TW202540154A - Rna compositions for delivery of incretin agents - Google Patents

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

RNA compounds used for delivering intestinal insulin agents

RNA compounds used for delivering intestinal insulin agents

Obesity is the most prevalent chronic disease worldwide, currently affecting approximately 650 million adults. Obesity is considered a starting point and key contributing factor to prediabetes, type 2 diabetes (T2D, and its complications), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease and kidney disease, and premature death. It is estimated that by 2030, the number of obese people (BMI > 30 kg/ ^m² ) will exceed one billion, with approximately 10% suffering from severe type III obesity (BMI > 40 kg/ ^m² ). Half of all obese individuals live in just nine countries: the United States, China, India, Brazil, Mexico, Russia, Egypt, Germany, and Turkey. Furthermore, childhood obesity is rising rapidly worldwide. T2D, NAFLD, NASH, cardiovascular disease, and kidney disease are also prevalent and unrelated to obesity. There is a need to develop further therapies for the treatment and/or prevention of obesity and other related diseases.

The use of incretins and incretin analogs, particularly GLP1 and GIP receptor agonists or combinations thereof, for the treatment of obesity and type 2 diabetes has shown significant benefits to individuals with these conditions. However, due to production and cost limitations, the availability of this new class of active agents is restricted, leaving millions without access to necessary treatment. This disclosure recognizes these drawbacks and provides a novel therapeutic approach that eliminates the need for the body to produce incretins by using polynucleotide precursors of these incretins to deliver incretin analogs. This disclosure specifically provides polynucleotide precursors of such incretins as molecular entities, their production, formulation, and administration for the treatment of obesity and its sequelae, including type 2 diabetes (T2D), early type 1 diabetes (T1D), cardiovascular disease, nephropathy, NASH, and NAFLD. This disclosure also provides methods for using these agents to treat diseases, including obesity-independent T2D, early type 1 diabetes (T1D), cardiovascular disease, nephropathy, NASH, and NAFLD. This disclosure further provides methods for using these agents to treat the sequelae of NASH, including liver fibrosis and cirrhosis.

This disclosure also recognizes that this treatment approach, namely the delivery of polynucleotide precursors of incretins, presents additional benefits over current treatments, including, but not limited to, broader accessibility to obese populations who would otherwise be unable to access current products due to limited supply, high prices, or lack of health insurance; the formulation requiring a lower injection volume compared to commercially available peptide-based products; a lower frequency of treatment interruptions due to factors such as gastrointestinal side effects; and the nature of the improvements, such as improved pharmacokinetic profile. In some embodiments, the improved pharmacokinetic profile benefits from the lower dosing frequency of long-acting formulations.

In one embodiment, this disclosure provides a composition comprising a polynucleotide encoding an incretin agent. In some embodiments, the incretin agent is a GLP1 receptor agonist. In some embodiments, the incretin agent is a GIP receptor agonist. In some embodiments, the incretin agent is a GLP1/GIP dual receptor agonist. In some embodiments, the incretin agent is a GLP1/GCG dual receptor agonist. In some embodiments, the incretin agent is a GLP1/GIP/GCG 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 NO: 5-7, 63-64, 69-70, and 74-75. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NO: 8-9, 62, and 72. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NO: 12-14. In some embodiments, the incretin agent comprises an incretin peptide having an amino acid sequence according to SEQ ID NO: 15.

In some embodiments, the incretin peptide is fused to the signal peptide via the N-terminus of the incretin peptide, or via a linker, as appropriate. In some embodiments, the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 16-39 and 65-67. In some embodiments, the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 16-21 and 65-67. In some embodiments, the signal peptide has an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the signal peptide has an amino acid sequence according to SEQ ID NO: 65. In some embodiments, the signal peptide has an amino acid sequence according to SEQ ID NO: 66. In some embodiments, the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 41-45, 52-61 and 108-152.

In some embodiments, the incretin agent comprises, where appropriate, an incretin peptide fused to one or more additional incretin peptides via one or more linkers. In some embodiments, the one or more linkers comprise an amino acid sequence according to any one of SEQ ID NO: 1-5, 68, or 156. In some embodiments, the incretin agent comprises an incretin peptide fused to two or more incretin peptides. In some embodiments, the incretin agent comprises at least one GLP1 receptor agonist and at least one GIP receptor agonist. In some embodiments, the incretin agent comprises at least two GLP1 receptor agonists. In some implementations, incretins contain at least two GIP receptor agonists.

In some embodiments, the incretin agent includes one or more furin cleavage sites. In some embodiments, the one or more furin cleavage sites are located between adjacent incretin peptides. In some embodiments, the one or more furin cleavage sites include the amino acid sequence according to SEQ ID NO: 153. In some embodiments, the incretin agent comprises one or more units, each of which comprises, from the N-terminus to the C-terminus, a GLP1 receptor agonist-linker-furin cleavage site-GIP receptor agonist, for example, wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 76, 77, 78, 79, 80, 81), two units (e.g., SEQ ID NO: 82), or four units (e.g., SEQ ID NO: 83). In some embodiments, the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 76-83, 94-97, 102-107.

In some embodiments, the incretin agent includes a half-life extension portion. In some embodiments, the half-life extension portion includes albumin (e.g., human serum albumin). In some embodiments, human serum albumin includes an amino acid sequence having at least 90%, 95%, or 99% identity with SEQ ID NO: 159. In some embodiments, human serum albumin includes an amino acid sequence according to SEQ ID NO: 159. In some embodiments, the incretin agent comprises albumin (e.g., human serum albumin) fused to one or more units, each of which comprises, from the N-terminus to the C-terminus,: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 98); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 100); (iii) a GLP1 receptor agonist-linker-furin cleavage site (e.g., SEQ ID NO: 102); or (iv) a GLP1 receptor agonist-linker-furin cleavage site-GIP receptor agonist, for example, wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 104), two units (e.g., SEQ ID NO: 106), or four units (e.g., SEQ ID NO: 104). 107). In some embodiments, the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 98, 100, 102, 104, 106, 107, or any combination thereof.

In some embodiments, the half-life extension portion includes an albumin-binding domain (ABD). In some embodiments, the ABD is derived from protein G of Streptococcus strain GI48 and/or protein PAB of Finegoldia magna , such as ABD035 and SA21. In some embodiments, the half-life extension portion includes an ABD that binds to domain II of human serum albumin without overlapping with or interfering with binding to FcRn binding sites on albumin. In some embodiments, the half-life extension portion includes ABDCon. In some embodiments, the half-life extension portion includes albumin-binding domains (ABDs) of bacterial proteins Sso7d derived from the hyperthermophilic archaeon *Sulfolobus solfataricus* , such as M11.12 and M18.2.5. In some embodiments, the half-life extension portion includes albumin-binding DARPin.

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

In some embodiments, the half-life extension portion does not contain an Fc domain, such as that derived from human IgG, or, depending on, from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the half-life extension portion contains an Fc domain, such as that derived from human IgG, or, depending on, from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the human IgG is human IgG4. In some embodiments, the incretin agent includes an IgG4 Fc domain fused to a unit comprising, from the N-terminus to the C-terminus: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 10, 89, 90, 91); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 92, 93); or (iii) a GLP1 receptor agonist-linker-furin protease-GIP receptor agonist-linker (e.g., SEQ ID NO: 94, 95, 96, 97). In some embodiments, the IgG4 Fc domain includes an amino acid sequence that is at least 90%, 95%, or 99% identical to that of SEQ ID NO: 155. In some embodiments, the IgG4 Fc domain includes an amino acid sequence according to SEQ ID NO: 155. In some embodiments, the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 10 and 89-97.

In some embodiments, the Fc domain contains one or more mutations in one or both of the constant Fc domains that increase the half-life of incretins and/or induce dimerization. In some embodiments, one or more mutations include one or more mutations in the CH3 domain. In some embodiments, one or more mutations that induce dimerization include: (i) according to EU designations Y349C, T366S, L368A and/or Y407V; or (ii) according to EU designations S354C and/or T366W. In some embodiments, one or more mutations include, according to EU designations, Y349C, T366S, L368A and Y407V (“FcKIH-b”); or according to EU designations, S354C and T366W (“FcKIH-a”). In some embodiments, the incretin agent comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an incretin peptide fused to a first Fc domain, wherein the first Fc domain comprises mutants Y349C, T366S, L368A and Y407V (“FcKIH-b”) according to EU designations, and wherein the second polypeptide chain comprises an incretin peptide fused to a second Fc domain, wherein the second Fc domain comprises mutants S354C and T366W (“FcKIH-a”) according to EU designations.

In some embodiments, one or more mutations that increase the half-life of the incretin agent include those according to EU designations M428L and N434S (“LS”). In some embodiments, the incretin agent includes an Fc domain having an FcKIH-a mutation on a first polypeptide chain and an Fc domain having an FcKIH-b mutation on a second polypeptide chain, wherein the Fc domains on each polypeptide chain are independently fused to one or more units, which, from the N-terminus to the C-terminus, include: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 84, 85, 86, 87); or (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 88). In some embodiments, the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 84-88.

In some embodiments, the Fc domain contains one or more mutations that eliminate the effectiveon activity of the Fc domain (e.g., binding to an Fcγ receptor or C1q). In some embodiments, one or more mutations that eliminate the effectiveon activity of the Fc domain (e.g., binding to an Fcγ receptor or C1q) include the following mutations: according to EU designations, L234S, L235T, and G236R (“STR”). In some embodiments, one or more mutations that eliminate the effectiveon activity of the Fc domain (e.g., binding to an Fcγ receptor or C1q) include the following mutations: according to EU designations, L234A and L235A (“LALA”). In some embodiments, one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to the Fcγ receptor or C1q) include the following mutations: according to EU designation, L234A/L235A/P329G (“LALAPG”).

In some embodiments, the half-life extension portion includes a VNAR that binds to albumin. In some embodiments, the half-life extension portion includes an XTEN sequence.

In some embodiments, the polynucleotide has a ribonucleic acid sequence that is at least 90% identical to any one of SEQ ID NO: 177-185 and 224-256. In some embodiments, the polynucleotide has a ribonucleic acid sequence according to any one of SEQ ID NO: 177-185 and 224-256.

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

In some embodiments, the polynucleotide includes, in the 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.

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

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

In some embodiments, the polynucleotide comprises at least one modified ribonucleotide. In some embodiments, the polynucleotide comprises a modified nucleoside replacing uridine. In some embodiments, the polynucleotide comprises a modified nucleoside replacing 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).

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

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

In some embodiments, the polynucleotide is formulated or intended to be formulated into lipid particles. In some embodiments, the polynucleotide is formulated or intended to be formulated into lipid nanoparticles. In some embodiments, the polynucleotide is encapsulated within lipid nanoparticles. In some embodiments, the lipid nanoparticles are lipid nanoparticles targeting the pancreas and/or targeting the intestine. In some embodiments, the lipid nanoparticles are cationic lipid nanoparticles.

In some embodiments, the lipids forming the lipid nanoparticles include a. polymer-coupled lipids; b. cationic lipids; and c. neutral lipids. In some embodiments, the polymer-coupled lipids are PEG-coupled lipids. In some embodiments, the cationic lipids are ionizable lipid-like materials (lipid-like substances).

In some embodiments, cationic lipids have one of the following structures: X-1 X-2 X-3 X-4

In some embodiments, neutral lipids include co-lipids such as 1,2-distearyl-sn-glycerol-3-phosphate choline (DPSC) and/or cholesterol.

In some embodiments, the cationic lipids are selected from cationic lipids X-2, X-3, or X-4, and the neutral lipids include auxiliary lipids (such as DOTAP, DOPE, or PS) and cholesterol.

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

In some embodiments, the lipid nanoparticles comprise: i) about 30 mol% to about 50 mol% cationic lipids; ii) about 1 mol% to 5 mol% PEG-coupled lipids; iii) about 30 mol% to about 50 mol% auxiliary lipids; and iv) about 20 mol% to about 40 mol% cholesterol.

In some embodiments, the lipid nanoparticles contain approximately 35 mol% cationic lipids; approximately 40 mol% co-lipids; approximately 22.5 mol% cholesterol; and approximately 2.5 mol% PEG-coupled lipids.

In some embodiments, the lipid nanoparticles contain approximately 35 mol% of cationic lipids X-2, X-3 or X-4, approximately 40 mol% of DOTAP, DOPE or PS, approximately 22.5 mol% of cholesterol, and approximately 2.5 mol% of C14-PEG2000.

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

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

In another embodiment, this disclosure provides a method for treating a disease condition in an individual of need, comprising administering to the individual a therapeutically effective amount of a combination comprising one or more of the polynucleotides described herein.

In some embodiments, the method further includes administering one or more DPP-4 inhibitors. In some embodiments, one or more DPP-4 inhibitors and their combination are administered simultaneously. In some embodiments, one or more DPP-4 inhibitors and their combination are administered sequentially. In some embodiments, one or more DPP-4 inhibitors are administered before the combination. In some embodiments, one or more DPP-4 inhibitors are administered after the combination. In some embodiments, one or more DPP-4 inhibitors include sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin, alogliptin, trelagliptin, omarigliptin, evogliptin, gosogliptin, dutogliptin, neogliptin, retagliptin, denagliptin, cofrogliptin, fotagliptin, prusogliptin, berberine, or any combination thereof. In some embodiments, one or more DPP-4 inhibitors are administered orally.

In some embodiments, the disease status is obesity or obesity-related conditions. In some embodiments, obesity-related conditions include prediabetes, type 2 diabetes (T2D), early type 1 diabetes (T1D), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease, kidney disease, or an increased risk of premature death. In some embodiments, cardiovascular (CV) disease includes major cardiovascular events (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 the individual's weight management. In some embodiments, the method reduces the individual's weight gain or induces weight loss. In some embodiments, the disease state is diabetes. In some embodiments, the method improves the individual's glycemic control. In some embodiments, the method lowers the individual's HbA1c. In some embodiments, the diabetes is prediabetes, type 2 diabetes (T2D), or early type 1 diabetes (T1D).

In some embodiments, the disease state is cardiovascular (CV) disease. In some embodiments, cardiovascular disease includes major cardiovascular events (MACE), including CV death, nonfatal myocardial infarction, nonfatal stroke, and/or heart failure with preserved ejection fraction (HfpEF). In some embodiments, the method improves the individual's blood pressure and/or blood lipids.

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

In some embodiments, administration of the compound to an individual involves administering one or more doses of the compound to the individual. In some embodiments, one or more doses of the compound are administered to an individual daily, every other day, or weekly. In some embodiments, one or more doses of the compound are administered to an individual at a frequency less than once weekly. In some embodiments, one or more doses of the compound are administered to an individual every 2, 3, or 4 weeks. In some embodiments, the compound is administered by injection. In some embodiments, the compound is administered subcutaneously, intravenously, intramuscularly, or intraperitoneally. In some embodiments, the compound is administered intraperitoneally. In some embodiments, the compound is administered noninvasively (e.g., orally or nasally). In some embodiments, administration of the compound resulted in the expression of the incretin in the individual. In some embodiments, the compound was administered in a volume of less than 0.5 mL.

In another embodiment, this disclosure provides the use of any combination of one or more of the polynucleotides described herein for the treatment of disease conditions in an individual in need.

In another embodiment, this disclosure provides a method for producing an incretin, comprising administering to cells a composition comprising the polynucleotides described herein, such that the cells express and secrete the incretin.

