyribonucleotide solutions can be mixed at room temperature by pumping each solution at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and a RNA solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid- lipid particles are formed as the ethanolic lipid solution is diluted with aqueous polyribonucleotides. The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged RNA.

[0339] In some embodiments, a solution comprising RNA-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and / or filtration.

[0340] In some embodiments, RNA-encapsulated lipid nanoparticles can be processed through filtration.

[0341] In some embodiments, particle size and / or internal structure of lipid nanoparticles (with or without RNAs) may be monitored by appropriate techniques such as, e.g., small-angle X-ray scattering (SAXS) and / or transmission electron cryomicroscopy (CryoTEM). H. Pharmaceutical Compositions

[0342] The present disclosure provides compositions, e.g., pharmaceutical compositions comprising one or more polyribonucleotides described herein. Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleteriousAttorney Docket No.: 2013237-0773 manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0343] In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0344] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0345] General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0346] In some embodiments, pharmaceutical compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

[0347] Pharmaceutical compositions described herein can be administered by appropriate methods known in the art. As will be appreciated by a skilled artisan, the route and / or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and / or pharmacokinetics and / or pharmacodynamics of pharmaceutical compositions described herein.Attorney Docket No.: 2013237-0773

[0348] In some embodiments, pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intraperitoneal, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular, or intraarticular injection and infusion. In preferred embodiments, pharmaceutical compositions described herein are formulated for intraperitoneal, intravenous, intramuscular, or subcutaneous administration.

[0349] In some embodiments, pharmaceutical compositions described herein are formulated for intraperitoneal administration. In some embodiments, pharmaceutically acceptable excipients that may be useful for intraperitoneal administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.

[0350] In some embodiments, pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable excipients that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.

[0351] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as 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 an agent that delays absorption, for example, monostearate salts and gelatin.

[0352] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination ofAttorney Docket No.: 2013237-0773 ingredients enumerated above, as required, followed by sterilization and / or microfiltration. In some embodiments, pharmaceutical compositions can be prepared as described herein and / or methods known in the art.

[0353] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, 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 pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0354] Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active agents into association with a diluent or another excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0355] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and / or method described herein.

[0356] Relative amounts of polyribonucleotides encapsulated in lipid nanoparticles, a pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition can vary, depending upon the subject to be treated, target cells, diseases or disorders, and may also further depend upon the route by which the composition is to be administered.

[0357] In some embodiments, pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods knownAttorney Docket No.: 2013237-0773 to those of skill in the art. Actual dosage levels of the active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of 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 being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions 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.

[0358] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician could start doses of active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0359] In some embodiments, a pharmaceutical composition described herein is formulated (e.g., but not limited to, for intravenous, intramuscular, or subcutaneous administration) to deliver an active dose that confers a plasma concentration of an incretin agent encoded by at least one polyribonucleotide (e.g., ones described herein) that mediates pharmacological activity via its dominant mode of action, agonists of GLP1 and / or GIP.

[0360] In some embodiments, a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions. Examples of additives may include but are not limited to salts, buffer substances, preservatives, and carriers. For example, in some embodiments, a pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and / or an aqueous buffered solution, which may in some embodiments include one or more salts, including, e.g., alkali metal salts or alkaline earth metal salts such as, e.g., sodium salts, potassium salts, and / or calcium salts.Attorney Docket No.: 2013237-0773

[0361] In some embodiments, a pharmaceutical composition described herein may further comprises one or more active agents other than at least one polyribonucleotide encoding an integrin agent. For example, in some embodiments, such an other active agent may be or comprise another known treatment for obesity or a condition or disease related to obesity. In some embodiments, an exemplary treatment may be one included in Table 1 herein.

[0362] The present disclosure provides the recognition that incretins may be useful in combination with polyribonucleotides and / or compositions provided herein, for example, for treating or preventing obesity and diseases or disorders associated with obesity. Exemplary integrins that can be used with compositions described herein include, but are not limited to, those provided in Table 1, fragments thereof, or combinations thereof.

[0363] 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 integrin agents via delivery of polyribonucleotides can achieve the same or substantially the same efficacy as known incretins and incretin mimetics that are peptide-based products (e.g., those in Table 1) but with a lower injection volume per administration.

[0364] In some embodiments, a pharmaceutical composition provided herein is a preservative-free, sterile RNA-lipid nanoparticle dispersion in an aqueous buffer for intravenous or intramuscular administration.

[0365] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation.Attorney Docket No.: 2013237-0773 I. Patient Populations

[0366] Technologies provided herein can be useful for treatment and / or prevention of obesity, or a disease or disorder associated with obesity. As described herein, technologies include polyribonucleotides encoding incretin agents, immunoglobulin chains thereof, or fragments thereof. Accordingly, the present disclosure provides pharmaceutical compositions for treatment and / or prevention of obesity and diseases or disorders associated with obesity (e.g., Type 2 Diabetes (T2D), early T1D (e.g., within 3 months after diagnosis of T1D), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease, or renal disease). In some embodiments, a pharmaceutical composition comprises a polyribonucleotides as described herein.

[0367] In some embodiments, a subject is one suffering from and / or susceptible to obesity or a disease or disorder associate with obesity. In some embodiments, a subject may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, and / or prior exposure to therapy.

[0368] In some embodiments, a subject may be determined to be classified as needing a pharmaceutical composition described herein in accordance with the screening tools for obesity and diseases and disorders associated with obesity. For example, in some embodiments, a subject may be determined to be classified as needing a pharmaceutical composition described herein according to the results obtained in enzyme immunoassays (EIA), western blot, and / or PCR test, and / or body weight, and / or waist circumference, and / or body mass index.

[0369] In some embodiments, a subject is a model organism. In preferred embodiments, a subject is a human. In some embodiments, a subject is between 18-65 years of age. In some embodiments, a subject is an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55Attorney Docket No.: 2013237-0773 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old, or from about 95 to about 100 years old.

[0370] In some embodiments, a subject is a human infant. In some embodiments, a subject is a human toddler. In some embodiments, a subject is a human child. In some embodiments, a subject is a human adult. In some embodiments, a subject is an elderly human.

[0371] In some embodiments, a subject is not currently considered obese. In some embodiments, a subject has or it as risk of developing obesity.

[0372] In some embodiments, a subject is suffering from and / or susceptible to obesity, pre-diabetes, type 2 diabetes (T2D, with its complications), early T1D, non- alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease (e.g., characterized by, e.g., a major cardiovascular event (MACE), including CV death, non-fatal myocardial infarction, non-fatal stroke, heart failure with preserved ejection fraction (HFpEF)), renal disease, or elevated risk of premature mortality.

[0373] In some embodiments, a subject is suffering from and / or susceptible to additional co-morbidities related or unrelated to obesity, including any of the following: pre- diabetes, T2D, early T1D, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease (e.g., characterized by, e.g., a major cardiovascular event (MACE), including CV death, non-fatal myocardial infarction, non- fatal stroke, heart failure with preserved ejection fraction (HFpEF)), renal disease, and elevated risk of premature mortality.

[0374] In some embodiments, a subject has not previously received treatment for obesity or a disease related to obesity.

[0375] In some embodiments, a subject suffering from and / or susceptible to obesity or a disease related to obesity may have received or is currently receiving other therapies for obesity. In some embodiments, a subject is currently receiving or has received one or more treatments listed in Table 1. In some embodiments, a subject suffering from and / orAttorney Docket No.: 2013237-0773 susceptible to obesity or a disease related to obesity may have received or is currently receiving a lifestyle intervention, e.g., reduced calorie intake and / or increased physical activity.