In another embodiment, this disclosure provides an incretin agent comprising an incretin peptide fused to a signal peptide. In some embodiments, the incretin peptide is fused to the signal peptide via a linker, where applicable. In some embodiments, the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 16-39 and 65-67. In some embodiments, the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 16-21 and 65-67. In some embodiments, the signal peptide has an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the signal peptide has an amino acid sequence according to SEQ ID NO: 65. In some embodiments, the signal peptide has an amino acid sequence according to SEQ ID NO: 66. In some embodiments, the incretin agent comprises an incretin peptide fused to a signal peptide, which comprises an amino acid sequence according to any one of SEQ ID NO: 41-45, 52-61 and 108-152.

In some embodiments, the incretin agent comprises, where appropriate, an incretin peptide fused to one or more additional incretin peptides via one or more linkers. In some embodiments, the one or more linkers comprise an amino acid sequence according to any of SEQ ID NO: 1-5, 68, or 156. In some embodiments, the incretin agent comprises an incretin peptide fused to two or more incretin peptides.

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

In some embodiments, the incretin agent includes a half-life extension portion. In some embodiments, the half-life extension portion includes albumin (e.g., human serum albumin). In some embodiments, human serum albumin includes an amino acid sequence having at least 90%, 95%, or 99% identity with SEQ ID NO: 159. In some embodiments, human serum albumin includes an amino acid sequence according to SEQ ID NO: 159.

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

In some embodiments, the half-life extension portion includes an albumin-binding domain (ABD). In some embodiments, the ABD is derived from protein G of Streptococcus strain GI48 and/or protein PAB of *Gynostemma pentaphyllum* , such as ABD035 and SA21. In some embodiments, the half-life extension portion includes an ABD that binds to domain II of human serum albumin without overlapping or interfering with binding to FcRn binding sites on albumin. In some embodiments, the half-life extension portion includes ABDCon. In some embodiments, the half-life extension portion includes an ABD derived from bacterial protein Sso7d of the hyperthermophilic archaea *Sulphozoa sulfolithoides* , such as M11.12 and M18.2.5. In some embodiments, the half-life extension portion includes albumin-binding DARPin. In some embodiments, the ABD includes an immunoglobulin domain or a fragment thereof that binds to albumin. In some embodiments, the ABD includes a fully human domain antibody (dAb) that binds to albumin, such as AlbudAb. In some embodiments, the ABD includes an albumin-binding Fab, such as dsFv CA645.

In some embodiments, the ABD comprises a heavy-chain-only (VHH) antibody that binds to albumin, such as a nano-antibody. In some embodiments, the VHH antibody comprises a VHH domain having complementary determinant region (CDR) sequences HCDR1, HCDR2, and/or HCDR3 according to SEQ ID NO: 191 (GFTLDYYA), SEQ ID NO: 192 (IASSGGST), and/or SEQ ID NO: 193 (AAAVLECRTVVRGYDY), respectively. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 90%, 95%, or 99% identity with SEQ ID NO: 154. In some embodiments, the VHH antibody comprises an amino acid sequence according to SEQ ID NO: 154. In some embodiments, the incretin agent comprises a VHH antibody bound to an albumin fused to a unit comprising, from the N-terminus to the C-terminus: (i) a GLP1-linker (e.g., SEQ ID NO: 99); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 101); or (iii) a GLP1 receptor agonist-linker-furin protease-GIP receptor agonist-linker (e.g., SEQ ID NO: 103 or 105). In some embodiments, the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 99, 101, 103, 105.

In some embodiments, the half-life extension portion does not contain an Fc domain, such as that derived from human IgG, or, depending on, from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the half-life extension portion contains an Fc domain, such as that derived from human IgG, or, depending on, from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the human IgG is human IgG4. In some embodiments, the incretin agent includes an IgG4 Fc domain fused to a unit comprising, from the N-terminus to the C-terminus: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 10, 89, 90, 91); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 92, 93); or (iii) a GLP1 receptor agonist-linker-furin protease-GIP receptor agonist-linker (e.g., SEQ ID NO: 94, 95, 96, 97). In some embodiments, the IgG4 Fc domain includes an amino acid sequence that is at least 90%, 95%, or 99% identical to that of SEQ ID NO: 155. In some embodiments, the IgG4 Fc domain includes an amino acid sequence according to SEQ ID NO: 155. In some embodiments, the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 10, 89-97.

In some embodiments, the Fc domain contains one or more mutations in one or both of the constant Fc domains that increase the half-life of incretins and/or induce dimerization. In some embodiments, one or more mutations include one or more mutations in the CH3 domain. In some embodiments, one or more mutations that induce dimerization include: (i) according to EU designations Y349C, T366S, L368A and/or Y407V; or (ii) according to EU designations S354C and/or T366W.

In some embodiments, one or more mutations include, according to EU designations, Y349C, T366S, L368A and Y407V (“FcKIH-b”); or according to EU designations, S354C and T366W (“FcKIH-a”). In some embodiments, the incretin agent comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an incretin peptide fused to a first Fc domain, wherein the first Fc domain comprises mutants Y349C, T366S, L368A and Y407V (“FcKIH-b”) according to EU designations, and wherein the second polypeptide chain comprises an incretin peptide fused to a second Fc domain, wherein the second Fc domain comprises mutants S354C and T366W (“FcKIH-a”) according to EU designations.

In some embodiments, one or more mutations that increase the half-life of the incretin agent include those according to EU designations M428L and N434S (“LS”). In some embodiments, the incretin agent includes an Fc domain having an FcKIH-a mutation on a first polypeptide chain and an Fc domain having an FcKIH-b mutation on a second polypeptide chain, wherein the Fc domains on each polypeptide chain are independently fused to one or more units, which, from the N-terminus to the C-terminus, include: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 84, 85, 86, 87); or (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 88). In some embodiments, the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 84-88.

In some embodiments, the Fc domain contains one or more mutations that eliminate the effectiveon activity of the Fc domain (e.g., binding to an Fcγ receptor or C1q). In some embodiments, one or more mutations that eliminate the effectiveon activity of the Fc domain (e.g., binding to an Fcγ receptor or C1q) include the following mutations: according to EU designations, L234S, L235T, and G236R (“STR”). In some embodiments, one or more mutations that eliminate the effectiveon activity of the Fc domain (e.g., binding to an Fcγ receptor or C1q) include the following mutations: according to EU designations, L234A and L235A (“LALA”). In some embodiments, one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to the Fcγ receptor or C1q) include the following mutations: according to EU designation, L234A/L235A/P329G (“LALAPG”).

In some embodiments, the half-life extension portion includes VNAR bound to albumin.

In some embodiments, the half-life extension portion includes the XTEN sequence.

In another embodiment, this disclosure provides an incretin agent comprising: a husec signaling peptide; an incretin peptide comprising a GLP1 incretin peptide or a fragment or variant thereof; wherein the GLP1 incretin peptide comprises an amino acid sequence having an A8G substitution mutation compared to the wild-type GLP1 amino acid sequence.

In another embodiment, this disclosure provides a polynucleotide encoding an incretin agent comprising a husec signaling peptide; an incretin peptide comprising a GLP1 incretin peptide or a fragment or variant thereof; wherein the GLP1 incretin peptide comprises an amino acid sequence having an A8G substitution mutation compared to the wild-type GLP1 amino acid sequence.

In another embodiment, this disclosure provides an incretin agent comprising: a husec signaling peptide; an incretin peptide comprising a GIP incretin peptide or a fragment or variant thereof; wherein the GIP incretin peptide comprises an amino acid sequence having an A2G substitution mutation compared to the wild-type GIP amino acid sequence.

In another embodiment, this disclosure provides a polynucleotide encoding an incretin agent comprising: a husec signaling peptide; an incretin peptide comprising a GIP incretin peptide or a fragment or variant thereof; wherein the GIP incretin peptide comprises an amino acid sequence having an A2G substitution mutation compared to the wild-type GIP amino acid sequence.

Related applications

This application claims priority and interests in U.S. Provisional Patent Application No. 63/662,890, filed June 21, 2024, and PCT Application No. PCT/IB2023/059007, filed September 11, 2023, the entire contents of which are hereby incorporated by reference. Definitions

Approximately : When used in this document to refer to a value, the term "approximately" refers to a value similar to the mentioned value in the context. Generally, those skilled in the art will understand the degree of relevant variation covered by "approximately" in that context once they are familiar with the context. For example, in some embodiments, the term "approximately" may cover a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the mentioned value.

Agent : As used herein, the term "agent" may refer to a physical entity. In some embodiments, an agent may be characterized by specific features and/or effects. For example, as used herein, the term "therapeutic agent" refers to a physical entity that has a therapeutic effect and/or induces 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, small molecules, peptides, nucleic acids, carbohydrates, lipids, metals, or combinations or complexes thereof.

Aliphatic : The term "aliphatic" refers to a fully saturated or branched, substituted or unsubstituted hydrocarbon chain containing one or more unsaturated units, or a fully saturated or bicyclic hydrocarbon (also referred to herein as "cyclic aliphatic") containing one or more unsaturated units but not aromatic, having a single or more than one bond site with the remainder of the molecule. Unless otherwise stated, 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, the aliphatic group contains 1-4 aliphatic carbon atoms (e.g., _C1-4 ). In other embodiments, the aliphatic group contains 1-3 aliphatic carbon atoms (e.g., _C1-3 ), and in still other embodiments, the aliphatic group contains 1-2 aliphatic carbon atoms (e.g., _C1-2 ). Suitable aliphatic groups include, but are not limited to, straight-chain or branched, substituted or unsubstituted alkyl, alkenyl or ynyl groups and their hybrids. Preferred aliphatic groups are _C1-6 alkyl groups.

Alkyl : The term "alkyl" as used alone or as part of a larger part refers to a saturated, substituted, straight-chain or branched hydrocarbon group having _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.

Alkyl group : The term "alkyl group" refers to a divalent alkyl group. In some embodiments, "alkyl group" is a divalent straight-chain or branched alkyl group. In some embodiments, "alkyl group" is a polymethylene group, i.e., -( _CH2 ) _n- , where n is a positive integer, such as 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. A substituted alkyl group is, where appropriate, a polymethylene group with one or more methylene hydrogen atoms substituted by substituents. Suitable substituents include those described below for substituted aliphatic groups, and also those described in this specification. It will be understood that two substituents of an alkyl group can together form a ring system. In some embodiments, two substituents can together form a 3 to 7-membered ring. Substituents can be on the same or different atoms. The suffix "-extrin" or "-extrinyl" when attached to certain groups herein is intended to refer to the bifunctional portion of that group. For example, when "-extrin" or "-extrinyl" is attached to "cyclopropyl", it becomes "cyclopropylene" or "cyclopropylenyl" and is intended to refer to a bifunctional cyclopropyl group, such as... .

Alkenyl : The term "alkenyl," used alone or as part of a larger part, refers to a linear or branched or cyclic hydrocarbon group having at least one double bond and, as appropriate, 2-12, 2-10, _2-8 , _2-6 , 2-4, or 2-3 carbon atoms (e.g., C _2-12 , C 2-10, C _2-8 , C 2-6, C _2-4 , or C _2-3 ). Exemplary alkenyl groups include vinyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms, as appropriate, substituted. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

Alynyl group : The term "alkynyl" used alone or as part of a larger group refers to a linear or branched hydrocarbon group having at least one triple bond and, where applicable, 2-12, 2-10, _2-8 , 2-6, _2-4 , or 2-3 carbon atoms (e.g., C _2-12 , C _2-10 , C 2-8, C 2-6, C _2-4 , or C _2-3 ). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentyynyl, hexynyl, and heptynyl.

Amino acid : In its broadest sense, as used herein, the term "amino acid" refers to, for example, a compound and/or substance that can be incorporated into, is incorporated into, or has been incorporated into a polypeptide chain by forming one or more peptide bonds. In some embodiments, an amino acid has the general structure _H₂NC (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. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. In some embodiments, the amino acids (including carboxyl-terminal and/or amino-terminal amino acids in the polypeptide) may contain structural modifications compared to the general structure described above. For example, in some embodiments, the amino acids may be modified by methylation, amination, acetylation, PEGylation, glycosylation, phosphorylation, and/or substitution (e.g., amino groups, carboxylic acid groups, one or more protons and/or hydroxyl groups) compared to the general structure. In some embodiments, such modifications may, for example, alter the cyclic half-life of the polypeptide containing the modified amino acid compared to a polypeptide containing otherwise identical unmodified amino acids. In some embodiments, such modifications do not significantly alter the relevant activity of the polypeptide containing the modified amino acid compared to a polypeptide containing otherwise identical unmodified amino acids. As will be clear from the context, in some embodiments the term "amino acid" may be used to refer to free amino acids; in other embodiments the term "amino acid" may be used to refer to the amino acid residues of a polypeptide.

Aryl : The term "aryl" refers to a monocyclic or bicyclic system having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and each ring in the system contains three to seven ring members. In some embodiments, "aryl" contains a total of six to twelve ring members (e.g., C6-C12). The term "aryl" is used interchangeably with the term "aryl ring." In some embodiments, "aryl" refers to an aromatic ring system that may have one or more substituents, including but not limited to phenyl, biphenyl, naphthyl, anthracene, and similar groups. Unless otherwise stated, "aryl" is a hydrocarbon. In some embodiments, the "aryl" ring system is an aromatic ring (e.g., phenyl) fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings include fused aryl rings, including... , and .

Relevance : When used in this text, two events or entities are “related” to each other if the existence, level, degree, type, and/or form of one event or entity is related to the existence, level, degree, type, and/or form of another event or entity. For example, an entity is considered related to a disease, condition, or disorder if the existence, level, and/or form of a particular entity (e.g., polypeptide, genetic imprint, metabolite, microorganism, etc.) is related to the incidence, susceptibility, severity, stage, etc., of that disease, condition, or disorder (e.g., in the relevant population). In some embodiments, entities are physically “attached” to each other if two or more entities interact directly or indirectly such that they are physically close to each other and/or remain close to each other. In some embodiments, two or more physically bonded entities are covalently bonded to each other; in other embodiments, two or more physically bonded entities are not covalently bonded to each other, but are non-covalently bonded, for example by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

Co-doping : As used herein, the term "co-doping" refers to the use of a combination described herein (e.g., a pharmaceutical combination) and one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents comprise at least one polynucleotide encoding another therapeutic agent (e.g., an incretin). The combined use of the combination described herein (e.g., a pharmaceutical combination) and the additional therapeutic agent may be performed simultaneously or separately (e.g., sequentially in any order). In some embodiments, the combination described herein (e.g., a pharmaceutical combination) and the additional therapeutic agent may be combined in a pharmaceutically acceptable excipient, or may be placed in a separate excipient and delivered to target cells or administered to an individual at different times. It is anticipated that in such cases the components fall within the meaning of "co-administration" or "combination" when the combination (e.g., a pharmaceutical combination) and the additional treatment are delivered or administered in sufficiently close temporal proximity such that there is at least some temporal overlap in the biological effects produced by each on the target cells or the treated individuals.

Combination therapy : As used herein, the term "combination therapy" refers to situations where an individual is simultaneously exposed to two or more treatment regimens (e.g., two or more agents of action (e.g., two or more incretin agents)). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all doses of the first regimen are administered before any dose of the second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, administration of combination therapy may involve administering one or more agents or methods to an individual receiving other agents or methods in the combination. For clarity, combination therapy does not require the individual agents to be administered together in a single combination (or even at the same time), although in some embodiments, two or more agents or their active portions may be administered together in a combination combination. In some embodiments, combination therapy comprises a polynucleotide encoding two or more incretin agents.