[0376] In some embodiments, a subject has been receiving one or more 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, a subject is responsive to another treatment at the time a polyribonucleotide, composition or pharmaceutical composition described herein is administered. In some embodiments, a subject is not responsive to another treatment at the time a polyribonucleotide, composition or pharmaceutical composition described herein is administered.

[0377] In some embodiments, a subject has previously received one or more treatments for obesity or a disease related to obesity (e.g., one or more treatments listed in Table 1). In some embodiments, a subject received prior treatment for obesity 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, a subject was responsive to the prior treatment. In some embodiments, a subject was not responsive to the prior treatment.

[0378] In some embodiments, a subject is characterized by any one of the following characteristics: a BMI of 30 or higher, a BMI of 40 or higher, waist circumference of greater than 35 inches (89 cm) (in women) or greater than 40 inches (102 cm) (in men), high blood pressure, high glucose levels and / or high cholesterol in a blood sample, high HbA1c level, underactive thyroid, liver problems and / or diabetes.

[0379] In some embodiments, a subject has received no other treatments for obesity or a disease related to obesity in the last month. In some embodiments, a subject has received no other treatments for obesity or a disease related to obesity in the last year. InAttorney Docket No.: 2013237-0773 some embodiments, a subject has received no treatments for obesity or a disease related to obesity in the last 2 years. J. Treatment Methods

[0380] In some embodiments, a pharmaceutical composition described herein can be taken up by cells for production of an encoded incretin agent at therapeutically relevant serum concentrations. Accordingly, the present disclosure provides methods of using pharmaceutical compositions described herein. For example, in some embodiments, a method provided herein comprises administering a pharmaceutical composition described herein to a subject.

[0381] As used herein, the term “administering” or “administration” typically refers to the administration of a composition to a subject to achieve delivery of an agent (e.g., at least one polyribonucleotide encoding an incretin agent described herein) that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. Administration may be, for example, bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In preferred embodiments, administration may be intramuscular, intraperitoneal, intravenous, or subcutaneous.

[0382] In some embodiments, administration of a pharmaceutical composition results in delivery of one or more polyribonucleotides as described herein (e.g., encoding an incretin agent) to a subject. In some embodiments, administering a pharmaceutical composition to a subject results in expression in the subject of an incretin agent encoded by an administered polyribonucleotide. In some embodiments, administering a pharmaceutical composition to a subject results in expression in the subject of an incretin agent encoded by an administered polyribonucleotide.Attorney Docket No.: 2013237-0773

[0383] In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve administration of a fixed number of doses.

[0384] In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0385] In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0386] Those skilled in the art are aware that therapies can be administered in dosing cycles. In some embodiments, pharmaceutical compositions described herein are administered in one or more dosing cycles.

[0387] In some embodiments, one dosing cycle is at least 3 or more days (including, e.g., at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 days). In some embodiments, one dosing cycle is at least 21 days.

[0388] In some embodiments, one dosing cycle may involve multiple doses, e.g., according to a pattern such as, for example, a dose may be administered daily within aAttorney Docket No.: 2013237-0773 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, monthly, every 2 months within a cycle.

[0389] In some embodiments, multiple dosing cycles may be administered. For example, in some embodiments, at least 2 dosing cycles (including, e.g., 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) can be administered. In some embodiments, the number of dosing cycles to be administered may vary with types of treatment (e.g., monotherapy vs. combination therapy). In some embodiments, at least 3-8 dosing cycles may be administered.

[0390] In some embodiments, there may be a “rest period” between dosing cycles; in some embodiments, there may be no rest period between dosing cycles. In some embodiments, there may be sometimes a rest period and sometimes no rest period between dosing cycles.

[0391] In some embodiments, a rest period may have a length within a range of several days to several months. For example, in some embodiments, a rest period may have a length of at least 3 days or more, including, e.g., 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, a rest period may have a length of at least 1 week or more, including, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, or more.

[0392] Dosage of pharmaceutical compositions described herein may vary with a number of factors including, e.g., but not limited to body weight of a subject to be treated, cancer types and / or cancer stages, and / or monotherapy or combination therapy. In some embodiments, a dosing cycle involves administration of a set number and / or pattern of doses. For example, in some embodiments, a pharmaceutical composition described herein is administered at least one dose per dosing cycle, including, e.g., at least two doses per dosing cycle, at least three doses per dosing cycle, at least four doses per dosing cycle, or more.

[0393] In some embodiments, a dosing cycle involves administration of a set cumulative dose, e.g., over a particular period of time, and optionally via multiple doses,Attorney Docket No.: 2013237-0773 which may be administered, for example, at set interval(s) and / or according to a set pattern. In some embodiments, a set cumulative dose may be administered via multiple doses at set intervals such that there is at least some temporal overlap in biological and / or pharmacokinetics effects generated by such multiple doses on a target cell or on a subject being treated. In some embodiments, a set cumulative dose may be administered via multiple doses at set intervals such that biological and / or pharmacokinetics effects generated by such multiple doses on a target cell or on a subject being treated may be additive. By way of example only, in some embodiments, a set cumulative dose of X mg may be administered via two doses with each dose of X / 2 mg, wherein such two doses are administered sufficiently close in time such that biological and / or pharmacokinetics effects generated by each X / 2 mg dose on a target cell or on a subject being treated may be additive.

[0394] In some embodiments, dosing may be adjusted based on response of a subject receiving the therapy. For example, in some embodiments, dosing may involve administration of a higher dose followed later by administration of a lower dose if one or more parameters for safety pharmacology assessment indicates that the prior dose may not satisfy the medical safety requirement according to a physician. In some embodiments, dose escalation may be performed at one or more of the levels. Without wishing to be bound by any particular theory, the present disclosure, among other things, provides an insight that a pharmaceutically guided dose escalation (PGDE) method may be applied to determine an appropriate dose of pharmaceutical compositions described herein.

[0395] In some embodiments, pharmaceutical compositions described herein can be administered to subjects as monotherapy.

[0396] In some embodiments, a pharmaceutical composition provided herein may be administered as part of combination therapy. In some embodiments, a pharmaceutical composition provided herein may be administered as part of a combination therapy comprising the pharmaceutical composition and one or more incretin agents. In some embodiments, one or more incretin agents can comprise any one of the incretin peptides as shown in Table 2, or a combination thereof.Attorney Docket No.: 2013237-0773

[0397] In some embodiments, a pharmaceutical composition provided herein may be administered as part of a combination therapy comprising the pharmaceutical composition and another therapy such as those described in Table 2.

[0398] In some embodiments, subjects receiving a composition provided herein (e.g., a pharmaceutical composition) may be monitored periodically over a dosing regimen to assess efficacy of the administered treatment. For example, in some embodiments, efficacy of an 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. K. Methods of Manufacture

[0399] Individual polyribonucleotides can be produced by methods known in the art. For example, in some embodiments, polyribonucleotides can be produced by in vitro transcription, for example, using a DNA template. A plasmid DNA used as a template for in vitro transcription to generate a polyribonucleotide described herein is also within the scope of the present disclosure.

[0400] A DNA template is used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase (e.g., a recombinant RNA-polymerase such as a T7 RNA- polymerase) with ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP). In some embodiments, polyribonucleotides (e.g., ones described herein) can be synthesized in the presence of modified ribonucleotide triphosphates. By way of example only, in some embodiments, pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP). In some embodiments, pseudouridine (ψ) can be used to replace uridine triphosphate (UTP). In some embodiments, N1-methyl-pseudouridine (m1ψ) can be used to replace uridine triphosphate (UTP). In some embodiments, 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP).