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 each other, but are similar enough to allow for comparison between them, so that those skilled in the art will understand that reasonable conclusions can be drawn based on observed differences or similarities. In some embodiments, the characteristics of comparable sets of conditions, environments, individuals, or groups lie in a plurality of substantially consistent features and one or a few varying features. In the context, those skilled in the art will understand the degree of consistency required in any given environment for two or more such sets of agents, entities, situations, sets of conditions, etc., to be considered comparable. For example, those skilled in this technique will understand that when a sufficient number and type of substantially consistent features are used to represent the differences in results or observed phenomena in different groups of environments, individuals or groups, the differences are caused by or indicate reasonable conclusions about the variations of those features. In this way, the environments, individuals or groups of groups are comparable to each other.

As used herein , the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" can be used to specify the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., relative to a suitable reference compound or composition). For example, in some embodiments, monomeric residues in a polymer (e.g., amino acid residues in a polypeptide or nucleic acid residues in a polynucleotide) can be identified as "corresponding to" residues in a suitable reference polymer. For example, those familiar with this technique will understand that, for simplicity, residues in peptides are typically designated using a reference peptide specification numbering system, so that the amino acid "corresponding" to the residue at position 190 is, for example, not actually the 190th amino acid in a specific amino acid chain, but the residue found at position 190 in the reference peptide; those familiar with this technique will easily understand how to identify the "corresponding" amino acid. For example, those skilled in the art will recognize various sequence alignment strategies, including software programs that can be used, for instance, to identify “corresponding” residues in peptides and/or nucleic acids disclosed herein, 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. Those skilled in the art will also understand that, in some cases, the term “corresponding to” can be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., a suitable reference event or entity). To cite just one example, a gene or protein in one organism may be described as “corresponding” to a gene or protein from another organism, so that in some embodiments it is indicated to perform a similar function or perform a similar function and/or to exhibit a particular degree of sequence identity or homology, or to share specific characteristic sequence elements.

Cycloaliphatic : As used in this article, the term "cycloaliphatic" refers to monocyclic _C3-8 hydrocarbons or bicyclic _C6-10 hydrocarbons that are completely saturated or contain one or more unsaturated units but are not aromatic, and have one or more bonding sites with the rest of the molecule.

Cycloalkyl : As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms, which may be substituted. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

Derivative : In the context of an amino acid sequence (peptide or polypeptide) "derived from" a specified amino acid sequence (peptide or polypeptide), it refers to a structural analog of the specified amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially identical, or homologous to that particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of that particular sequence or a fragment thereof. For example, an incretin agent utilized according to this disclosure may include an amino acid sequence derived from two or more incretin agents (e.g., two or more naturally occurring incretins).

Detection : The term "detection" is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest in a sample or any form of measurement of the entity of interest. Thus, "detection" can include determining, measuring, evaluating, or verifying the presence or absence, level, quantity, and/or location of an entity of interest. This includes both quantitative and qualitative determination, measurement, or evaluation, including semi-quantitative determination. Such determination, measurement, or evaluation can be relative, such as when relative to a reference detected entity of interest, or absolute. Therefore, the term "quantification," when used in the context of quantifying an entity of interest, can refer to absolute quantification or relative quantification. Absolute quantification can be accomplished by relating the detected level of the entity of interest to a known reference standard (e.g., by generating a standard curve). Alternatively, relative quantification can be accomplished by comparing the detected levels or quantities between two or more different entities of interest to provide relative quantification of each of the two or more different entities of interest (i.e., relative to each other).

Dosage regimen : Those familiar with this technique will know that the term "dosage regimen" (or "treatment regimen") can be used to refer to a series of unit doses (usually more than one unit dose) administered to an individual at regular intervals. In some embodiments, the prescribed treatment has a recommended dosing regimen, which may involve one or more doses.

Encoding : As used herein, the term "encode" or "encoding" refers to the sequence information of a first molecule produced from a second molecule having a defined nucleotide sequence (e.g., a polynucleotide) or a defined amino acid sequence. For example, a DNA molecule can encode an RNA molecule (e.g., through a transcription process involving a DNA-dependent RNA polymerase). An RNA molecule can encode a polypeptide (e.g., through a translation process). Thus, if the transcription and translation of RNA corresponding to a gene produces a polypeptide in a cell or other biological system, then that gene, cDNA, or RNA molecule encodes that polypeptide. In some embodiments, the coding region of a polynucleotide encoding a target antigen refers to a coding strand whose nucleotide sequence is identical to the polynucleotide sequence of such a target antigen. In some embodiments, the coding region of the polynucleotide encoding the target antigen refers to the non-coding portion of the target antigen, which can be used as a template for gene or cDNA transcription.

Engineered : Generally speaking, the term "engineered" refers to a state of artificial manipulation. For example, when two or more sequences that are not linked together in nature are artificially manipulated to directly link with each other in an engineered polynucleotide, and/or when a specific residue in the polynucleotide is not naturally occurring and/or is linked to an entity or part of it that is not linked to it in nature through artificial manipulation, the polynucleotide is considered "engineered".

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

Aliphatic : As used herein, the term "aliphatic" or "aliphatic group" refers to a hydrocarbon moiety having one to five heteroatoms, which may be substituted, in addition to carbon atoms. These heteroatoms may be straight-chain (i.e., unbranched), branched, or cyclic ("heterocyclic") and may be fully saturated or contain one or more unsaturated units, but are not aromatic. The term "heteroatom" means nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternary ammonium form of basic nitrogen. The term "nitrogen" also includes substituted nitrogen. Unless otherwise stated, aliphatic groups contain 1 to 10 carbon atoms, of which 1 to 3 carbon atoms are, as appropriate and independently, substituted with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the aliphatic group contains 1-4 carbon atoms, wherein 1-2 carbon atoms are, as appropriate, independently replaced by a heteroatom selected from oxygen, nitrogen, and sulfur. In some embodiments, the aliphatic group contains 1-3 carbon atoms, wherein 1 carbon atom is, as appropriate, independently replaced by a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable aliphatic groups include, but are not limited to, straight-chain or branched heteroalkyl, heteroalkenyl, and heteroynyl groups. For example, aliphatic groups of 1 to 10 atoms include the following exemplary groups: -O- _CH3 , _-CH2 -O- _CH3 , -O- _CH2 - _CH2 -O- _CH2 - _CH2 -O- _CH3 , and similar groups.

Heteroaryl: The terms “heteroaryl” and “heteroaryl-” used alone or as part of a larger part (e.g., “heteroarylalkyl” or “heteroarylalkoxy”) refer to a monocyclic or bicyclic group having 5 to 10 ring atoms (e.g., 5 to 6-membered monocyclic heteroaryl or 9 to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π electrons shared in a ring array; and having one to five heteroatoms in addition to carbon atoms. The heteroaryl group includes, but is not limited to, thiophene, furanyl, pyrrole, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, dazopyridine, pyrimidinyl, pyrazinyl, indazinyl, purine, naphridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, pyrrolopyridinyl, pyrrolopyrazinyl, thiophenopyrimidinyl, triazolopyridinyl, and benzoisooxazolyl. As used herein, the terms “heteroaryl” and “heteroary-” also include groups in which a heteroaryl ring is fused to one or more aryl, cycloaliphatic or heterocyclic rings, wherein the linking group or linking point is on the heteroaryl ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, dibenzofuranyl, inzolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzooxazolyl, quinolinyl, isoquinolinyl, phenolinyl, phthalazinyl, quinazolinyl, quinoxolinyl, 4H-quinazinyl, carbazolyl, acridineyl, phenazinyl, phenthiazolyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3( 4H )-one, 4H-thieno[3,2-b]pyrrole, and benzoisooxazolyl. The term "hybrid" is used interchangeably with the terms "hybrid ring", "hybrid" or "hybrid family", any of which includes, where appropriate, a substituted ring.

heteroatom : As used herein, the term “heteroatom” means nitrogen, oxygen or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternary ammonium form of basic nitrogen.

Heterocycle : As used herein, the terms "heterocycle,""heterocyclicgroup,""heterocyclicradical," and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 8-membered monocyclic, 6- to 10-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more heteroatoms as defined above, such as one to four, in addition to a carbon atom. When referring to the ring atom of a heterocycle, the term "nitrogen" includes substituted nitrogen. As examples, in saturated or partially unsaturated rings having 0-3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen can be N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolidinyl), or NR ⁺ (as in N-substituted pyrrolidinyl). The heterocycle can be attached to its side group at any heteroatom or carbon atom that produces a stable structure, and any ring atom can be substituted, if applicable. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, aziridine, oxadiazinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, pyrazinyl, dioxalyl, dioxopentyl, diazapinel, oxonitrilepinel, thionitrilepinel, morpholinyl, and thiomorpholinyl. The heterocyclic group can be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, and more preferably monocyclic or bicyclic. Bicyclic heterocyclic groups also include groups in which the heterocycle is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indololinyl, isoindololinyl, benzodioxanecyclopentenyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. Bicyclic heterocycles can also be spirocyclic systems (e.g., 7- to 11-membered spirocyclic fused heterocycles having one or more heteroatoms as defined above (e.g., one, two, three, or four heteroatoms) in addition to a carbon atom). Bicyclic heterocycles can also be bridging ring systems (e.g., 7- to 11-membered bridging heterocycles having one, two, or three bridging atoms).

Homology : As used herein, the term "homology" or "homogeneity" refers to the overall similarity between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered "homological" 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 and/or RNA molecules) and/or polypeptide molecules are considered "homologous" 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 relevant chemical properties at corresponding positions). For example, as is well known to those skilled in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids that are similar to each other, and/or as having "polar" or "nonpolar" side chains. The substitution of one amino acid for another of the same type can generally be considered a "homologous" substitution.

Consistency : As used herein, the term "consistency" refers to the overall correlation between polynucleotide molecules (e.g., DNA and/or RNA molecules) and/or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA and/or RNA molecules) and/or polypeptide molecules are considered "substantially identical" if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. The percentage consistency of two nucleic acid or polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second sequences, and non-identical sequences can be ignored for comparison purposes). In some embodiments, the length of the sequence compared for comparison purposes is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or substantially 100% of the length of the reference sequence. Then, the nucleotides at corresponding positions are 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, the molecule is consistent at that position. Taking into account the number of vacancies introduced for optimal alignment of the two sequences and the length of each vacancy, the percentage consistency between the two sequences is a function of the number of consistent positions shared by the sequences. Sequence comparison and determination of the percentage consistency between two sequences can be performed using mathematical algorithms. For example, the percentage similarity 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 performed using the ALIGN program utilize the PAM120 weighted residual table, a 12-void length penalty, and a 4-void penalty. Alternatively, the percentage similarity between two nucleotide sequences can be determined using the GAP program in the GCG software package that utilizes the NWSgapdna.CMP matrix.

Increase, induce, or decrease : As used herein, these terms or grammatically comparable comparative terms indicate a value relative to a comparable reference measurement. For example, in some embodiments, an assessment value achieved with the provided composition (e.g., a pharmaceutical composition) may be "increased" relative to an assessment value obtained with a comparable reference composition. Alternatively or additionally, in some embodiments, the assessment achieved in an individual may be "increased" relative to assessments obtained in the same individual under different conditions (e.g., before or after the event; or with or without the event, such as administration of a combination as described herein (e.g., a pharmaceutical combination)), or in different comparable individuals (e.g., in comparable individuals who are different from those previously exposed to the disease (e.g., in the absence of administration of a combination as described herein (e.g., a pharmaceutical combination)). In some embodiments, the term "comparative" refers to statistically relevant differences (e.g., differences with a generality and/or magnitude sufficient to achieve statistical correlation). Those skilled in this art will recognize or be able to readily determine, within a given context, the degree of difference and/or generality required or sufficient to achieve such statistical significance. In some embodiments, the term "reduction" or equivalent term, compared to a comparable reference, refers to a reduction in the level of the assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or higher. In some embodiments, the term "reduction" or equivalent term refers to complete or substantially complete suppression, i.e., reduction to zero or substantially to zero. In some embodiments, the term "increase" or "inducement," compared to a comparable reference, refers to an increase in the level of the 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.

In order : As used herein, with respect to polynucleotides or polynucleotides, "in order" refers to the order of features along the 5' to 3' of the polynucleotide or polynucleotide. As used herein, with respect to polypeptides, "in order" refers to the order of features along the polypeptide from the N-terminus—most features move to the C-terminus—most features. "In order" does not imply that additional features cannot exist among the listed features. For example, if features A, B, and C of a polynucleotide are described herein as "feature A, feature B, and feature C in order," this description does not exclude, for example, feature D located between features A and B.

Ionizable : The term "ionizable" refers to a compound, group, or atom that carries a charge at a specific pH. In the context of ionizable amino lipids, such lipids or their functional groups or atoms carry a positive charge at a specific pH. In some embodiments, ionizable amino lipids carry a positive charge at acidic pH. In some embodiments, ionizable amino lipids are primarily neutral at physiological pH values (e.g., about 7.0–7.4 in some embodiments) but become positively charged at lower pH values. In some embodiments, ionizable amino lipids may have a pKa in the range of about 5 to about 7.

Isolated : The term "isolated" means altered or removed from its natural state. For example, nucleic acids or peptides naturally present in living animals are not "isolated," but identical nucleic acids or peptides partially or completely separated from their natural coexisting material are "isolated." Isolated nucleic acids or proteins can exist in a substantially purified form or in non-natural environments (such as, for example, host cells).

Lipids : As used herein, the terms “lipids” and “lipid-like materials” are broadly defined as molecules that contain one or more hydrophobic moieties or groups and, where appropriate, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are also commonly referred to as amphiphiles.

RNA -lipid nanoparticles : As used herein, the term "RNA-lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and an RNA molecule, such as one or more polynucleotides as provided herein. In some embodiments, the RNA-lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, the RNA-lipid nanoparticle comprises at least one cationic amino lipid, at least one cofactor lipid, and at least one polymer-coupled lipid (e.g., PEG-coupled lipid). In various embodiments, the RNA-lipid nanoparticles described herein may have an average size (e.g., Z-mean) 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 disclosed herein, the RNA lipid nanoparticles may have a particle size (e.g., Z-mean) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, the average size of the lipid nanoparticles is determined by measuring the average particle size. In some embodiments, the RNA lipid nanoparticles may be prepared by mixing lipids with the RNA molecules described herein.

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

Nucleic Acids/Polynucleotides : As used herein, the term "nucleic acid" refers to a polymer of at least 10 nucleotides or more. In some embodiments, nucleic acids are or contain DNA. In some embodiments, nucleic acids are or contain RNA. In some embodiments, nucleic acids are or contain peptide nucleic acids (PNAs). In some embodiments, nucleic acids are or contain single-stranded nucleic acids. In some embodiments, nucleic acids are or contain double-stranded nucleic acids. In some embodiments, nucleic acids contain both single-stranded and double-stranded portions. In some embodiments, nucleic acids contain a backbone comprising one or more phosphodiester bonds. In some embodiments, nucleic acids contain a backbone comprising both phosphodiester bonds and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may comprise a backbone comprising one or more phosphate thioester bonds or 5'-N-phosphine bonds and/or one or more peptide bonds, as in "peptide nucleic acid". In some embodiments, the nucleic acid comprises one or more or all of the natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more or all of the non-natural residues. In some embodiments, the non-natural residue includes nucleoside analogues (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazoadenosine, 7-deazoguanidine, 8-sideoxyadenosine, 8-sideoxyguanidine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the non-natural residue contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (such as RNA or polypeptide). In some embodiments, the nucleic acid has a nucleotide sequence containing one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on complementary templates, such as in vivo or in vitro ), replication in recombinant cells or systems, or chemical synthesis. In some implementations, the length of the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 350. 0, 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.