[0401] As will be clear to those skilled in the art, during in vitro transcription, an RNA polymerase (e.g., as described and / or utilized herein) typically traverses at least a portion of a single-stranded DNA template in the 3'→ 5' direction to produce a single- stranded complementary RNA in the 5'→ 3' direction.

[0402] In some embodiments where a polyribonucleotide comprises a polyA tail, one of skill in the art will appreciate that such a polyA tail may be encoded in a DNAAttorney Docket No.: 2013237-0773 template, e.g., by using an appropriately tailed PCR primer, or it can be added to a polyribonucleotide after in vitro transcription, e.g., by enzymatic treatment (e.g., using a poly(A) polymerase such as an E. coli Poly(A) polymerase). Suitable poly(A) tails are described herein above. In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 40).

[0403] In some embodiments, those skilled in the art will appreciate that addition of a 5' cap to an RNA (e.g., mRNA) can facilitate recognition and attachment of the RNA to a ribosome to initiate translation and enhances translation efficiency. Those skilled in the art will also appreciate that a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life. Methods for capping are known in the art; one of ordinary skill in the art will appreciate that in some embodiments, capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme- based capping system such as, e.g., capping enzymes of vaccinia virus). In some embodiments, a cap may be introduced during in vitro transcription, along with a plurality of ribonucleotide triphosphates such that a cap is incorporated into a polyribonucleotide during transcription (also known as co-transcriptional capping). In some embodiments, a GTP fed- batch procedure with multiple additions in the course of the reaction may be used to maintain a low concentration of GTP in order to effectively cap the RNA. Suitable 5' cap are described herein above. For example, in some embodiments, a 5' cap comprises m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0404] Following RNA transcription, a DNA template is digested. In some embodiments, digestion can be achieved with the use of DNase I under appropriate conditions.

[0405] In some embodiments, in-vitro transcribed polyribonucleotides may be provided in a buffered solution, for example, in a buffer such as HEPES, a phosphate buffer solution, a citrate buffer solution, an acetate buffer solution; in some embodiments, such solution may be buffered to a pH within a range of, for example, about 6.5 to about 7.5; in some embodiments approximately 7.0. In some embodiments, production of polyribonucleotides may further include one or more of the following steps: purification, mixing, filtration, and / or filling.Attorney Docket No.: 2013237-0773

[0406] In some embodiments, polyribonucleotides can be purified (e.g., in some embodiments after in vitro transcription reaction), for example, to remove components utilized or formed in the course of the production, like, e.g., proteins, DNA fragments, and / or or nucleotides. Various nucleic acid purifications that are known in the art can be used in accordance with the present disclosure. Certain purification steps may be or include, for example, one or more of precipitation, column chromatography (including, e.g., but not limited to anionic, cationic, hydrophobic interaction chromatography (HIC)), solid substrate- based purification (e.g., magnetic bead-based purification). In some embodiments, polyribonucleotides may be purified using magnetic bead-based purification, which in some embodiments may be or comprise magnetic bead-based chromatography. In some embodiments, polyribonucleotides may be purified using hydrophobic interaction chromatography (HIC) and / or diafiltration. In some embodiments, polyribonucleotides may be purified using HIC followed by diafiltration.

[0407] In some embodiments, dsRNA may be obtained as side product during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination. For example, in some embodiments, cellulose materials (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, for examples in some embodiments in a chromatographic format. In some embodiments, cellulose materials (e.g., microcrystalline cellulose) can be pretreated to inactivate potential RNase contamination, for example in some embodiments by autoclaving followed by incubation with aqueous basic solution, e.g., NaOH. In some embodiments, cellulose materials may be used to purify polyribonucleotides according to methods described in WO2017 / 182524, the entire content of which is incorporated herein by reference.

[0408] In some embodiments, a batch of polyribonucleotides may be further processed by one or more steps of filtration and / or concentration. For example, in some embodiments, polyribonucleotide(s), for example, after removal of dsRNA contamination, may be further subject to diafiltration (e.g., in some embodiments by tangential flow filtration), for example, to adjust the concentration of polyribonucleotides to a desirable RNA concentration and / or to exchange buffer to a drug substance buffer.

[0409] In some embodiments, polyribonucleotides may be processed through 0.2 μm filtration before they are filled into appropriate containers.Attorney Docket No.: 2013237-0773

[0410] In some embodiments, polyribonucleotides and compositions thereof may be manufactured in accordance with a process as described herein, or as otherwise known in the art.

[0411] In some embodiments, polyribonucleotides and compositions thereof may be manufactured at a large scale. For example, in some embodiments, a batch of polyribonucleotides can be manufactured at a scale of greater than 1 g, greater than 2 g, greater than 3 g, 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 higher.

[0412] In some embodiments, RNA quality control may be performed and / or monitored at any time during production process of 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 natures), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and / or monitored after each or certain steps of a polyribonucleotide manufacturing process, e.g., after in vitro transcription, and / or each purification step.

[0413] In some embodiments, the stability of polyribonucleotides (e.g., produced by in vitro transcription) and / or compositions comprising one or more RNAs can be assessed under various test storage conditions, for example, at room temperatures vs. fridge or sub- zero temperatures over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer). In some embodiments, polyribonucleotides (e.g., ones described herein) and / or compositions thereof may be stored stable at a fridge temperature (e.g., about 4°C to about 10°C) for at least 1 month or longer 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 longer. In some embodiments, polyribonucleotides (e.g., ones described herein) and / or compositions thereof may be stored stable at a sub-zero temperature (e.g., - 20°C or below) for at least 1 month or longer 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 longer. In some embodiments, polyribonucleotides (e.g., ones described herein) and / or compositionsAttorney Docket No.: 2013237-0773 thereof may be stored stable at room temperature (e.g., at about 25°C) for at least 1 month or longer.

[0414] In some embodiments, one or more assessments may be utilized during manufacture, or other preparation or use of polyribonucleotides (e.g., as a release test).

[0415] In some embodiments, one or more quality control parameters may be assessed to determine whether polyribonucleotides described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and / or release for distribution). 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 of skill in the art will recognize that in some embodiments, one or more analytical tests can 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 assay.

[0416] In some embodiments, a batch of polyribonucleotides may be assessed for one or more features as described herein to determine next action step(s). For example, a batch of polyribonucleotides can be designated for one or more further steps of manufacturing and / or formulation and / or distribution if RNA quality assessment indicates that such a batch of polyribonucleotides meet or exceed the relevant acceptance criteria. Otherwise, an alternative action can be taken (e.g., discarding the batch) if such a batch of polyribonucleotides does not meet or exceed the acceptance criteria.

[0417] In some embodiments, a batch of polyribonucleotides that satisfy assessment results can be utilized for one or more further steps of manufacturing and / or formulation and / or distribution. L. DNA Constructs

[0418] Among other things, the present disclosure provides DNA constructs, for example that may encode one or more incretin agents as described herein, or components thereof. In some embodiments, DNA constructs provided by and / or utilized in accordance with the present disclosure are comprised in a vector.Attorney Docket No.: 2013237-0773

[0419] Non-limiting examples of a vector include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). In some embodiments, a vector is an expression vector. In some embodiments, a vector is a cloning vector. In general, a vector is a nucleic acid construct that can receive or otherwise become linked to a nucleic acid element of interest (e.g., a construct that is or encodes a payload, or that imparts a particular functionality, etc.).

[0420] Expression vectors, which may be plasmid or viral or other vectors, typically include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked with one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in a system of interest. In some embodiments, a system is ex vivo (e.g., an in vitro transcription system); in some embodiments, a system is in vivo (e.g., a bacterial, yeast, plant, insect, fish, vertebrate, mammalian cell or tissue, etc.).

[0421] Cloning vectors are generally used to modify, engineer, and / or duplicate (e.g., by replication in vivo, for example in a simple system such as bacteria or yeast, or in vitro, such as by amplification such as polymerase chain reaction or other amplification process). In some embodiments, a cloning vector may lack expression signals.

[0422] In many embodiments, a vector may include replication elements such as primer binding site(s) and / or origin(s) of replication. In many embodiments, a vector may include insertion or modification sites such as restriction endonuclease recognition sites and / or guide RNA binding sites, etc.

[0423] In some embodiments, a vector is a viral vector (e.g., an AAV vector). In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.

[0424] Those skilled in the art are aware of a variety of technologies useful for the production of recombinant polynucleotides (e.g., DNA or RNA) as described herein. For example, restriction digestion, reverse transcription, amplification (e.g., by polymerase chain reaction), Gibson assembly, etc., are well established and useful tools and technologies.Attorney Docket No.: 2013237-0773 Alternatively or additionally, certain nucleic acids may be prepared or assembled by chemical and / or enzymatic synthesis. In some embodiments, a combination of known methods is utilized to prepare a recombinant polynucleotide.

[0425] In some embodiments, polynucleotide(s) of the present disclosure are included in a DNA construct (e.g., a vector) amenable to transcription and / or translation.

[0426] In some embodiments, an expression vector comprises a polynucleotide that encodes proteins and / or polypeptides of the present disclosure operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized to achieve a desired level of expression of a plurality of polynucleotides that encode a plurality proteins and / or polypeptides. In some embodiments, a plurality of recombinant proteins and / or polypeptides are expressed from the same vector (e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic). In some embodiments, a plurality of polypeptides are expressed, each of which is expressed from a separate vector.

[0427] In some embodiments, an expression vector comprising a polynucleotide of the present disclosure is used to produce a RNA and / or protein and / or polypeptide in a host cell. In some embodiments, a host cell may be in vitro (e.g., a cell line) – for example a cell or cell line (e.g., Human Embryonic Kidney (HEK cells), Chinese Hamster Ovary cells, etc.) suitable for producing polynucleotides of the present disclosure and proteins and / or polypeptides encoded by said polynucleotides.

[0428] In some embodiments, an expression vector is an RNA expression vector. In some embodiments, an RNA expression vector comprises a polynucleotide template used to produce a RNA in cell-free enzymatic mix. In some embodiments, an RNA expression vector comprising a polynucleotide template is enzymatically linearized prior to in vitro transcription. In some embodiments, a polynucleotide template is generated through PCR as a linear polynucleotide template. In some embodiments, a linearized polynucleotide is mixedAttorney Docket No.: 2013237-0773 with enzymes suitable for RNA synthesis, RNA capping and / or purification. In some embodiments, the resulting RNA is suitable for producing proteins encoded by the RNA.

[0429] A variety of methods are known in the art to introduce an expression vector into host cells. In some embodiments, a vector may be introduced into host cells using transfection. In some embodiments, transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection. In some embodiments, a vector may be introduced into a host cell using transduction. In some embodiments, transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides. In some embodiments, a 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 longer. Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected. A skilled artisan would recognize culture conditions for host cells selected are well known in the art. EXEMPLIFICATION Example 1: Generating polyribonucleotides encoding exemplary incretin agents

[0430] The present Example describes methods for generating polyribonucleotide sequences encoding an incretin agent. The present Example further describes the design of polyribonucleotides that can achieve transient in vivo incretin agent production following i.p. / i.v. / i.m. / s.c. delivery.

[0431] Methods of the present example include: (1) Cloning of DNA fragments encoding incretin agents (e.g., GLP1, GIP or variants thereof) into a DNA plasmid appropriate to use for RNA expression. An appropriate DNA plasmid 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. The DNA plasmid also typically includes restriction sites that enable cloning of DNA fragments encoding incretin agents downstream of regions encoding the 5’ UTR and Kozak sequence and upstream of the regionsAttorney Docket No.: 2013237-0773 encoding the 3’ UTR and polyA tail sequence. Examples of appropriate DNA plasmids can also be found in WO2021 / 214204, which is hereby incorporated by reference in its entirety. (2) Verification of selected clones by control digestion and optionally sequencing. (3) Linearization of DNA plasmids encoding incretin agents. (4) Synthesis of polyribonucleotides encoding incretin agents. (5) Biochemical characterization of polyribonucleotides encoding incretin agents. (6) Transfection of polyribonucleotides encoding incretin agents into HEK cells and quantification of incretin agent levels. Codon Optimization

[0432] For optimal expression of exemplary incretins, DNA sequences are generated based on the amino acid sequences of GLP1 (7-37), GIP (1-42) and truncation or mutation variants thereof fused to an exemplary signal peptide (SP) sequence (SP1-2, MRVLVLLACLAAASNA) as shown in Table 7: Table 7: Sequences of exemplary incretin agents Incretin agent Sequence (SP in bold) SEQ ID NO GLP1 (7-37) MRVLVLLACLAAASNAHAEGTFTSDVSSYL 41 EGQAAKEFIAWLVKGRG GLP1 (7-37) MRVLVLLACLAAASNAHAEGTFTSDVSSYL 42 (Arg34 mutant) EGQAAKEFIAWLVRGRG GLP1 (7-36) MRVLVLLACLAAASNAHAEGTFTSDVSSYL 43 EGQAAKEFIAWLVKGR GIP (1-42) MRVLVLLACLAAASNAYAEGTFISDYSIAM 44 DKIHQQDFVNWLLAQKGKKNDWKHNITQ GIP (1-30) MRVLVLLACLAAASNAYAEGTFISDYSIAM 45 DKIHQQDFVNWLLAQKAttorney Docket No.: 2013237-0773

[0433] The amino acid sequences are translated to DNA nucleotide sequences. Restriction sites, e.g., for Eam1104I (GAAGAG), BamHI (GGATCC), PstI (CTGCAG), SbfI (CCTGCAGG), XhoI (CTCGAG), SpeI (ACTAGT), BspEI (TCCGGA), SacI (GAGCTC), Ear1 (CTCTTCN^NNN) and NheI (GCTAGC) are optionally eliminated after optimization if any of these enzymes (or others) are used either for linearization or for cloning of the plasmid. Sequences are also examined for the presence of regions showing high homology to the T7 RNA polymerase termination signal sequence “ATCTGTT” followed by multiple “T” residues.