Pharmaceutically Effective Dose : The terms "pharmacologically effective dose" or "therapeuticly effective dose" refer to the amount, alone or in combination with other doses, that achieves the desired response or desired effect. In the treatment of a specific disease (e.g., obesity), in some embodiments, the desired response involves the inhibition of the disease (e.g., obesity) progression. In some embodiments, such inhibition may include slowing the progression of the disease (e.g., obesity) and/or intermittently or reversing the progression of the disease (e.g., obesity). In some embodiments, the desired response in the treatment of a disease (e.g., obesity) may be or include delaying or preventing the onset of the disease (e.g., obesity) or disorder (e.g., obesity-related disorders). The effective dose of the combination (e.g., pharmaceutical combination) described herein will depend on factors such as the disease (e.g., obesity) or condition to be treated (e.g., obesity-related conditions), the severity of the disease (e.g., obesity) or condition (e.g., obesity-related conditions), individual patient parameters (including, for example, age, physical condition, body size, and weight), duration of treatment, type of concomitant therapy (if present), specific route of administration, and similar factors. Therefore, the dosage of the combination (e.g., pharmaceutical combination) described herein may depend on various such parameters. In cases where the response in a patient is insufficient at the initial dose, a higher dose may be used (or an effective higher dose achieved through a different, more localized route of administration).

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 is naturally occurring. In some embodiments, a polypeptide has an amino acid sequence that is not naturally occurring. In some embodiments, a polypeptide has an engineered amino acid sequence because it is designed and/or produced manually. In some embodiments, a polypeptide may contain natural amino acids, non-natural amino acids, or both, or be composed of them. In some embodiments, a polypeptide may contain only natural amino acids, only non-natural amino acids, or be composed of them. In some embodiments, a polypeptide may contain D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may contain only D-amino acids. In some embodiments, a polypeptide may contain only L-amino acids. In some embodiments, the polypeptide may include one or more side groups or other modifications, such as modifying one or more amino acid side chains or attaching them to the N-terminus, C-terminus, or any combination thereof of the polypeptide. In some embodiments, such side groups or modifications include acetylation, amination, esterification, methylation, polyethylene glycolation, etc., including combinations thereof. In some embodiments, the polypeptide may be cyclic and/or may contain a cyclic moiety. In some embodiments, the polypeptide is not cyclic and/or does not contain any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or contain a nailed polypeptide. In some embodiments, the term "peptide" may be appended to the name of a reference peptide, activity, or structure; in such cases, it is used herein to refer to peptides that share a relevant activity or structure and are therefore considered members of the same peptide class or family. For each of these classes, this specification provides and/or those skilled in the art will recognize exemplary peptides within that class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary peptides are reference peptides of the peptide class or family. In some embodiments, members of a peptide class or family show significant sequence homology or similarity to the reference peptide of that class (and in some embodiments, all peptides within that class), share common sequence motifs (e.g., characteristic sequence elements), and/or share common activities (in some embodiments, at a comparable level or within a specified range). For example, in some embodiments, the member polypeptide and the reference polypeptide show an overall degree of sequence homology or identity of at least about 30%-40%, and generally greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or include at least one region showing very high sequence identity, generally greater than 90% or even 95%, 96%, 97%, 98% or 99% (e.g., in some embodiments, this may be or contain a conserved region of characteristic sequence elements). Such conserved regions typically encompass at least 3-4 and usually at most 35 or more amino acids; in some embodiments, the conserved region encompasses at least one segment of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive amino acids. In some embodiments, the related polypeptide may comprise or be composed of fragments of the parent polypeptide.

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

Reference : As used herein, the term "reference" describes a standard or comparison relative to which it is compared. For example, in some embodiments, the 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, the reference or control is tested and/or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or comparison, as appropriate, implemented in tangible media. Generally, as understood by those skilled in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those of the references or controls being evaluated. Those familiar with this technique will understand when there is sufficient similarity to justify relying on or comparing with a particular possible reference or comparison.

Ribonucleic acid (RNA) or polynucleotide : As used herein, the terms "ribonucleic acid,""RNA," or "polynucleotide" refer to a polymer of ribonucleotides. In some embodiments, RNA is single-stranded. In some embodiments, RNA is double-stranded. In some embodiments, RNA comprises both single-stranded and double-stranded portions. In some embodiments, RNA may contain a backbone structure as defined above in the definition of "nucleic acid/polynucleotide." RNA may be regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA (mRNA). In some embodiments, RNA is mRNA. In some embodiments, where RNA is mRNA, RNA typically contains a poly(A) region at its 3' end. In some embodiments, when the RNA is mRNA, the RNA typically includes a technically recognized cap structure at its 5' end, for example, to identify the mRNA and link it to the ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic synthesis and/or by chemical synthesis).

Ribonucleotides : As used herein, the term "ribonucleotide" encompasses both unmodified 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) terminal modifications, such as 5' modifications (e.g., phosphorylation, dephosphorylation, coupling, anti-coupling, etc.), 3' modifications (e.g., coupling, anti-coupling, etc.), (b) base modifications, such as substitution with a modified base, a stable base, a destabilized base, or a base or coupling base paired with an extended spectrum base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution, and (d) internucleotide bond modifications, including phosphodiester bond modifications or substitutions. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

Risk : As will be understood from the context, “risk” for a disease, condition, and/or disorder refers to the likelihood that a particular individual will develop a disease, condition, and/or disorder. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is expressed as risk relative to the risk associated with a reference sample or reference sample group. In some embodiments, the reference sample or reference sample group has a known risk of a disease, condition, disorder, and/or event. In some embodiments, the reference sample or reference sample group is derived from individuals comparable to the particular individual. In some embodiments, risk may reflect one or more genetic attributes, such as predispositions to (or non-predisposition to) a particular disease, condition, and/or disorder. In some implementations, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.

Specificity : When used herein to refer to an active agent, the term "specificity" should be understood by those skilled in the art to mean that the agent distinguishes a potential target entity, state, or cell. For example, in some embodiments, an agent is said to "specifically" bind to its target if it preferentially binds to one or more competing alternative targets in the presence of such targets. In many embodiments, specific interactions depend on the presence of specific structural features of the target entity (e.g., antigenic determinants, gaps, binding sites). It should be understood that specificity need not be absolute. In some embodiments, specificity can be assessed relative to the specificity of the target-binding portion to one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of the reference specificity combination component. In some embodiments, specificity is evaluated relative to the specificity of the reference nonspecificity combination component.

Substitutionalized or as appropriate : As described herein, compounds of the invention may contain a "substituted" moiety. Generally, the term "substituted" (whether preceded by the phrase "as appropriate") means that one or more of the specified moiety of hydrogen atoms are substituted with suitable substituents. "Substitutionalized" applies to self-structured (e.g., It means at least ;and It means at least , or The presence or absence of one or more hydrogen atoms is explicitly or implicitly implied in the designation. Unless otherwise indicated, a "substituted" group may have suitable substituents at each substituted position of the group, and when more than one position in any given structure is substituted by more than one substituent selected from the designated group, the substituents may be the same or different at each position. The substituent combinations contemplated in this invention are preferably combinations that result in the formation of stable or chemically viable compounds. As used herein, the term "stable" means a compound that is substantially unchanged when subjected to permission to produce, detect, and, in some embodiments, to be recovered, purified, and used for one or more of the purposes provided herein. A group described as "substituted" preferably has 1 to 4 substituents, more preferably 1 or 2 substituents. Groups described as "substituted as appropriate" can be unsubstituted or "substituted" as described above.

The appropriate monovalent substituent on the substituted carbon atom of the "substituent as appropriate" group is independently a halogen; -( _CH2 ) _0-4 R°; -( _CH2 ) _0-4 OR°; -O( _CH2 ) _0-4 ^Ro , -O-( _CH2 ) _0-4 C(O)OR°; -( _CH2 ) _0-4 CH(OR°) ₂ ; -( _CH2 ) _0-4 SR°; -( _CH2 ) _0-4 Ph, which can be substituted by R°; -( _CH2 ) _0-4 O( _CH2 ) _0-1 Ph, which can be substituted by R°; -CH=CHPh, which can be substituted by R°; -( _CH2 ) _0-4 O( _CH2 ) _0-1 -pyridyl, which can be substituted by R°; _-NO2 ; -CN; _-N3 ;-(CH ₂ ) _0-4 N(R°) ₂ ;-(CH ₂ ) _0-4 N(R°)C(O)R°;-N(R°)C(S)R°;-(CH ₂ ) _0-4 N(R°)C(O)NR° ₂ ;-N(R°)C(S)NR° ₂ ;-(CH ₂ ) _0-4 N(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°; -(CH ₂ ) _0-4 C(O)R°; C(S)R°; -(CH ₂ ) _0-4 C(O)OR°; -(CH ₂ ) _0-4 C(O)SR°;-(CH ₂ ) _0-4 C(O)OSiR° ₃ ;-(CH ₂ ) _0-4 OC(O)R°;-OC(O)(CH ₂ ) _0-4 SR°;-(CH ₂ ) _0-4 SC(O)R°;-(CH ₂ ) _0-4 C(O)NR° ₂ ;-C(S)NR° ₂ ; -C(S)SR°; -SC(S)SR°, -(CH ₂ ) _0-4 OC(O)NR° ₂ ; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH ₂ C(O)R°; -C(NOR°)R°; -(CH ₂ ) _0-4 SSR°; - (CH ₂ ) _0-4 S(O) ₂ R°;-(CH ₂ ) _0-4 S(O) ₂ OR°; -( _CH2 ) _O-4OS (O) _2R °; -S(O) _2NR ° ₂ ; - _(CH2 )O _-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-chain or branched alkyl)ON(R°) ₂ ; or -( _C1-4 straight-chain or branched alkyl)C(O)ON(R°) ₂ , wherein each R° may be substituted and independently represented as hydrogen, C as defined below. _1-6 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, -CH ₂ - (5 to 6 member heteroaryl rings), or having 3 to 6 saturated, partially unsaturated or aryl rings with 0 to 4 independent heteroatoms selected from nitrogen, oxygen or sulfur, or, despite the above definition, two independently occurring R° together with intercalated atoms to form 3 to 12 saturated, partially unsaturated or aryl monocyclic or bicyclic rings with 0 to 4 independent heteroatoms selected from nitrogen, oxygen or sulfur, which may be substituted as defined below.

The suitable monovalent substituents on R° (or a ring formed by two independently occurring R° and their intercalation atoms) are independently halogens, -( _CH2 ) _O-2Rl ^, -(halogen ^Rl ), -( _CH2 )O _-2OH , -( _CH2 )O ^- _2ORl , -( _CH2 )O- _2CH ( ^ORl ) ₂ , -O(halogen ^Rl ), -CN, _-N3 , -( _CH2 ) _O-2C (O) ^Rl , -( _CH2 )O _-2C (O)OH, -( _CH2 )O _-2C (O) ^ORl , -( _CH2 )O _-2SRl , -( _CH2 ) ^O _-2SH , -( _CH2 )O _{- 2NH2} , -(CH2)O _{-2 -0-2} NHR ^l , -(CH ₂ ) _O-2 NR ^l ₂ , -NO ₂ , -SiR ^l ₃ , -OSiR ^l ₃ , -C(O)SR ^l _, -(C _1-4 straight-chain or branched-chain alkyl)C(O)OR ^l or -SSR ^l , wherein each R ^l is unsubstituted or, if preceded by a "halogen", substituted by only one or more halogens, and independently selected from C _1-4 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _O-1 Ph, or has 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, with 3 to 6 saturated, partially unsaturated, or aryl rings. Suitable divalent substituents on the saturated carbon atom of R° include =O and =S.

Suitable divalent substituents on the saturated carbon atom of the "substituted as appropriate" group include the following: =O ("side-oxygen"), =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 independently appearing R ^* is selected from hydrogen, can be a substituted _C1-6 aliphatic group as defined below, or an unsubstituted 5 to 6 saturated, partially unsaturated or aryl ring having 0 to 4 independently selected heteroatoms from nitrogen, oxygen or sulfur. Suitable divalent substituents that bind to adjacent substituted carbons of a "substituted as appropriate" group include: -O(CR ^* ₂ ) _2-3O- , wherein each independently appearing R ^* is selected from hydrogen, a substituted _C1-6 aliphatic group as defined below, or an unsubstituted 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 independently selected heteroatoms from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R ^* include halogens, -R ^l , -(halogen R ^l ), -OH, -OR ^l , -O(halogen R ^l ), -CN, -C(O)OH, -C(O)OR ^l , -NH ₂ , -NHR ^l , -NR ^l ₂ or -NO ₂ , wherein each R ^l is unsubstituted or, if preceded by "halogen", is substituted by only one or more halogens, and is independently C _1-4 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, or has 0-4 3 to 6 saturated, partially unsaturated or aryl rings independently selected from nitrogen, oxygen or sulfur.

Suitable substituents on the substituted nitrogen of the "substitutable as appropriate" 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, and the substituted C can be defined as follows. _1-6 aliphatic, unsubstituted -OPh, or an unsubstituted 3-6 member saturated, partially unsaturated, or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, or, despite the above definition, two independently occurring R ^† together with intercalated atoms to form an unsubstituted 3-12 member saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R ^† are independently halogens, -R ^l , -(halogen R ^l ), -OH, -OR ^l , -O(halogen R ^l ), -CN, -C(O)OH, -C(O)OR ^l , -NH ₂ , -NHR ^l , -NR ^l ₂ or -NO ₂ , wherein each R ^l is unsubstituted or, if preceded by "halogen", is substituted by only one or more halogens, and is independently C _1-4 aliphatic, -CH ₂ Ph, -O(CH ₂ ) _0-1 Ph, or has 0-4 heteroatoms independently selected from nitrogen, oxygen or sulfur, with 3 to 6 saturated, partially unsaturated or aryl rings.

Individual : As used herein, the term "individual" means an organism intended to be administered the combination described herein, for example, for experimental, diagnostic, preventative, and/or therapeutic purposes. Typical individuals include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the individual is a human individual. In some embodiments, the individual suffers from a disease, condition, or disorder (e.g., obesity, obesity-related disorders, etc.). In some embodiments, the individual is susceptible to a disease, condition, or disorder (e.g., obesity, obesity-related disorders, etc.). In some embodiments, the individual exhibits one or more symptoms or features of a disease, condition, or disorder (e.g., obesity, obesity-related disorders, etc.). In some embodiments, the individual exhibits one or more nonspecific symptoms of a disease, condition, or disorder (e.g., obesity, obesity-related disorders, etc.). In some embodiments, the individual does not exhibit any symptoms or features of a disease, condition, or disorder (e.g., obesity, obesity-related disorders, etc.). In some embodiments, the individual is a person who has one or more features that indicate susceptibility or risk to a disease, condition, or disorder (e.g., obesity, obesity-related disorders, etc.). In some embodiments, the individual is a patient. In some embodiments, the individual is an individual who has received and/or has received a diagnosis and/or therapy.

" Having " : An individual who has been diagnosed with and/or exhibits one or more symptoms of a disease, condition and/or disorder (e.g., obesity, obesity-related disorders).

"Susceptible " : An individual "susceptible" to a disease, condition, and/or disorder (e.g., obesity, obesity-related disorders) is someone who has a higher risk of developing that disease, condition, and/or disorder than the general population. In some embodiments, an individual susceptible to a disease, condition, and/or disorder (e.g., obesity, obesity-related disorders) may not be diagnosed with that disease, condition, and/or disorder (e.g., obesity, obesity-related disorders). In some embodiments, an individual susceptible to a disease, condition, and/or disorder (e.g., obesity, obesity-related disorders) may exhibit symptoms of that disease, condition, and/or disorder (e.g., obesity, obesity-related disorders). In some embodiments, individuals susceptible to a disease, condition, and/or disorder (e.g., obesity, obesity-related disorders, etc.) may not exhibit symptoms of that disease, condition, and/or disorder (e.g., obesity, obesity-related disorders, etc.). In some embodiments, individuals susceptible to a disease, condition, and/or disorder (e.g., obesity, obesity-related disorders, etc.) will develop that disease, condition, and/or disorder (e.g., obesity, obesity-related disorders, etc.). In some embodiments, individuals susceptible to a disease, condition, and/or disorder (e.g., obesity, obesity-related disorders, etc.) will not develop that disease, condition, and/or disorder (e.g., obesity, obesity-related disorders, etc.).