[0434] Optimization is carried out with the GeneOptimizer® software provided by Life Technologies GmbH GeneArt®. This software adjusts the codon usage by using the most frequent codons and adapts the GC-content of an uploaded sequence for a chosen expression system, in this case Homo sapiens. At the same time, GeneOptimizer® removes sequence repeats, introns, cryptic splice sites, internal ribosome entry sites and RNA destabilizing sequence elements (e.g., UpA-dinucleotides), adds RNA stabilizing sequence elements (e.g., CpG-dinucleotides) and avoids stable RNA secondary structures as well as unwanted sequences such as restriction sites. The output sequence is then used for ordering of DNA fragment strings. Those of skill in the art would be aware that alternative methods for codon optimization are available. Moreover, additional information on codon optimization approaches is provided herein. Cloning

[0435] Each incretin sequence is cloned into the DNA plasmid, e.g., pST5. This can for example be done by in vivo assembly. Garcia-Nafria, “IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly,” Scientific Reports volume 6, Article number: 27459 (2016), which is incorporated herein by reference in its entirety. Plasmid DNA Preparation

[0436] Plasmid DNA is prepared by selecting clones for inoculation in culture media. Selected clones are optionally verified by control digestion and optionally sequencing. Cultures are grown and following cell harvest, purification is done, e.g., using the QIAGEN Plasmid Plus Maxi Kit according to the manufacturer’s instructions. DNAAttorney Docket No.: 2013237-0773 concentration may be determined by UV spectroscopy. DNA is stored in certified RNase- and DNase-free reaction tubes. Linearization and DNA purification

[0437] Linearization of plasmid DNA is performed using appropriate restriction enzymes, followed by purification of the linearized DNA template using magnetic beads, e.g., Dynabeads™ MyOne™ Carboxylic Acid according to the manufacturer’s protocol. DNA concentration is measured by UV spectroscopy, control digestion and optionally sequencing. In Vitro Transcription

[0438] RNA, optionally capped RNA, is then produced, e.g., following the process as disclosed, e.g., 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 pseudo-uridine may be used in the in vitro transcription reaction and incorporated into the produced RNA. Cellulose purification of the resulting RNA is performed to isolate single- stranded RNA, followed by concentration measurement by UV spectroscopy. RNA integrity is determined by microfluidic-based electrophoresis. Further biochemical characterization of the resulting RNA is optionally performed. Transfection and Expression

[0439] RNA encoding incretin agents is transfected into HEK cells, e.g., via electroporation and resulting incretin agent levels are quantified. HEK cells, e.g., HEK293T cells are washed with cooled medium. Electroporation is performed in pre-cooled cuvettes. Cells and RNA in each sample are at typical concentrations for RNA electroporation. Cells are incubated on ice after electroporation.

[0440] Cells are then transferred into expression medium, e.g., Expi293 Medium and counted. Cells are seeded at typical concentrations for expression and incubated, e.g., for 48 hours at 37°C. Supernatants are then harvested by centrifugation of the cells, followed by careful aspiration so as to not disturb the cell pellet, and then stored at 4°C.

[0441] Expression of incretin agents is quantified, e.g., via ELISA or Western blot analysis of the cell culture supernatants.Attorney Docket No.: 2013237-0773 Example 2: Generating reporter cell lines to monitor incretin activity

[0442] The present Example describes methods for generating reporter cell lines to monitor incretin activity.

[0443] Methods of the present example include: (1) Cloning of DNA fragments encoding incretin receptors (e.g., GLP1R and / or GIPR) into a DNA plasmid (e.g., pT2). (2) Stably transfecting HEK293 cells with the DNA plasmid encoding incretin receptors and mRNA transposase. (3) Sorting cells by FACS for high, medium and low incretin receptor expression, e.g., GLP1R and / or GIPR expression, optionally with a bulk sort followed by a single cell sort. (4) Confirming stable expression of incretin receptors, e.g., GLP1R and / or GIPR. (5) Generating a master cell bank. Cloning

[0444] Exemplary DNA sequences encoding incretin receptor sequences are shown in Table 8. These DNA sequences are cloned into a DNA plasmid, e.g., pT2. GLP1R_mutR and GLP1R_mutL encode slightly different variants of GLP1R (GLP1R_mutR encodes a GLP1R with a L260F mutation relative to the GLP1R encoded by GLP1R_mutL). This can for example be done by in vivo assembly. Garcia-Nafria, “IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly,” Scientific Reports volume 6, Article number: 27459 (2016), which is incorporated herein by reference in its entirety. Table 8: Sequences encoding exemplary incretin receptors Receptor Sequence SEQ ID NO GLP1R_mutL atggccggcgcccccggcccgctgcgccttgcgctgctgctgctcgggat 46 ggtgggcagggccggcccccgcccccagggtgccactgtgtccctctggAttorney Docket No.: 2013237-0773 gagacggtgcagaaatggcgagaataccgacgccagtgccagcgctccc tgactgaggatccacctcctgccacagacttgttctgcaaccggaccttcgat gaatacgcctgctggccagatggggagccaggctcgttcgtgaatgtcagc tgcccctggtacctgccctgggccagcagtgtgccgcagggccacgtgta ccggttctgcacagctgaaggcctctggctgcagaaggacaactccagcct gccctggagggacttgtcggagtgcgaggagtccaagcgaggggagaga agctccccggaggagcagctcctgttcctctacatcatctacacggtgggct acgcactctccttctctgctctggttatcgcctctgcgatcctcctcggcttcag acacctgcactgcacccggaactacatccacctgaacctgtttgcatccttca tcctgcgagcattgtccgtcttcatcaaggacgcagccctgaagtggatgtat agcacagccgcccagcagcaccagtgggatgggctcctctcctaccagga ctctctgagctgccgcctggtgtttctgctcatgcagtactgtgtggcggcca attactactggctcttggtggagggcgtgtacctgtacacactgctggccttct cggtcttAtctgagcaatggatcttcaggctctacgtgagcataggctgggg tgttcccctgctgtttgttgtcccctggggcattgtcaagtacctctatgaggac gagggctgctggaccaggaactccaacatgaactactggctcattatccgg ctgcccattctctttgccattggggtgaacttcctcatctttgttcgggtcatctg catcgtggtatccaaactgaaggccaatctcatgtgcaagacagacatcaaa tgcagacttgccaagtccacgctgacactcatccccctgctggggactcatg aggtcatctttgcctttgtgatggacgagcacgcccgggggaccctgcgctt catcaagctgtttacagagctctccttcacctccttccaggggctgatggtgg ccatcttatactgctttgtcaacaatgaggtccagctggaatttcggaagagct gggagcgctggcggcttgagcacttgcacatccagagggacagcagcat gaagcccctcaagtgtcccaccagcagcctgagcagtggagccacggcg ggcagcagcatgtacacagccacttgccaggcctcctgcagctgatgaTG gcgacctaggcatatgtgtacaacgcgtga GLP1R_mutF atggccggcgcccccggcccgctgcgccttgcgctgctgctgctcgggat 47 ggtgggcagggccggcccccgcccccagggtgccactgtgtccctctgg gagacggtgcagaaatggcgagaataccgacgccagtgccagcgctccc tgactgaggatccacctcctgccacagacttgttctgcaaccggaccttcgat gaatacgcctgctggccagatggggagccaggctcgttcgtgaatgtcagc tgcccctggtacctgccctgggccagcagtgtgccgcagggccacgtgta ccggttctgcacagctgaaggcctctggctgcagaaggacaactccagcct gccctggagggacttgtcggagtgcgaggagtccaagcgaggggagaga agctccccggaggagcagctcctgttcctctacatcatctacacggtgggct acgcactctccttctctgctctggttatcgcctctgcgatcctcctcggcttcag acacctgcactgcacccggaactacatccacctgaacctgtttgcatccttca tcctgcgagcattgtccgtcttcatcaaggacgcagccctgaagtggatgtat agcacagccgcccagcagcaccagtgggatgggctcctctcctaccagga ctctctgagctgccgcctggtgtttctgctcatgcagtactgtgtggcggcca attactactggctcttggtggagggcgtgtacctgtacacactgctggccttct cggtcttctctgagcaatggatcttcaggctctacgtgagcataggctggggt gttcccctgctgtttgttgtcccctggggcattgtcaagtacctctatgaggac gagggctgctggaccaggaactccaacatgaactactggctcattatccgg ctgcccattctctttgccattggggtgaacttcctcatctttgttcgggtcatctg catcgtggtatccaaactgaaggccaatctcatgtgcaagacagacatcaaa tgcagacttgccaagtccacgctgacactcatccccctgctggggactcatg aggtcatctttgcctttgtgatggacgagcacgcccgggggaccctgcgcttAttorney Docket No.: 2013237-0773 catcaagctgtttacagagctctccttcacctccttccaggggctgatggtgg ccatcttatactgctttgtcaacaatgaggtccagctggaatttcggaagagct gggagcgctggcggcttgagcacttgcacatccagagggacagcagcat gaagcccctcaagtgtcccaccagcagcctgagcagtggagccacggcg ggcagcagcatgtacacagccacttgccaggcctcctgcagctgatgaTG gcgacctaggcatatgtgtacaacgcgtga GIPR atggatgactacctctccgatcctgcagctgctgctgcggctctcactgtgcg 48 ggctgctgctccagagggcggagacaggctctaaggggcagacggcgg gggagctgtaccagcgctgggaacggtaccgcagggagtgccaggaga ccttggcagccgcggaaccgccttcaggcctcgcctgtaacgggtccttcg atatgtacgtctgctgggactatgctgcacccaatgccactgcccgtgcgtc ctgcccctggtacctgccctggcaccaccatgtggctgcaggtttcgtcctc cgccagtgtggcagtgatggccaatggggactttggagagaccatacaca atgtgagaacccagagaagaatgaggcctttctggaccaaaggctcatcttg gagcggttgcaggtcatgtacactgtcggctactccctgtctctcgccacact gctgctagccctgctcatcttgagtttgttcaggcggctacattgcactagaaa ctatatccacatcaacctgttcacgtctttcatgctgcgagctgcggccattct cagccgagaccgtctgctacctcgacctggcccctaccttggggaccagg cccttgcgctgtggaaccaggccctcgctgcctgccgcacggcccagatc gtgacccagtactgcgtgggtgccaactacacgtggctgctggtggaggg cgtctacctgcacagtctcctggtgctcgtgggaggctccgaggagggcca cttccgctactacctgctcctcggctggggggcccccgcgcttttcgtcattc cctgggtgatcgtcaggtacctgtacgagaacacgcagtgctgggagcgc aacgaagtcaaggccatttggtggattatacggacccccatcctcatgacca tcttgattaatttcctcatttttatccgcattcttggcattctcctgtccaagctgag gacacggcaaatgcgctgccgggattaccggctgaggctggctcgctcca cgctgacgctggtgcccctgctgggtgtccacgaggtggtgtttgctcccgt gacagaggaacaggcccggggcgccctgcgcttcgccaagctcggctttg agatcttcctcagctccttccagggcttcctggtcagcgtcctctactgcttcat caacaaggaggtgcagtcggagatccgccgtggctggcaccactgccgc ctgcgccgcagcctgggcgaggagcaacgccagctcccggagcgcgcc ttccgggccctgccctccggctccggcccgggcgaggtccccaccagcc gcggcttgtcctcggggaccctcccagggcctgggaatgaggccagccg ggagttggaaagttactgcttgccaactttcttgtacaaagttggcattataag aaagcattgcttatcaatttgttgcaacgaatgatgaTGgcgacctaggcat atgtgtacaacgcgtga Plasmid DNA Preparation