Therapy : The term "therapy" means the administration or delivery of an agent or intervention that has a therapeutic effect and/or induces a desired biological and/or pharmacological effect (e.g., one that has been shown to be statistically likely to have when administered to a relevant population). In some embodiments, a therapy or treatment is any substance that can be used to reduce, improve, alleviate, inhibit, prevent, disease, condition, and/or disorder (e.g., obesity, obesity-related disorders, etc.), delay its onset, reduce its severity, and/or reduce its incidence. In some embodiments, a treatment or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to reduce, alleviate, suppress, present one or more symptoms or features of a disease, condition and/or disorder, delay its onset, reduce its severity and/or reduce its incidence.

Treatment : As used herein, the terms “treat,” “treatment,” or “treating” mean any method used to partially or completely reduce, improve, alleviate, suppress, prevent, or delay the onset, severity, and/or incidence of one or more symptoms or features of a disease, condition, and/or disorder (e.g., obesity, obesity-related disorders). Treatment may be administered to individuals who do not exhibit signs of a disease, condition, and/or disorder (e.g., obesity, obesity-related disorders). In some embodiments, treatment may be administered to individuals exhibiting only early signs of a disease, symptom, and/or disorder (e.g., obesity, obesity-related disorders), for example, to reduce the risk of developing a pathology associated with that disease, symptom, and/or disorder. In some embodiments, treatment may be administered to individuals in the later stages of a disease, symptom, and/or disorder (e.g., obesity, obesity-related disorders).

The compounds disclosed herein include those generally described above and further described by the classes, subclasses and species disclosed herein. Unless otherwise indicated, the following definitions should apply as used herein. For the purposes of this disclosure, chemical elements are identified according to the periodic table, CAS version, Handbook of Chemistry and Physics, 75th edition. Furthermore, the general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999 and "March’s Advanced Organic Chemistry," 5th edition, edited by Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

Unless otherwise stated, the structures described herein are intended to include all stereoisomers (e.g., mirror or non-mirror isomers) of the structure, as well as all geometric or configurational isomers of the structure. For example, the R and S configurations of each stereocenter are considered as part of this disclosure. Therefore, single stereochemical isomers of the provided compounds, as well as mirror isomers, non-mirror isomers, and geometric (or configurational) mixtures, are within the scope of this disclosure. For example, in some cases, the provided compounds exhibit one or more stereoisomers, and unless otherwise indicated, each stereoisomer represents itself individually and/or as a mixture. Unless otherwise stated, all tautomerisms of the provided compounds are within the scope of this disclosure.

Unless otherwise indicated, the structures described herein are intended to include compounds differing only in the presence of one or more isotopically enriched atoms. For example, compounds having this structure (including those with hydrogen replaced by deuterium or tritium, or carbon replaced by carbon enriched in 13C or 14C) are within the scope of this disclosure. Methods of Embodiment: Incretins and their Use in the Treatment of Diseases

Incretins are peptide hormones released in the gastrointestinal (GI) tract in response to glucose consumption. They stimulate the pancreas to secrete insulin and reduce glucagon production, thereby lowering blood glucose levels. Incretins exert their effects by binding to their corresponding receptors on pancreatic β-cells, leading to insulin release. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are two incretins whose roles in postprandial insulin secretion have been identified. GIP is primarily responsible for releasing insulin in response to glucose ingestion. GLP-1 stimulates satiety, slows gastric emptying, reduces glucagon secretion, and decreases food intake, leading to weight loss. It has been shown that when insulin is secreted, the agonist GIP and GLP1 receptors have an additive effect. See Chim, US Pharm . 2022; 47(10):18-22, which is incorporated herein by reference in its entirety.

Due to their roles in controlling blood sugar and satiety, incretins and incretin analogs have the potential to treat a variety of diseases, including obesity, prediabetes, type 2 diabetes (T2D, and its complications), early-stage type 1 diabetes (e.g., within 3 months of a T1D diagnosis), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) diseases (e.g., characterized by major cardiovascular events (MACE), including CV death, non-fatal myocardial infarction, non-fatal stroke, or heart failure with preserved ejection fraction (HFpEF)), kidney disease, and an increased risk of premature death. These chronic diseases are prevalent worldwide and often coexist as comorbidities. Obesity

Obesity is the most prevalent chronic disease worldwide, affecting approximately 650 million adults. Obesity is considered a starting point and key contributing factor to prediabetes, type 2 diabetes (T2D and its complications), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease and kidney disease, and premature death. Obesity imposes a significant economic burden, including additional direct healthcare costs, productivity costs (absence, attendance, disability support, premature death), transportation costs (including increased _CO2 footprint), and human capital accumulation costs (school absences, highest level of education achieved). It is estimated that by 2030, the number of obese people (BMI > 30 kg/ ^m² ) will exceed one billion, of whom approximately 10% will suffer from severe type III obesity (BMI > 40 kg/ ^m² ). Half of all obese men live in just nine countries: the United States, China, India, Brazil, Mexico, Russia, Egypt, Germany, and Turkey. Childhood obesity is also rising sharply worldwide.

Obesity was only declared a disease by the American Association of Clinical Endocrinologists (AACE) in 2011, and is managed based on severity, starting with lifestyle/behavioral interventions and increasing physical activity, followed by drug therapy, and finally weight loss surgery.

Based on short-term studies, it is recommended that overweight or obese individuals with type 2 diabetes mellitus (T2D) lose weight. The Look AHEAD study, which investigated the long-term effects of weight loss on cardiovascular disease in more than 5,100 people with T2D, was discontinued in 2012 after nearly 10 years because it showed that intensive lifestyle interventions focused on weight loss did not reduce the rate of cardiovascular events in overweight or obese adults with T2D.

In summary, drug therapy has had limited success for obesity to date, producing only moderate placebo-corrected weight loss, such as 3% for Xenical® (orlistat) and 4-5% for Belviq® (lorcaserin) and Contrave® (naltrexone SR/bupropion SR). It also carries the social restriction side effects of Xenical® and the adverse central nervous system effects of centrally acting agents. The FDA rejected Acomplia® (rimonabant) due to concerns that its use might increase suicidal thoughts and depression.

The most effective intervention for obesity remains weight-loss surgery; however, due to perceived serious complications (including mortality), only 1% of eligible patients undergo this procedure. Weight-loss surgery has been shown to substantially alter the release of endocrine hormones, leading to interest in targeting the pathway of endogenous nutrient-stimulating hormones, essentially simulating the effects of weight-loss surgery using chemotherapeutic agents called "incretin analogs." Current Treatments Using Incretin Analogs

Current therapies using incretin analogues include glucagon-like peptide-1 (GLP-1) receptor agonists, such as Trulicity® (dulaglutide), Byetta® (exenatide), Ozempic®/Rybelsus® (semaglutide, injectable/oral), Victoza® (liraglutide), and Suliqua® (lixisenatide, in combination with glargine only). These receptor agonists are approved for lowering blood glucose levels in individuals with type 2 diabetes without the need for continuous blood glucose monitoring. Additional benefits include weight loss (2-4%) and positive effects on cardiovascular and renal parameters. Recent studies have explored the combination of GLP1 receptor agonists with GIP receptor agonists and/or glucagon (GCG) receptor agonists (dual/triple agonists) to achieve better blood glucose control and greater weight loss. The GLP1/GCG receptor dual agonist SAR425899 demonstrated blood glucose control and weight loss; however, this program was discontinued in 2019 due to unacceptable gastrointestinal side effects. Recently, the GLP1/GIP receptor dual agonist tirzepatide (now marketed as Mounjaro®) was approved as an injectable medication for adults with type 2 diabetes (T2D), used in conjunction with diet and exercise to improve blood glucose levels. GIP functions by regulating energy balance through signal transduction on cell surface receptors in the brain and adipose tissue. The SURPASS-2 study demonstrated that telpolide is non-inferior to smegglutinin and superior in lowering blood sugar. However, weight loss is only a secondary endpoint.

Phase 3 trials with weight loss as the primary endpoint have been conducted in non-diabetic, overweight/obese patients – see the SCALE (liraglutide), STEP-1 (semaglutide), and SURMOUNT-1 (telpovide) trials. The SCALE trial demonstrated a 5.4% placebo-corrected weight loss and a lower incidence of prediabetes with a once-daily dose of 3 mg liraglutide plus lifestyle intervention. The STEP-1 trial produced a 12.4% weight loss in overweight or obese participants receiving a once-weekly dose of 2.4 mg semaglutide plus lifestyle intervention. The SURMOUNT-1 trial showed a 17.8% placebo-corrected weight loss at the highest once-weekly dose of 15 mg telpovide. Cardiac metabolic measures improved in all studies. Gastrointestinal side effects were as expected, especially at the start of treatment, and were manageable. Approved obesity products now include Saxenda® (liraglutide 3 mg daily injection) and Wegovy® (semaglutide once weekly injection). In October 2022, the FDA granted semaglutide Fast Track designation for the treatment of adults who are obese or overweight with weight-related comorbidities, which could lead to approval for the indication in early 2024 based on rolling submissions of further data from the SURMOUNT study series. Topline results recently published from the SELECT trial, which tested Wegovy® in patients with pre-existing cardiovascular disease and who were overweight/obese but without type 2 diabetes (T2D), showed that 2.4 mg semaglutide reduced the risk of major adverse cardiovascular events by 20% in overweight or obese adults. Another study using an incretin analogue was OASIS-1, which investigated the oral administration of 25 or 50 mg semagravitide in non-diabetic overweight/obese patients. A 14 mg maintenance dose of oral semagravitide has been approved as Rybelsus® for type 2 disease (T2D). Recently, Phase 2 results for the triple receptor agonist (GIP/GLP1/GCG receptor) retratutide LY3437943 were published, showing placebo-corrected weight loss in 22.1% of obese patients after 48 weeks of treatment. Semagravitide has also recently shown benefit in early T1D (within 3 months of T1D diagnosis). In a small study, all participants no longer needed mealtime insulin, and most participants no longer needed basal insulin.

Exemplary treatments using incretin models for obesity/T2D are shown in Table 1 below. Table 1: Exemplary Incretin Models for the Treatment of Obesity/T2D Drug Name Target Indications Trulicity® (dulaglutide) GLP1 receptor agonists T2D Byetta® (exenatide) GLP1 receptor agonists T2D Ozempic® (Smigratide) GLP1 receptor agonists T2D (injectable) Rybelsus® (Smigrape peptide) GLP1 receptor agonists T2D (oral) Victoza (Liraglutide) GLP1 receptor agonists T2D Saxenda® (liraglutide) GLP1 receptor agonists Obesity/weight loss Suliqua® (Lixinatide) GLP1 receptor agonists T2D Wegovy® (Smigrape Peptide) GLP1 receptor agonists obesity Mounjaro® (Telbopeptide) GLP1/GIP dual receptor agonists T2D

This disclosure specifically recognizes current problems in the market for incretin analogues used to treat obesity, prediabetes, type 2 diabetes, early type 1 diabetes, NAFLD, NASH, cardiovascular disease, kidney disease, and increased risk of premature death, including but not limited to limited supply, high prices, lack of health insurance coverage for such treatments, frequent injections (e.g., once a week), large injection volumes, and gastrointestinal side effects.

This disclosure provides, in particular, a more effective and cost-efficient method for using incretins and incretin mimics (understood by the term "incretin agent") encoded by one or more polynucleotides to treat obesity, prediabetes, type 2 diabetes, early type 1 diabetes, NAFLD, NASH, cardiovascular disease, kidney disease, and increased risk of premature death. In some embodiments, polynucleotides encoding incretin agents provide therapeutic treatment for obesity and/or prediabetes, type 2 diabetes, early type 1 diabetes, NAFLD, NASH, cardiovascular disease, kidney disease, and increased risk of premature death (e.g., obesity-related diseases) with improved properties compared to known incretin mimicry therapies, including requiring fewer injections (no more than once a week), smaller injection volumes (e.g., no more than 0.5 ml), and fewer or less severe side effects. Additionally, polynucleotides used to deliver incretin agents provide incretin agents with therapeutically relevant levels of performance in cells, comparable to the dosage of current peptide-based therapies. Polynucleotides used to deliver incretin agents

This disclosure utilizes RNA technology, in particular, as a modality for the direct expression in vivo of incretins as a novel class of therapeutic agents that activate GLP1, GIP, and/or GCG receptors to effectively treat disease conditions such as obesity, prediabetes, type 2 diabetes, early type 1 diabetes, NAFLD, NASH, cardiovascular disease, kidney disease, and/or an increased risk of premature death. In some embodiments, the polynucleotides described herein encode naturally occurring incretins or fragments or variants thereof. In some embodiments, the polynucleotides described herein encode incretins or fragments or variants thereof modified from their natural form.

As used herein, the term "incretin preparation" refers to a preparation containing incretin or incretin mimics (collectively referred to herein as "incretin peptides"). Exemplary incretins include GLP1, GIP, and GCG. Exemplary incretin mimics are shown, for example, in Table 1. In some embodiments, the incretin preparation is a biologically active portion or fragment of an incretin or incretin mimic. In some embodiments, the incretin preparation contains an incretin peptide as part of a fusion compound. For example, in some embodiments, the incretin is an incretin peptide fused to another peptide moiety (e.g., a half-life extension (HLE) domain).

In some embodiments, the incretin agent includes a GLP-1 receptor agonist, such as GLP-1. In some embodiments, the incretin agent includes a GIP receptor agonist, such as GIP. In some embodiments, the incretin agent includes dual GIP and GLP-1 receptor agonists. In some embodiments, the incretin agent includes triple GIP, GLP-1, and GCG receptor agonists. Exemplary Incretin Peptide

In some embodiments, the incretin agent comprises a wild-type (i.e., unmutated) incretin peptide sequence or a fragment thereof. For example, in some embodiments, the incretin agent comprises any of the incretin peptides shown in SEQ ID NO: 5-15 and 62-64.

In some embodiments, the incretin agent comprises an incretin peptide or fragment thereof having at least one mutated amino acid residue compared to a wild-type reference sequence. In some embodiments, the mutated amino acid residue comprises a native amino acid residue substituted with another native amino acid residue. In some embodiments, the incretin agent comprises any of the incretin peptides shown in SEQ ID NO: 5-10. In some embodiments, the mutated amino acid residue may endow the incretin agent with dual or triple agonistic properties. For example, in some embodiments, one or more amino acid substitutions are introduced into the GLP1, GIP, or GCG peptide sequence (e.g., as shown in SEQ ID NO: 12-15 and 62-64) to endow two or more of the GLP1, GIP, and/or GCG receptors with binding properties.

In some embodiments, the incretin agent has an amino acid sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to any of the incretin peptides detailed in Table 2. In some embodiments, the incretin agent comprises any of the incretin peptides detailed in Table 2 below, or combinations or variants thereof. Table 2: Exemplary Incretin Peptides (Mutations are shown in bold) Incretins SEQ ID NO sequence GLP1 (7-37) 5 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG GLP1 with K34R mutation (7-37) 6 HAEGTFTSDVSSYLEGQAAKEFIAWLV R GRG GLP1 (7-36) 7 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR GIP (1-42) 8 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GIP (1-30) 9 YAEGTFISDYSIAMDKIHQQDFVNWLLAQK Exenatide (GLP1R agonist) 11 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSAPPPS Telborpeptide (GLP1R/GIPR dual agonist) 12 YAEEGTFTSDYSIYLDKIAQKAFVQWLIAGGPSSGAPPPS NNC0090-2746 (GLP1R/GIPR dual agonist) 13 YAEEGTFTSDYSIYLDKQAAKEFVNWLLAGGPSSGAPPPS MAR701 (GLP1R/GIPR dual agonist) 14 YAEGTFTSDYSIYLDKQAAKEFVCWLLAGGPSSGAPPPS Retalutide (GLP1R/GIPR/GCGR triple agonist) 15 YXQGTFTSDYSIYLDKKAQXAFIEYLLEGGPSSGAPPPS Where X at position 2 is A or S, and X at position 20 is K or Q. GIPs with A2G mutations (1-42) 62 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP1 with A8G mutation (7-37) 63 H G EGTFTSDVSSYLEGQAAKEFIAWLVKGRG GLP1 with H7Y and A8G mutations (7-37) 64 YG EGTFTSDVSSYLEGQAAKEFIAWLVKGRG GLP1 with A8G and R36G mutations (7-37) 69 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GLP1 with H7Y, A8G and R36G mutations (7-37) 70 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GIPs with A2G mutations (1-30) 72 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQK GLP1 with A8G and K34R mutations (7-37) 74 H G EGTFTSDVSSYLEGQAAKEFIAWLV R GRG GLP1 with H7Y, A8G and K34R mutations (7-37) 75 YG EGTFTSDVSSYLEGQAAKEFIAWLV R GRG connector

In some embodiments, the incretin agents described herein comprise a single incretin peptide (this configuration is referred to herein as "I:1x") (see, for example, Figure 3 ). In some embodiments, the incretin peptide is fused to another peptide (e.g., a half-life extension (HLE) domain) via a linker (see, for example, Figures 4-14 ). In some embodiments, the linker contains at least one Gly (G) amino acid residue. Suitable connectors can be easily selected and can have various lengths, such as 1 amino acid (e.g., Gly) to 25 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids). Exemplary linkers include glycine polymers (G) _n , glycine-serine polymers (including, for example, (GS) _n , (GGGGS: SEQ ID NO: 1) _n , and (GGGS: SEQ ID NO: 2) _n , where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as neutral links between components. Glycine is even closer to a significantly larger phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11:173-142 (1992)). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 3 (GGGGSGGGGSGGGGSGGGS or "(G4S)4" linker) or SEQ ID NO: 4 (GGGGSGGGGSGGGGSGGGSGGGS or "(G4S)5" linker). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 68 (GGGGSGGGGS or "(G4S)2" linker), SEQ ID NO: 156 (GGGSGGGS or "(G3S)2" linker), SEQ ID NO: 157 (GGGGSGGGGSGGGS or "(G4S)3" linker) or GGGGSGGGS (SEQ ID NO: 186).

In some embodiments, the incretin agent comprises an incretin peptide linked to another peptide (e.g., the HLE domain described herein) using a (G4S)3 linker. In some embodiments, the incretin agent described herein comprises an incretin peptide linked to another incretin peptide using a (G4S)2 linker. Cleavage site

In some embodiments, the incretin peptide is fused to another peptide (e.g., a peptide including motif R-X-K/R-R SEQ ID NO: 158, such as SEQ ID NO: 160 RRKR or SEQ ID NO: 153 NVRRKR) via a protease cleavage site, such as a furin cleavage site (e.g., a peptide including motif R-X-K/R-R SEQ ID NO: 158, such as SEQ ID NO: 160 RRKR or SEQ ID NO: 153 NVRRKR or SEQ ID NO: 189 RKKR or SEQ ID NO: 190 RMQR or SEQ ID NO: 191 VFRR or SEQ ID NO: 191) via a protease cleavage site (e.g., a furin cleavage site). The terms "furin cleavage site" and "furin recognition site" are used interchangeably in this article and refer to the sequence that promotes furin cleavage.

In some embodiments, the furin cleavage site is operatively linked to a linker (e.g., a glycine linker, such as a (G4S)2 linker) (e.g., on its C-terminus). In some embodiments, the incretin agent comprises multiple (e.g., 2, 3, 4 or more) incretin peptides, each separated from the furin cleavage site and, where applicable, a linker (e.g., a (G4S)2 linker located on the N-terminus of the furin cleavage site).

The incretin formulations described herein include furin cleavage sites, particularly in cases where an incretin peptide is fused to another peptide (e.g., another incretin peptide and/or a half-life extension (HLE) domain), to promote appropriate cleavage of the incretin peptide from the other peptide and to allow the incretin peptide to be fully processed and functional after its expression.

Without being bound by any theory, in the context of the polynucleotides encoding incretins, the type of cleavage sites and recognition sites is important for ensuring the correct processing of the N-terminus of incretin peptides. Certain furin cleavage and recognition sites, and the placement of these sites relative to the incretin peptide within the incretin, can create alternative processing or cleavage sites, ultimately altering the final amino acid sequence of the mature incretin peptide. In relatively small peptides such as GLP1 or GIP (or their variants, and other peptides of similar size/properties), any change in amino acid residues can affect the peptide's biological activity. In some embodiments, furin recognition/cleavage sites are selected and located within the incretin agent to promote proper cleavage of the N-terminus of the incretin peptide, or in other words, to produce a "scarless" N-terminus of the incretin peptide to maintain its biological activity. Figures 20 and 21 illustrate schematic diagrams of the theoretical cleavage site locations of some exemplary signaling peptides. Figure 20 shows that the A8G mutation promotes the correct N-terminal processing of the GLP1 incretin peptide with the husec signaling peptide. Figure 21 shows that the A2G mutation promotes the correct N-terminal processing of the GIP incretin peptide with the husec signaling peptide.

This concept and utilization of the furin cleavage site at the N-terminus of an incretin peptide linked to another peptide can also be applied to other incretin peptides (e.g., glucagon) and/or other peptides of similar size/properties to GLP1 and GIP described herein. This is particularly important in the context of delivering incretins (or other similar peptides) as encoding one or more polynucleotides of incretins. Such delivery requires proper translation of intracellular proteins, in addition to post-translational processing (including the proper cleavage of one incretin peptide from another). In some embodiments, incretins comprising one or more incretin peptides fused to another peptide as described herein have been designed and generated to include a protease cleavage site within the incretin, such that the cleavage of the incretin peptide from the other peptide is accurate and does not affect the amino acid sequence of the mature peptide (i.e., producing a scarless N-terminus). The term "scarless" N-terminus as used herein includes a peptide that has been cleaved from another peptide by a cleavage site, wherein the cleavage occurs in such a manner that no residual amino acids are not part of the mature peptide and all amino acids of the mature peptide are retained at the N-terminus of the peptide. Scarless N-termini of incretin peptides (and other similar peptides, such as other incretins, e.g., glucagon) allow the peptide to function appropriately after processing into the mature peptide.

In some embodiments, the furin cleavage site is adjacent to the 5' position of the second incretin peptide in the incretin agent to ensure that the cleavage produces a scarless N-terminus on the second incretin peptide. This type of cleavage can be important for maintaining the function of mature incretin peptides.

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

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

In some embodiments, mutations are introduced into the incretin peptides described herein to promote the cleavage of the signal peptide and produce mature incretin peptides with unscarred N-termini. In some embodiments, such mutations include the A2G mutation in GIP incretin peptides (e.g., GIP (1-42) incretin peptides). In some embodiments, such mutations include the A8G mutation in GLP1 (7-37) incretin peptides. In some embodiments, such mutations may also increase the half-life of incretin agents, for example, by preventing the proteolysis of an amino acid at the second position of the incretin peptide (leaving behind a mature incretin peptide that has been improperly cleaved at its N-terminus and truncated by one or two amino acids). In some embodiments, the mutation is selected to increase the probability of correct cleavage (i.e., cleavage at the N-terminus that prevents the mature incretin peptide from being truncated). In some embodiments, the compatibility of the signal peptide and cleavage site sequences used in the incretin formulations described herein depends on the amino acid sequences of specific adjacent incretin peptides, particularly the amino acid residue at the N-terminus. Incretin formulations containing multiple incretin peptides

In some embodiments, the incretin formulation comprises a single incretin peptide. In some embodiments, the incretin formulation comprises one or more incretin peptides. In some embodiments, the two or more incretin peptides included in the incretin formulation described herein are the same or derived from the same incretin peptide (e.g., a GLP1 receptor agonist). In some embodiments, the two or more incretin peptides included in the incretin formulation described herein are different or derived from different incretin peptides.

In some embodiments, the incretin agent comprises a combination of incretin peptides, such as fused to a single polypeptide chain. For example, in some embodiments, the incretin agent comprises a GLP1 receptor agonist (e.g., a GLP1 peptide or a fragment or variant thereof) and a GIP receptor agonist (e.g., a GIP peptide or a fragment or variant thereof). In some embodiments, the incretin agent comprises one or more incretin peptides selected from SEQ ID NO: 5-15 and 62-64 and one or more incretin peptides selected from SEQ ID NO: 5-15 and 62-64. In some embodiments, the incretin agent comprises a GLP1 receptor agonist (e.g., a GLP1 peptide or a fragment or variant thereof), a GIP receptor agonist (e.g., a GIP peptide or a fragment or variant thereof), and a GCG receptor agonist (e.g., a fragment of a GCG peptide or a variant thereof). In some embodiments, the incretin agent comprises more than one GLP1 receptor agonist (e.g., a GLP1 peptide or a fragment or variant thereof), which may be the same or different. In some embodiments, the incretin agent comprises more than one copy of the same incretin peptide and/or a combination of different incretin peptides.

In some embodiments, the incretin peptide is fused to another incretin peptide via a linker. In some embodiments, the linker contains at least one Gly (G) amino acid residue. Suitable connectors can be easily selected and can have various lengths, such as 1 amino acid (e.g., Gly) to 25 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids). Exemplary linkers include glycine polymers (G) _n , glycine-serine polymers (including, for example, (GS) _n , (GGGGS: SEQ ID NO: 1) _n , and (GGGS: SEQ ID NO: 2) _n , where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as neutral links between components. Glycine is even closer to a significantly larger phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11:173-142 (1992)). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 3 (GGGGSGGGGSGGGGSGGGS or "(G4S)4" linker) or SEQ ID NO: 4 (GGGGSGGGGSGGGGSGGGSGGGS or "(G4S)5" linker). In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 68 (GGGGSGGGGS or "(G4S)2" linker), SEQ ID NO: 156 (GGGSGGGS or "(G3S)2" linker) or SEQ ID NO: 157 (GGGGSGGGGSGGGGS or "(G4S)3" linker).

In some embodiments, the incretin peptide is fused to another incretin peptide via a protease cleavage site, such as a furin cleavage site (e.g., a peptide comprising motif R-X-K/R-R SEQ ID NO: 158, such as RRKR SEQ ID NO: 160 or SEQ ID NO: 153 NVRRKR) and, where appropriate, any of the aforementioned linkers. In some embodiments, the protease cleavage site (e.g., a furin cleavage site) comprises any of SEQ ID NO: 160 RRKR, SEQ ID NO: 153 NVRRKR, SEQ ID NO: 189 RKKR, SEQ ID NO: 190 RMQR, or SEQ ID NO: 191 VFRR. In some embodiments, the furin cleavage site is operatively linked to a linker (e.g., a glycine linker, such as a (G4S)2 linker) (e.g., its 3'). In some embodiments, the incretin agent comprises multiple (e.g., 2, 3, 4 or more) incretin peptides, each separated from the furin cleavage site and, where applicable, a linker (e.g., a (G4S)2 linker located at the 5' of the furin cleavage site). As described herein, in some embodiments, the sequence and placement of the cleavage site (e.g., the furin cleavage site) within the incretin agent are important to facilitate proper cleavage of the peptide and to produce a scarless N-terminus of the incretin peptide.

In some embodiments, the polynucleotide encoding an incretin agent comprising a signal peptide and a single incretin peptide (this configuration is referred to herein as "I:1x") (see, for example, Figure 3 ). In some embodiments, the polynucleotide encoding an incretin agent comprising a signal peptide and two incretin agents separated by a linker and a cleavage site (e.g., a furin cleavage site) (this configuration is referred to herein as "I:2x") (see, for example, Figure 4 ). This design allows for the cleavage of the first incretin peptide from the second incretin peptide after the polynucleotide-translated peptide. In some embodiments, the polynucleotide encodes an incretin agent comprising a signal peptide and four separate incretin peptides (this configuration is referred to herein as "I:4x") separated by individual linkers and cleavage sites (e.g., furin cleavage sites) (see, for example, Figure 5 ). This design allows for the cleavage of four individual incretin peptides after the incretin peptides are translated from the polynucleotide. Those skilled in the art will understand that although incretin agents comprising 1, 2, and 4 incretin peptides are specifically described herein, incretin agents having different numbers of incretin peptides separated by cleavage sites and linkers can be used. In any of the designs shown in Figures 3-5 , the incretin peptide can be any incretin peptide described herein (e.g., GLP1 or GIP incretin peptides, such as any of the incretin peptides shown in Table 2).

In some embodiments, the polynucleotide described herein encodes a GLP1 intestinal peptide (e.g., any of the GLP1 intestinal peptides described in Table 2) upstream or 5' upstream. In some embodiments, the order of the incretin peptides (N-terminus to C-terminus direction) is determined by the expected cleavage pattern of the incretin peptides, so that the incretin peptides maintain their amino acid sequence and scarless N-terminus.