[0445] Plasmid DNA is prepared by selecting clones for inoculation in culture media. Selected clones are optionally verified by control digestion and optionally sequencing. Cultures are grown and following cell harvest, purification is done, e.g., using the QIAGEN Plasmid Plus Maxi Kit according to the manufacturer’s instructions. DNAAttorney Docket No.: 2013237-0773 concentration may be determined by UV spectroscopy. DNA is stored in certified RNase- and DNase-free reaction tubes. Transfection

[0446] Plasmid DNA encoding incretin receptor(s) and RNA encoding a transposase, e.g., SB100X transposase are transfected into HEK cells, e.g., via electroporation. HEK cells, e.g., HEK293 cells are washed with cooled medium. Electroporation is performed in pre-cooled cuvettes. Cells, plasmid DNA and RNA in each sample are at typical concentrations for electroporation. Cells are incubated on ice after electroporation. Cell Sorting

[0447] Cells are then sorted by FACS, e.g., using a BD FACSMelody™ Cell Sorter for high, medium and low incretin receptor expression, e.g., GLP1R and / or GIPR expression, optionally with a bulk sort followed by a single cell sort. Stable expression of incretin receptors is confirmed for selected clones and master cell banks are generated. Resulting cell lines may include one or more of the following: HEK293_GLP1Rhi, HEK293_GLP1Rmed, HEK293_GLP1Rlow, HEK293_GIPRhi, HEK293_GIPRmed, HEK293_GIPRlow, HEK293_GLP1Rhi / GIPRhi, HEK293_GLP1Rmed / GIPRmed, and HEK293_GLP1Rlow / GIPRlow. Example 3: Assessing functionality of polyribonucleotide encoded incretins

[0448] The present Example describes methods for assessing the functionality of polyribonucleotide encoded incretins.

[0449] Methods of the present example include: (1) Transfecting HEK293_GLP1R / GIPR reporter cells of Example 2 with GLP1 or GIP RNA of Example 1. Reporter cells may be incubated for, e.g., 24 hours after transfection before supernatant is collected. (2) Quantifying downstream signal induction by measuring cAMP release, e.g., in the supernatant, e.g., using a cAMP-Glo™ assay or an ELISA.Attorney Docket No.: 2013237-0773

[0450] Methods of the present example also include: (1) Transfecting wild type HEK293 cells with GLP1 or GIP RNA of Example 1. (2) Collecting supernatant containing GLP1 and GIP. (3) Incubating HEK293_GLP1R / GIPR reporter cells of Example 2 with supernatant. Reporter cells may be incubated for, e.g., 24 hours before supernatant is collected. (4) Quantifying downstream signal induction by measuring cAMP release, e.g., in the supernatant, e.g., using a cAMP-Glo™ assay or an ELISA. EQUIVALENTS

[0451] 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 present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

Attorney Docket No.: 2013237-0773 CLAIMS We claim:

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 GLP1 / GIP dual receptor agonist.

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

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

6. The composition of claim 2, wherein the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NOs: 5-7.

7. The composition of claim 3, wherein the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NOs: 8-9.

8. The composition of claim 4, wherein the incretin agent comprises an amino acid sequence according to SEQ ID NO:

10.

9. The composition of claim 5, wherein the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NOs: 11-15.

10. The composition of any one of claims 6-7 or 9, wherein the incretin peptide is fused to a signal peptide, optionally via the N-terminus of the incretin peptide, optionally via a linker.

11. The composition of claim 10, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NOs: 16-39.

12. The composition of claim 10, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NOs: 16-21.Attorney Docket No.: 2013237-0773 13. The composition of claim 10, wherein the signal peptide has an amino acid sequence according to SEQ ID NO:

17.

14. The composition of claim 1, wherein the incretin agent comprises an incretin peptide fused to a signal peptide that comprises an amino acid sequence according to any one of SEQ ID NOs: 41-45.

15. The composition of any one of claims 1-14, wherein the incretin agent comprises an incretin peptide fused to a half-life extending moiety, optionally via the C-terminus or N- terminus of the incretin peptide, optionally via a linker.

16. The composition of claim 15, wherein the half-life extending moiety comprises a peptide moiety that binds albumin.

17. The composition of claim 16, wherein the half-life extending moiety comprises an albumin binding domain (ABD) derived from protein G of Streptococcus strain GI48 and / or from protein PAB of Finegoldia magna, such as ABD035 and SA21.