In some embodiments, the polynucleotides described herein encode incretins having an amino acid sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to any of the incretins detailed in Table 3. In some embodiments, the polynucleotides described herein encode incretins having an amino acid sequence according to any of the incretins detailed in Table 3. Table 3: Exemplary incretins comprising more than one incretin peptide (mutations are shown in bold, linkers are underlined, and furin cleavage sites are shown in italics), wherein examples x2 and x4 include linkers between the repeating units and furin cleavage sites. Incretins SEQ ID NO sequence GLP1 (7-37), connecton, furin protease , and GIP (1-42) with A8G and R36G mutations. 76 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP1 (7-37), connecton, furin protease , and GIP (1-42) with A8G, K34R, and R36G mutations. 77 H G EGTFTSDVSSYLEGQAAKEFIAWLV R G G G GGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP1 (7-37) with A8G and R36G mutations, connecton, furin protease, and GIP (1-42) with A2G mutation. 78 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP1 (7-37), connecton, furin protease, and GIP (1-42) with H7Y, A8G, and R36G mutations. 79 YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP1 (7-37), connecton, furin protease, and GIP (1-42) with H7Y, A8G, K34R, and R36G mutations. 80 YG EGTFTSDVSSYLEGQAAKEFIAWLV R G G G GGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP1 (7-37) with H7Y, A8G, and R36G mutations, connecton, furin protease, and GIP (1-42) with A2G mutation. 81 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ [GLP1 (7-37) with H7Y, A8G, and R36G mutations, linkers, furin protease, GIP (1-42) with A2G mutation] x2 82 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ [GLP1 (7-37) with H7Y, A8G, and R36G mutations, connecton, furin protease, GIP (1-42) with A2G mutation] x4 83 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GLP1 (7-37) with A8G and R36G mutations, connective tissue, furin, GIP (1-42), connective tissue, and Dula_IgG4 94 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37), linker, furin, GIP (1-42), linker, and Dula_IgG4 with H7Y, A8G, and R36G mutations 95 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37) with A8G and R36G mutations, connective tissue, furin protease; GIP (1-42) with A2G mutation, connective tissue; Dula_IgG4 96 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37) with H7Y, A8G, and R36G mutations, connective tissue, furin protease; GIP (1-42) with A2G mutation, connective tissue; Dula_IgG4 97 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37), linker, furin, GIP (1-42), linker, and hAlbumin with H7Y, A8G, and R36G mutations 102 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS GLP1 (7-37), linker, furin protease, GIP (1-42), linker, and aHSA-VHH with H7Y, A8G, and R36G mutations 103 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS GLP1 (7-37) with H7Y, A8G, and R36G mutations, connective tissue, furin; GIP (1-42) with A2G mutation, connective tissue, hAlbumin 104 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS GLP1 (7-37) with H7Y, A8G, and R36G mutations, linker, furin protease; GIP (1-42) with A2G mutation, linker, aHSA-VHH 105 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS [GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin, GIP (1-42) with A2G mutation] x2, linker, hAlbumin 106 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS [GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin, GIP (1-42) with A2G mutation] x4, linker, hAlbumin 107 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS GLP1 (7-37) with H7Y, A8G, and R36G mutations, connective tissue, furin protease ; GIP (1-42) with A2G mutation, connective tissue; Dula_IgG4 (LS) 170 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG GIPs (1-42) with the A2G mutation, connective tissue, furin protease ; GLP1 (7-37) with the H7Y, A8G, and R36G mutations, connective tissue; Dula_IgG4 (LS) 171 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG [GLP1 (7-37) with H7Y, A8G, and R36G mutations, connective tissue, furin , GIP (1-42) with A2G mutation] x4, connective tissue, Dula_IgG4 (LS) 172 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG

In some embodiments, this disclosure provides one or more polynucleotides encoding an incretin agent comprising a combination of incretin peptides. In such embodiments, one or more polynucleotides encode an incretin agent. In some embodiments, a first polynucleotide encodes a first incretin peptide of the incretin agent, and a second polynucleotide encodes a second incretin peptide of the incretin agent. Hyper-life extension (HLE) domain

In some embodiments, the polynucleotides described herein encode incretin formulations comprising one or more incretin peptides fused to a half-life extension (HLE) domain (see, for example, incretin formulations shown in Figures 7-14 ). In some embodiments, where the incretin formulation comprises more than one incretin peptide, the HLE domain may be included in the incretin formulation described herein to increase the half-life of one or more of the incretin peptides. Human serum albumin (HSA)

In some embodiments, the incretin agent comprises one or more incretin peptides fused to an HLE domain comprising albumin (e.g., human serum albumin (HSA)). In some embodiments, the half-life extension portion comprises albumin, e.g., human serum albumin. In some embodiments, the human serum albumin (HSA) sequence is identical to that according to SEQ ID NO: 159. (DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPE LLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAA The amino acid sequence of DFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL) is at least 90%, 95%, or 99% identical to a fragment or variant thereof. In some embodiments, the HSA sequence comprises or consists of an amino acid sequence according to SEQ ID NO: 159. In some embodiments, the HSA sequence comprises or is composed of an amino acid sequence, which is a variant of the wild-type HSA (i.e., SEQ ID NO: 159) containing one or more amino acid mutations. In some embodiments, the one or more mutations include the mutation at position 573 of SEQ ID NO: 159. In some embodiments, the K residue at position 573 of SEQ ID NO: 159 is substituted with any of the following amino acid residues: A, C, D, F, G, H, I, L, M, N, P, Q, R, S, V, W, and Y (SEQ ID NO: 187). In some embodiments, the K residue at position 573 of SEQ ID NO: 159 is substituted with a P residue (SEQ ID NO: 188). In some embodiments, an HSA variant includes any HSA variant disclosed in U.S. Patent No. 8,748,380, which is hereby incorporated by reference in its entirety.

In some embodiments, the polynucleotides described herein encode and fuse to the HLE domain (e.g., HSA or HSA variants as described herein), as shown in Figure 7 , include I:1x, I:2x, or I:4x configurations (i.e., 1, 2, or 4 intestinal insulin peptides). In some embodiments, the intestinal insulin peptides may be GLP1 or GIP intestinal insulin peptides as described herein, or variants thereof. In some embodiments, where the incretin agent contains more than one incretin peptide, the incretin peptide adjacent to the HLE domain will remain fused to the HLE domain after post-translational processing, while the incretin peptide not adjacent to the HLE domain will cleave from its adjacent incretin peptide and the HLE domain. This design can be used when it is desirable to administer multiple incretin peptides with various half-lives. Such a design could also be desirable if one of the incretin peptides intended to cross the blood-brain barrier (i.e., when the HLE domain is undesirable) and if one of the incretin peptides intended to remain in circulation for a longer period of time (i.e., when the HLE remains connected).

In some embodiments, the incretin agent comprises more than one incretin peptide separated by a linker and a protease cleavage site (e.g., a furin cleavage site), one of which is the GLP1 peptide described herein adjacent to the HLE domain (e.g., HSA or an HSA variant), and the other is the GIP peptide not adjacent to the HLE (e.g., at the N-terminus of the polypeptide chain). In such embodiments, when the incretin agent is expressed from the polynucleotide described herein, the GIP peptide will cleave from the GLP1 peptide linked to the HLE domain, such that the GLP1 incretin peptide will have a longer half-life than the GIP incretin peptide. In some embodiments, the incretin agent comprises more than one incretin peptide separated by a linker and a protease cleavage site (e.g., a furin cleavage site), one of which is the GIP incretin peptide described herein adjacent to the HLE domain (e.g., HSA or an HSA variant), and the other is the GLP1 peptide not adjacent to the HLE (e.g., at the N-terminus of the polypeptide chain). In such embodiments, when the incretin agent is expressed from the polynucleotide described herein, the GLP1 incretin peptide will cleave from the GIP peptide linked to the HLE domain, such that the GIP incretin peptide will have a longer half-life than the GLP1 incretin peptide (see, for example, Figure 9 ).

In some embodiments, the polynucleotide encoding described herein is an incretin agent as shown in Figure 8A , wherein the incretin agent has a signal peptide (“SP”), a GLP1 incretin peptide, a linker, a second GLP1 incretin peptide, a second linker (GGGS) ₃ , and a half-life extension (HLE) domain of human serum albumin (HSA) or an HSA variant. Additionally, the incretin agent may include protease cleavage sites (e.g., furin cleavage sites) between the GLP1 incretin peptides. In this embodiment, once the incretin agent is expressed, the first (N-terminal) GLP1 incretin peptide is cleaved from the second incretin GLP1 incretin peptide adjacent to the HLE domain. The resulting GLP1 incretin peptide will have two different half-lives (i.e., the GLP1 incretin peptide that remains linked to the HLE domain will have a longer half-life than the cleaved GLP1 incretin peptide).

In some embodiments, the polynucleotides described herein encode an incretin agent as shown in Figure 9 , wherein the incretin agent includes a signal peptide (SP), a first GLP1 incretin peptide, a linker (GGGS) ₂ , a first GIP incretin peptide, a second linker (GGGS) ₂ , a second GLP1 incretin peptide, a third linker (GGGS) ₂ , a second GIP incretin agent, a fourth linker (GGGS) ₃ , and a half-life extension (HLE) domain of human serum albumin (HSA) or an HSA variant. The furin and SP cleavage sites within the incretin agent are indicated by arrows. In this embodiment, when an incretin agent is expressed, the first GLP1 incretin peptide, the first GIP incretin peptide, and the second GLP1 incretin peptide are cleaved, while the second GIP incretin peptide remains fused to the HLE domain. The resulting second GIP-HLE fusion will have a longer half-life than other incretin peptides.

In some embodiments, the polynucleotide-encoded incretins described herein contain an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the incretin sequences listed in Table 4. In some embodiments, the incretins comprise any of the incretins detailed in Table 4 below, or combinations or variants thereof. Table 4: Exemplary incretins including hAlbumin (mutations are shown in bold, linkers are underlined, and furin cleavage sites are shown in italics), where examples x2 and x4 include linkers between each repeating unit and furin cleavage sites. Incretins SEQ ID NO sequence GLP1 (7-37), connector, and hAlbumin with H7Y, A8G, and R36G mutations 98 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGSGGGGS GIPs (1-42) with A2G mutation, connectors, and hAlbumin 100 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS GLP1 (7-37), linker, furin, GIP (1-42), linker, and hAlbumin with H7Y, A8G, and R36G mutations 102 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS GLP1 (7-37) with H7Y, A8G, and R36G mutations, connective tissue, furin; GIP (1-42) with A2G mutation, connective tissue, hAlbumin 104 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS [GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin, GIP (1-42) with A2G mutation] x2, linker, hAlbumin 106 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS [GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin, GIP (1-42) with A2G mutation] x4, linker, hAlbumin 107 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS albumin-binding domain

In some embodiments, the incretin peptide is fused to a half-life extension portion of albumin-binding protein. Various albumin-binding portions (i.e., albumin-binding protein domains) can be used as half-life extension portions of the incretin agents described herein (see, for example, Zorzi et al., MedChemComm , 2019, 10.7, 1068-1081, which are incorporated herein by reference in their entirety). In some embodiments, the albumin-binding protein domain comprises an albumin-binding domain (ABD) derived from protein G of Streptococcus strain GI48 and/or protein PAB derived from Corydalis flavoids , such as ABD035 and SA21 (as described in Levy et al., PLoS One , 2014, 9(2), e87704, which is incorporated herein by reference in its entirety) and ABD094 (NCT02690142) (as described in Frejd and Kim, Exp. Mol. Med ., 2017, 49(3), e306, which is incorporated herein by reference in its entirety). In some embodiments, the ABD binds to domain II of human serum albumin without overlapping with or interfering with binding to FcRn binding sites on albumin.

In some embodiments, ABD includes ABDCon (a triple-helix bundle albumin-binding domain), as described in Jacobs et al., Protein Eng., Des. Sel., 2015, 28(10), 385–393, which is incorporated herein by reference in its entirety. In some embodiments, ABD is derived from the bacterial protein Sso7d of the hyperthermophilic archaea *Sulphozoa sulfothioides* , such as M11.12 and M18.2.5 (as described in Gao et al., Nat. Struct. Biol., 1998, 5(9), 782–786 and Traxlmayr et al., J. Biol. Chem., 2016, 291(43), 22496–22508, which are incorporated herein by reference in their entirety). In some embodiments, ABD includes DARPin, as described in Pluckthun, Annu. Rev. Pharmacol. Toxicol ., 2015, 55, 489–511, which is incorporated herein by reference in its entirety.

In some embodiments, the ABD contains an immunoglobulin domain or a fragment thereof. In some embodiments, the ABD contains a fully human domain antibody (dAb). For example, in some embodiments, the ABD contains AlbudAb, as described in Holt et al., Protein Eng., Des. Sel., 2008, 21(5), 283–288, which is incorporated herein by reference in its entirety. In some embodiments, the ABD contains Fab, such as dsFv CA645, as described in Adams et al., mAbs, 2016, 8(7), 1336–1346, which is incorporated herein by reference in its entirety.

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

In some embodiments, the polynucleotide encoding described herein is shown in Figure 8B as an incretin agent, which displays an incretin agent having a signal peptide (SP), a first GLP1 incretin peptide, a linker, a second GLP1 incretin peptide, a second linker (GGGS) ₃ , and an incretin agent having a half-life extension (HLE) domain that binds to the VHH domain of HSA. The furin and SP cleavage sites within the incretin agent are indicated by arrows. In this embodiment, when the incretin agent is expressed, the first GLP1 incretin peptide is cleaved from the second GLP1 incretin peptide, and the second GLP1 incretin peptide remains fused to the HLE domain (anti-HSA VHH domain). Therefore, the second GLP1 incretin peptide will have a longer half-life than the first GLP1 incretin peptide. In some embodiments, the incretin is the incretin shown in Figure 8B , wherein one or both of the GLP1 incretin peptides may instead be different incretins (e.g., the GIP incretin peptide described herein). In some embodiments, a single incretin peptide is fused to a VHH domain that binds to the HSA.

Other albumin-binding domains are known in this art, see, for example, Zorzi et al., MedChemComm , 2019, 10.7, 1068-1081, which are incorporated herein by reference in their entirety.

In some embodiments, the polynucleotide-encoded incretins described herein contain an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the incretin sequences listed in Table 5. In some embodiments, the incretins comprise any of the incretins detailed in Table 5 below, or combinations thereof, or variants thereof. Table 5: Exemplary Incretins Including the aHSA-VHH Domain (Mutations are shown in bold, connectives are underlined) Incretins SEQ ID NO sequence GLP1 (7-37) with H7Y, A8G and R36G mutations, connectors, aHSA-VHH 99 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS GIP (1-42) with A2G mutation, connector, aHSA-VHH 101 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS EVQLLESGGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS GLP1 (7-37), linker, furin protease , GIP (1-42), linker, and aHSA-VHH with H7Y, A8G, and R36G mutations 103 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS GLP1 (7-37) with H7Y, A8G, and R36G mutations, connective tissue, furin protease ; GIP (1-42) with A2G mutation, connective tissue, aHSA-VHH 105 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS XTEN

In some embodiments, the half-life extension is the XTEN sequence as described in U.S. Patent No. 8,673,860 and Podust et al., Journal of Controlled Release , 2016, 240, 52-66, which are incorporated herein by reference in their entirety.

In some embodiments, the XTEN sequence comprises about 100 to about 3000 amino acid residues, preferably 400 to about 3000 residues, wherein at least about 80%, or at least about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99% to about 100% of the sequence consists of multiple units of two or more non-overlapping sequence motifs selected from the amino acid sequences in Table 6 or Table 7. In some cases, the XTEN comprises non-overlapping sequence motifs, wherein approximately 80%, or at least approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99% to approximately 100% of the sequences consist of two or more non-overlapping sequences selected from a single motif family from Table 6 or Table 7, thereby producing a "family" sequence in which the overall sequence remains substantially non-repeating. Therefore, in these embodiments, the XTEN sequence may comprise multiple units of non-overlapping sequence motifs from the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family of the sequences in Table 6. In other cases, the XTEN comprises motif sequences from two or more motif families from Table 6. In other cases, XTENs comprise motif sequences from one or more motif families listed in Table 7. Table 6: XTEN Sequence Motifs and Motif Families for the 12 Amino Acids Motif family SEQ ID NO: motif sequence AD 194 GESPGGSSGSES AD 195 GSEGSSGPGESS AD 196 GSSESGSSEGGP AD 197 GSGGEPSESGSS AE, AM 198 GSPAGSPTSTEE AE, AM, AQ 199 GSEPATSGSETP AE, AM, AQ 200 GTSESATPESGP AE, AM, AQ 201 GTSTEPSEGSAP AF, AM 202 GSTSESPSGTAP AF, AM 203 GTSTPESGSASP AF, AM 204 GTSPSGESSTAP AF, AM 205 GSTSSTAESPGP AG, AM 206 GTPGSGTASSSP AG, AM 207 GSSTPSGATGSP AG, AM 208 GSSPSASTGTGP AG, AM 209 GASPGTSSTGSP AQ 210 GEPAGSPTSTSE AQ 211 GTGEPSSTPASE AQ 212 GSGPSTESAPTE AQ 213 GSETPSGPSETA AQ 214 GPSETSTSEPGA AQ 215 GSPSEPTEGTSA BC 216 GSGASEPTSTEP BC 217 GSEPATSGTEPS BC 218 GTSEPSTSEPGA BC 219 GTSTEPSEPGSA BD 220 GSTAGSETSTEA BD 221 GSETATSGSETA BD 222 GTSESATSESGA BD 223 GTSTEASEGSAS Table 7: XTEN sequence motifs and motif families of the 12 amino acids Motif family SEQ ID NO: motif sequence AE, AM 198 GSPAGSPTSTEE AE, AM, AQ 199 GSEPATSGSETP AE, AM, AQ 200 GTSESATPESGP AE, AM, AQ 201 GTSTEPSEGSAP It has an Fc domain with multiple polypeptide chains and is an incretin agent.