18. The composition of claim 16, wherein the half-life extending moiety comprises an albumin binding domain (ABD) that binds to domain II of human serum albumin and does not overlap or interfere with binding to the FcRn-binding site on albumin.

19. The composition of claim 16, wherein the half-life extending moiety comprises ABDCon.

20. The composition of claim 16, wherein the half-life extending moiety comprises an albumin binding domain (ABD) derived from the bacterial protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus, such as M11.12 and M18.2.

5.

21. The composition of claim 15, wherein the half-life extending moiety comprises a DARPin that binds albumin.

22. The composition of claim 15, wherein the half-life extending moiety comprises an immunoglobulin domain or fragment thereof.

23. The composition of claim 15, wherein the half-life extending moiety comprises a fully human domain antibody (dAb) that binds albumin, such as AlbudAb.Attorney Docket No.: 2013237-0773 24. The composition of claim 15, wherein the half-life extending moiety comprises a Fab that binds albumin, such as dsFv CA645.

25. The composition of claim 15, wherein the half-life extending moiety comprises a Fc domain, such as from a human IgG, optionally from a human IgG1, IgG2, IgG3, or IgG4.

26. The composition of claim 15, wherein the half-life extending moiety does not comprise a Fc domain, such as from a human IgG, optionally from a human IgG1, IgG2, IgG3, or IgG4.

27. The composition of claim 16, wherein the half-life extending moiety comprises a heavy chain only (VHH) antibody, such as a nanobody, that binds albumin.

28. The composition of claim 16, wherein the half-life extending moiety comprises a VNAR that binds albumin.

29. The composition of claim 15, wherein the half-life extending moiety comprises an XTEN sequence.

30. The composition of any one of claims 15-29, wherein the incretin peptide is fused to the half-life extending moiety via a linker.

31. The composition of claim 30, wherein the linker is (a) fused to the C-terminus of the incretin peptide and the N-terminus of the half-life extending moiety or (b) fused to the C- terminus of the half-life extending moiety and the N-terminus of the incretin peptide.

32. The composition of claim 30 or 31, wherein the linker has an amino acid sequence of any one of SEQ ID NOs: 1-5.

33. The composition of any one of claims 1-32, wherein the polyribonucleotide comprises at least one non-coding sequence element that enhances RNA stability and / or translation efficiency.

34. The composition of claim 33, wherein the at least one non-coding sequence element comprises a 5’ cap structure, a 5’ UTR, a 3’ UTR, and / or a polyA tail.Attorney Docket No.: 2013237-0773 35. The composition of claim 34, wherein the polyribonucleotide comprises, in a 5’ to 3’ direction: a. a 5’ UTR; b. a signal peptide-coding sequence; c. an incretin peptide-coding sequence; d. a 3’ UTR; and e. a polyA tail.

36. The composition of claim 34, wherein the polyribonucleotide comprises, in a 5’ to 3’ direction: (1) a. a 5’ UTR; b. a signal peptide-coding sequence; c. an incretin peptide-coding sequence; d. a linker-coding sequence; e. a half-life extending moiety-coding sequence; f. a 3’ UTR; and g. a polyA tail; or (2) a. a 5’ UTR; b. a signal peptide-coding sequence; c. a half-life extending moiety-coding sequence; d. a linker-coding sequence; e. an incretin peptide-coding sequence; f. a 3’ UTR; and g. a polyA tail.

37. The composition of any one of claims 1-36, wherein the incretin peptide is encoded by a coding sequence which is codon-optimized and / or the G / C content of which is increased compared to wild type coding sequence, wherein the codon-optimization and / orAttorney Docket No.: 2013237-0773 the increase in the G / C content does not change the sequence of the encoded amino acid sequence.

38. The composition of any one of claims 1-37, wherein the polyribonucleotide comprises at least one modified ribonucleotide.

39. The composition of claim 38, wherein the polyribonucleotide comprises a modified nucleoside in place of uridine.

40. The composition of claim 39, wherein the polyribonucleotide comprises a modified nucleoside in place of each uridine.

41. The composition of claim 40, wherein the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

42. The composition of any one of claims 39-41, wherein the modified nucleoside is N1- methyl-pseudouridine (m1ψ).

43. The composition of any one of claims 1-42, wherein the polyribonucleotide comprises a 5’ cap structure.

44. The composition of any one of claims 1-43, wherein the polyribonucleotide comprises a 5' UTR.

45. The composition of any one of claims 1-44, wherein the polyribonucleotide comprises a 3' UTR.

46. The composition of any one of claims 1-45, wherein the polyribonucleotide comprises a polyA tail.

47. The composition of claim 46, wherein the polyA tail comprises at least 100 nucleotides.

48. The composition of any one of claims 1-47, wherein the polyribonucleotide is mRNA.

49. The composition of any one of claims 1-48, wherein the polyribonucleotide is formulated as a liquid, formulated as a solid, or a combination thereof.Attorney Docket No.: 2013237-0773 50. The composition of any one of claims 1-49, wherein the polyribonucleotide is formulated for injection.

51. The composition of any one of claims 1-50, wherein the polyribonucleotide is formulated for intraperitoneal or intravenous administration.

52. The composition of any one of claims 1-51, wherein the polyribonucleotide is formulated or is to be formulated as lipid particles.

53. The composition of claim 52, wherein the polyribonucleotide is formulated or is to be formulated as lipid nanoparticles.

54. The composition of claim 53, wherein the polyribonucleotide is encapsulated within the lipid nanoparticles.

55. The composition of claim 53 or 54, wherein the lipid nanoparticles are pancreas- targeting and / or gut-targeting lipid nanoparticles.

56. The composition of any one of claims 53-55, wherein the lipid nanoparticles are cationic lipid nanoparticles.

57. The composition of claim 56, wherein lipids that form the lipid nanoparticles comprise: a. a polymer-conjugated lipid; b. a cationic lipid; and c. a neutral lipid.

58. The composition of claim 57, wherein the polymer-conjugated lipid is a PEG- conjugated lipid.

59. The composition of claim 57 or 58, wherein the cationic lipid is an ionizable lipid- like material (lipidoid).

60. The composition of claim 59, wherein the cationic lipid has one of the following structures:Attorney Docket No.: 2013237-077361. The composition of any one of claims 57-60, wherein the neutral lipid comprises a helper lipid such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DPSC) and / or cholesterol.

62. The composition of any one of claims 57-61, wherein the cationic lipid is selected from cationic lipid X-2, X-3, or X-4 and the neutral lipid comprises a helper lipid such as DOTAP, DOPE, or PS, and cholesterol.

63. The composition of claim 62, wherein the polymer-conjugated lipid is C14- PEG2000.Attorney Docket No.: 2013237-0773 64. The composition of any one of claims 58-63, wherein the lipid nanoparticles comprise: i) about 30 mol% to about 50 mol% of a cationic lipid; ii) about 1 mol% to 5 mol% of a PEG-conjugated lipid; iii) about 30 mol% to about 50 mol% of a helper lipid; and iv) about 20 mol% to about 40 mol% of cholesterol.

65. The composition of any one of claims 58-63, wherein the lipid nanoparticles comprise about 35 mol% of a cationic lipid; about 40 mol% of a helper lipid, about 22.5 mol% of cholesterol, and about 2.5 mol% of a PEG-conjugated lipid.

66. The composition of claim 65, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-2, X-3, or X-4, about 40 mol% of DOTAP, DOPE, or PS, about 22.5 mol% of cholesterol, and about 2.5 mol% of C14-PEG2000.

67. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-2; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol, and about 2.5 mol% of C14-PEG2000.

68. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-3; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

69. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-4; about 40 mol% of DOTAP; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

70. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-2; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

71. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-3; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

72. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-4; about 40 mol% of DOPE; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.Attorney Docket No.: 2013237-0773 73. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-2; about 40 mol% of PS; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

74. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-3; about 40 mol% of PS; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

75. The composition of claim 66, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipid X-4; about 40 mol% of PS; about 22.5 mol% of cholesterol; and about 2.5 mol% of C14-PEG2000.

76. The composition of any one of claims 53-75, wherein the lipid nanoparticles are formulated for intraperitoneal (i.p.) delivery.

77. The composition of any one of claims 53-76, wherein the lipid nanoparticles have an average size of about 50-150 nm.

78. The composition of any one of claims 1-77, further comprising one or more pharmaceutically acceptable carriers, diluents and / or excipients.

79. The composition of claim 78, further comprising a cryoprotectant.

80. The composition of claim 79, wherein the cryoprotectant is sucrose.

81. The composition of any one of claims 78-80, further comprising an aqueous buffered solution.

82. The composition of claim 81, wherein the aqueous buffered solution includes sodium ions.

83. A method of treating a disease-state in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a composition according to any one of claims 1-82.

84. The method of claim 83, wherein the disease-state is obesity or an obesity-related disorder.Attorney Docket No.: 2013237-0773 85. The method of claim 84, wherein the obesity-related disorder is pre-diabetes, type 2 diabetes (T2D), early type 1 diabetes (T1D), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), a cardiovascular (CV) disease, a renal disease, or elevated risk of premature mortality.

86. The method of claim 85, 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).

87. The method of claim 84, wherein the method improves weight management in said subject. 88 The method of claim 84, wherein the method reduces weight gain or induces weight loss in said subject.

89. The method of claim 83, wherein the disease-state is diabetes.

90. The method of claim 89, wherein the method improves glycemic control in said subject.

91. The method of claim 89, wherein the method lowers HbA1c in said subject.

92. The method of claim 89, wherein the diabetes is pre-diabetes, type 2 diabetes (T2D), or early type 1 diabetes (T1D).

93. The method of claim 83, wherein the disease-state is a cardiovascular (CV) disease.

94. The method of claim 93, wherein the cardiovascular 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).

95. The method of claim 93, wherein the method improves a subject’s blood pressure and / or blood lipids in said subject.

96. The method of claim 83, wherein the disease-state is a renal disease.

97. The method of claim 83, wherein the disease-state is non-alcoholic fatty liver disease (NAFLD).Attorney Docket No.: 2013237-0773 98. The method of claim 83, wherein the disease-state is non-alcoholic steatohepatitis (NASH) and optionally its sequelae, liver fibrosis and cirrhosis.

99. The method of any one of claims 83-98, wherein administering the composition to the subject comprises administering one or more doses of the composition to the subject.

100. The method of claim 99, wherein the one or more doses of the composition are administered to the subject daily, every other day or once a week.

101. The method of claim 99, wherein the one or more doses of the composition are administered to the subject less frequently than once a week.

102. The method of claim 99, wherein the one or more doses of the composition are administered to the subject once every 2, 3 or 4 weeks.

103. The method of any one of claims 83-102, wherein the composition is administered via injection.

104. The method of claim 103, wherein the composition is administered subcutaneously, intravenously, intramuscularly, or intraperitoneally 105. The method of claim 104, wherein the composition is administered intraperitoneally.

106. The method of any one of claims 83-102, wherein the composition is administered non-invasively (e.g., orally or nasally).

107. The method of any one of claims 83-106, wherein administration of the composition results in expression of the incretin agent in the subject.

108. The method of any one of claims 83-107, wherein the composition is administered in a volume that is less than 0.5 mL.

109. Use of the composition of any one of claims 1-82 for the treatment of a disease-state in a subject in need thereof.

110. A method of producing an incretin agent comprising administering to cells the composition of any one of claims 1-82 so that the cells express and secrete the incretin agent.Attorney Docket No.: 2013237-0773 111. An incretin agent that comprises an incretin peptide fused to a signal peptide.

112. The incretin agent of claim 111, wherein the incretin peptide is fused to the signal peptide via the N-terminus of the incretin peptide, optionally via a linker.

113. The incretin agent of claim 111 or 112, wherein the incretin peptide is also fused to a signal peptide 114. The incretin agent of any one of claims 111-113, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NOs: 16-39.

115. The incretin agent of claim 114, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NOs: 16-21.

116. The incretin agent of claim 114, wherein the signal peptide has an amino acid sequence according to SEQ ID NO:

17.

117. The incretin agent of claim 111, wherein the incretin agent comprises an incretin peptide fused to a signal peptide that comprises an amino acid sequence according to any one of SEQ ID NOs: 41-45.

118. The incretin agent of any one of claims 111-117, wherein the incretin peptide is also fused to a half-life extending moiety via the C-terminus or N-terminus of the incretin peptide, optionally via a linker.

119. The incretin agent of claim 118, wherein the half-life extending moiety comprises a peptide moiety that binds albumin.

120. The incretin agent of claim 119, wherein the half-life extending moiety comprises an albumin binding domain (ABD) derived from protein G of Streptococcus strain GI48 and / or from protein PAB of Finegoldia magna, such as ABD035 and SA21.

121. The incretin agent of claim 119, wherein the half-life extending moiety comprises an albumin binding domain (ABD) that binds to domain II of human serum albumin and does not overlap or interfere with binding to the FcRn-binding site on albumin.Attorney Docket No.: 2013237-0773 122. The incretin agent of claim 119, wherein the half-life extending moiety comprises ABDCon.

123. The incretin agent of claim 119, wherein the half-life extending moiety comprises an albumin binding domain (ABD) derived from the bacterial protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus, such as M11.12 and M18.2.

5.

124. The incretin agent of claim 118, wherein the half-life extending moiety comprises a DARPin that binds albumin.

125. The incretin agent of claim 118, wherein the half-life extending moiety comprises an immunoglobulin domain or fragment thereof.

126. The incretin agent of claim 118, wherein the half-life extending moiety comprises a fully human domain antibody (dAb) that binds albumin, such as AlbudAb.

127. The incretin agent of claim 118, wherein the half-life extending moiety comprises a Fab that binds albumin, such as dsFv CA645.

128. The incretin agent of claim 118, wherein the half-life extending moiety comprises a Fc domain, such as from a human IgG, optionally from a human IgG1, IgG2, IgG3, or IgG4.

129. The incretin agent of claim 118, wherein the half-life extending moiety does not comprise a Fc domain, such as from a human IgG, optionally from a human IgG1, IgG2, IgG3, or IgG4.

130. The incretin agent of claim 119, wherein the half-life extending moiety comprises a heavy chain only (VHH) antibody, such as a nanobody, that binds albumin.

131. The incretin agent of claim 119, wherein the half-life extending moiety comprises a VNAR that binds albumin.

132. The incretin agent of claim 118, wherein the half-life extending moiety comprises an XTEN sequence.

133. The incretin agent of any one of claims 118-132, wherein the incretin peptide is fused to the half-life extending moiety via a linker.Attorney Docket No.: 2013237-0773 134. The incretin agent of claim 133, wherein the linker is (a) fused to the C-terminus of the incretin peptide and the N-terminus of the half-life extending moiety or (b) fused to the C-terminus of the half-life extending moiety and the N-terminus of the incretin peptide.

135. The incretin agent of claim 133 or 134, wherein the linker has an amino acid sequence of any one of SEQ ID NOs: 1-5.

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