In some embodiments, the half-life extension portion is or includes the Fc domain of, for example, human IgG (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the half-life extension portion does not include the Fc domain of, for example, human IgG (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the half-life extension portion includes the Fc domain of human IgG4 or a variant thereof (e.g., as included in dulaglutide). In some embodiments, the Fc domain of the IgG4 sequence is at least 90%, 95%, 96%, 97%, 97%, or 99% identical to SEQ ID NO: 155 (AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG). In some embodiments, the Fc domain of the IgG4 sequence is or contains the amino acid sequence according to SEQ ID NO: 155. Homodimeric incretin agent

In some embodiments of an incretin agent comprising more than one incretin peptide, the incretin agent comprises two or more incretin peptides on a single polypeptide chain. In some embodiments of an incretin agent comprising more than one incretin peptide, the incretin agent comprises one or more incretin peptides on a single polypeptide chain.

In some embodiments, the individual polypeptide chains are polymerized (e.g., dimerized). In some embodiments of incretin agents that comprise one or more incretin peptides on individual polypeptide chains, the individual polypeptide chain comprises two polypeptide chains, each containing an immunoglobulin constant domain, and the two polypeptide chains are dimerized by two constant domains, which combine to create an Fc domain.

In some embodiments, the Fc domain includes an IgG4 Fc domain (e.g., as included in dulaglutide). In some embodiments, the Fc domain includes an IgG1 Fc domain. Exemplary designs of incretin agents comprising multiple polypeptide chains are shown, for example, in Figure 10 , where the polypeptide chains include Fc domains such that the polypeptide chains dimerize. The designs in Figure 10 may include incretin peptides configured in 1:1x, 1:2x, or 1:4x (or other numbers of incretin peptides may be used), and each may be a GLP1 or GIP incretin peptide (or any of the variants described herein). In Figure 10 , two Fc domains are identical. The incretins on each polypeptide chain may be the same or different (or, in the case of multiple incretins on a single chain, may contain different combinations of incretin peptides). An exemplary incretin agent comprising two polypeptide chains forming a homodimer via dimerization of the Fc domains on each polypeptide chain is shown in Figure 11. In Figure 11 , the incretin peptide comprises two polypeptide chains, each including a signal peptide (SP), a GLP1 incretin peptide, a linker (GGGGS) ₃ , and an Fc domain. In some embodiments, each polypeptide chain may contain two or more incretin peptides (see, for example, Figure 12 ). In some embodiments, the two or more incretin peptides may be the same incretin peptide. In some embodiments, the two or more incretin peptides may be different incretin peptides (see, for example, Figure 12 ). When two or more incretin peptides are included on each polypeptide chain, cleavage sites can be introduced to cleave the incretin peptides, thereby leaving one incretin peptide linked to the Fc domain. Those skilled in the art will understand that the incretin peptide remaining linked to the Fc domain will have a longer half-life and different activity than the other cleaved incretin peptides.

The Fc domain in the incretin formulations described in this article not only allows for dimerization of the two polypeptide chains but also increases the half-life of the incretin peptide. Other mutations can also be introduced into the Fc domain to increase the half-life of the incretin formulation. Fc mutations in HLE .

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

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

In some embodiments, the Fc domain of the incretin includes one or more mutated amino acid residues that increase the half-life. In some embodiments, the Fc domain includes one of the following mutated amino acid residues: M252Y, S254T, and T256E (“YTE”) according to the EU numbering scheme, to increase the half-life. In some embodiments, the Fc domain includes a combination of the following mutated amino acid residues: M252Y, S254T, and T256E according to the EU numbering scheme, to increase the half-life.

In some embodiments, the Fc domain includes one of the following mutated amino acid residues: T250Q and M428L (“QL”) according to the EU designation scheme, to increase the half-life. In some embodiments, the Fc domain includes one of the following mutated amino acid residues: H433K and N434F (“KF”) according to the EU designation scheme, to increase the half-life. In some embodiments, the second Fc domain includes one of the following mutated amino acid residues: T307A, E380A, and N434A (“AAA”) according to the EU designation scheme, to increase the half-life. In some embodiments, the Fc domain includes one of the following mutated amino acid residues: V308P according to the EU designation scheme, to increase the half-life. In some embodiments, the Fc domain includes one of the following mutated amino acid residues according to the EU designation scheme: M252Y, V308P, and N434Y (“YPY”) to increase the half-life. In some embodiments, the Fc domain includes one of the following mutated amino acid residues according to the EU designation scheme: H285D, T307Q, and A378V (“DQV”) to increase the half-life. In some embodiments, the Fc domain includes one of the following mutated amino acid residues according to the EU designation scheme: L309D, Q311H, and N434S (“DHS”) to increase the half-life. Exemplary Fc mutations are described, for example, in Liu et al., Antibodies 9.4: 64 (2020), which are hereby incorporated by reference in their entirety.

In some embodiments, the polynucleotide-encoded incretins described herein contain an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the incretin sequences listed in Table 8. In some embodiments, the incretins comprise any of the incretins detailed in Table 8 below, or combinations or variants thereof. Table 8: Exemplary incretins including the IgG4 Fc domain (mutations are shown in bold, linkers are underlined, and furin cleavage sites are shown in italics), where x4 examples include linkers between repeating units and furin cleavage sites. Incretins SEQ ID NO sequence Dulaglutide (GLP1 (7-37) with A8G and R36G mutations, linker, Dula_IgG4) 10 H G EGTFTSDVSSYLEEQAAKEFIAWLVKG G GGGGGSGGGGSGGGGSA ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37), linker, and Dula_IgG4 with A8G and R36G mutations 89 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37), linker, and Dula_IgG4 with A8G, K34R, and R36G mutations 90 H G EGTFTSDVSSYLEGQAAKEFIAWLV R G G G GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37), linker, and Dula_IgG4 with H7Y, A8G, and R36G mutations 91 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GIP (1-42), ligands, Dula_IgG4 92 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GIP (1-42) with A2G mutation, linker, Dula_IgG4 93 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQG GGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37) with A8G and R36G mutations, connective tissue, furin, GIP (1-42), connective tissue, and Dula_IgG4 94 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37), linker, furin, GIP (1-42), linker, and Dula_IgG4 with H7Y, A8G, and R36G mutations 95 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGGSGGGGS NVRRKR YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37) with A8G and R36G mutations, connective tissue, furin protease; GIP (1-42) with A2G mutation, connective tissue; Dula_IgG4 96 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37) with H7Y, A8G, and R36G mutations, connective tissue, furin protease; GIP (1-42) with A2G mutation, connective tissue; Dula_IgG4 97 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG GLP1 (7-37), linker, and Dula_IgG4 (LS) with H7Y, A8G, and R36G mutations 168 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG GIPs (1-42) with A2G mutation, linkers, and Dula_IgG4 (LS) 169 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG GLP1 (7-37) with H7Y, A8G, and R36G mutations, connective tissue, furin protease ; GIP (1-42) with A2G mutation, connective tissue; Dula_IgG4 (LS) 170 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG GIPs (1-42) with the A2G mutation, connective tissue, furin protease ; GLP1 (7-37) with the H7Y, A8G, and R36G mutations, connective tissue; Dula_IgG4 (LS) 171 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG [GLP1 (7-37) with H7Y, A8G, and R36G mutations, connecton, furin protease , GIP (1-42) with A2G mutation] x4 Dula_IgG4 (LS) 172 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGS NVRRKR YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGS NVRRKR Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS AESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG Eliminating mutations in Fc effect subfunctions

In some embodiments, the Fc domain includes one or more mutations that eliminate the function of the Fc domain included in the incretin agent. By eliminating the Fc effector function of the incretin agent, the delivery of the incretin agent is less likely to induce an undesirable immune response due to cytotoxicity and other effector activities triggered by immune cells. In some embodiments, the Fc domain of the molecule includes one or more mutations that silence the effector function.

In some embodiments, one or more mutations in the Fc domain include "STR" modification, or, according to the EU numbering scheme, combinations of mutations including L234S, L235T, and G236R. When such mutations are introduced into the Fc domain, the Fc domain exhibits minimal or undetectable binding to Fcγ receptors or C1q and does not promote inflammatory intercytokine responses (see, for example, Wilkinson et al., (2021) PLoS One 16.12: e0260954, which is incorporated herein by reference in its entirety). Exemplary incretins containing STR modification are illustrated, for example, in Figure 14 .

In some embodiments, modifications that reduce or silence the effector function of the Fc domain included in the incretins described herein comprise one or more of the following mutations: L234A, L235A, P329G, P329A, N297A, or N297D, according to the EU designation scheme. In some embodiments, modifications that silence the effector function of the Fc domain comprise the following mutated amino acid residues: L234A and L235A, according to the EU designation scheme (“LALA”). In some embodiments, mutations used to eliminate the effector function of the Fc domain include: L234A/L235A/P329G, according to the EU designation (“LALAPG”). In some embodiments, the modification of the effector function of the silencing Fc domain includes the following mutated amino acid residues: L234A, L235A, and P329A (“LALAPA”) according to the EU designation scheme. In some embodiments, the modification of the effector function of the silencing Fc domain further includes N297A or N297D according to EU designation. In some embodiments, the modification of the effector function of the silencing Fc domain includes the following Fc mutations: L234A/L235A/P329G and N297A according to EU designation. In some embodiments, the modification of the effector function of the silencing Fc domain includes the following Fc mutations: L234A/L235A/P329G and N297D according to EU designation. In some embodiments, the modification of the effect subfunction of the silencing Fc domain includes the following Fc mutations: according to EU designations L234A, L235A, and N297A. In some embodiments, the modification of the effect subfunction of the silencing Fc domain includes the following Fc mutations: according to EU designations L234A, L235A, and N297D. In some embodiments, the modification of the effect subfunction of the silencing Fc domain includes the following Fc mutations: according to EU designations L234A, L235A, P329A, and N297A. In some embodiments, the modification of the effect subfunction of the silencing Fc domain includes the following Fc mutations: according to EU designations L234A, L235A, P329A, and N297D.

In some embodiments, modifications that reduce or silence the effector function of the Fc domain included in the incretins described herein comprise the following mutations: L234F/L235E/P331S (“FES”) according to the EU designation scheme. In some embodiments, modifications that reduce or silence the effector function of the Fc domain included in the incretins described herein comprise the following mutations: L234F/L235Q/K322Q (“FQQ”) according to the EU designation scheme. In some embodiments, modifications that reduce or silence the effector function of the Fc domain included in the incretins described herein comprise the following mutations: A330S/P331S according to the EU designation scheme.

Those familiar with this technique will understand that other modifications known in this technique can be used to eliminate the function of the effector. Heterodimeric incretins

In some embodiments, the one or more polynucleotides described herein encode an incretin agent containing more than one incretin peptide on a single polypeptide chain. In some embodiments, the single polypeptide chain is polymerized (e.g., dimerized). In some embodiments where the incretin agent contains one or more incretin peptides on a single polypeptide chain, the single polypeptide chain comprises two polypeptide chains, each containing an immunoglobulin constant domain, and the two polypeptide chains are dimerized by two constant domains, which combine to create an Fc domain.

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

One method for induced dimerization is called the "mortar and pestle structure technique" (KIH), which aims to force two distinct constant domains to pair by modifying the contact interface through the introduction of a mutation into the CH3 domain. In one CH3 domain, a bulk amino acid is replaced by an amino acid with a short side chain to produce a "mortar," and an amino acid with a large side chain is introduced into the other CH3 domain to produce a "pestle." The co-existence of these two constant domains induces dimerization. In some embodiments, the Fc domain described herein utilizes the KIH technique as described, for example, in WO1998/050431, which is incorporated herein by reference in its entirety. As described herein, the Fc domain of an incretin agent may contain certain mutations utilizing KIH technology, including but not limited to CH3 modification. In some embodiments, the Fc domain of the incretin agent includes a CH3 domain containing one or more of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU designations). In some embodiments, the Fc domain of the incretin agent includes a CH3 domain containing each of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU designations). This combination of mutations is referred to herein as "FcKIH-b". In some embodiments, the Fc domain of the incretin agent includes a CH3 domain comprising one or more mutations selected from S354C and T366W (according to EU designations). In some embodiments, the Fc domain of the incretin agent includes a CH3 domain comprising each of the following mutations: S354C and T366W (according to EU designations). This combination of CH3 mutations is referred to herein as "FcKIH-a". In some embodiments, the incretin agent includes an Fc domain comprising both the FcKIH-a and FcKIH-b sequences.

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

Therefore, in some embodiments, the incretin agent encoded by one or more polynucleotides as described herein comprises one or more incretin peptides fused to the Fc domain, wherein the CH3 domain of the Fc domain comprises one or more of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU designations). In some embodiments, the incretin agent encoded by one or more polynucleotides as described herein comprises one or more incretin peptides fused to the Fc domain, wherein the Fc domain comprises a CH3 domain comprising one or more mutations selected from the following: S354C and T366W (according to EU designations).

In some embodiments, the incretin agent comprises a heterodimer, as shown in Figures 13 or 14. Figure 13 illustrates an exemplary design of two polypeptide chains comprising an incretin peptide fused to an Fc domain. In each polypeptide chain (incretin-Fc fusion), a signal peptide (SP) and one, two, or four incretin peptides (I:1x, I:2x, or I:4x) fused to the Fc domain are present, and each incretin agent includes an Fc mutation that induces heterodimerization (e.g., a club-and-socket structure mutation). The Fc domain may also include modifications that eliminate effector function and/or increase half-life, as described herein. When one or more polynucleotides of the two polypeptide chains (top) in Figure 13 are expressed, the two polypeptide chains condense and form a heterodimeric incretin (bottom). An exemplary incretin designed according to Figure 13 is shown in Figure 14. Specifically, each polypeptide chain of the incretin in Figure 14 has a signal peptide (SP), a GLP1 or GIP incretin peptide, a linker (GGGS) ₃ , and an Fc domain. One or both of the Fc domains contain an "LS" mutation (M428L/N434S) that prolongs the half-life of the incretin, a "STR" mutation that silences the function of the Fc effector, and a "mortar" mutation that promotes heterodimerization. In some embodiments, instead of LS and/or STR mutations, or in addition to LS and/or STR mutations, any mutation described herein may be included. When two peptide chains are expressed, they assemble to form a heterodimeric structure containing two peptide chains with different incretin peptides. SP cleavage sites within the incretin are indicated by arrows.

In some embodiments, the incretin agent comprises two polypeptide chains that are conjugated and form a heterodimeric incretin agent, one polypeptide chain comprising the sequence SEQ ID NO: 300 (DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK) ("FcKIH-a (LS and STR)") and the other polypeptide chain comprising SEQ ID NO: 301(DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK) ("FcKIH-b (LS and STR)").

In some embodiments, the polynucleotide-encoded incretins described herein contain an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the incretin sequences listed in Table 9. In some embodiments, the incretins comprise any of the incretins detailed in Table 9 below, or combinations thereof, or variants thereof. Table 9: Exemplary incretins forming heterodimers including FcKIH-a or FcKIH-b domains (mutations are shown in bold, linkers are underlined). Incretins SEQ ID NO sequence GLP1 (7-37) with A8G and R36G mutations, connectors, and FcKIH-a (LS and STR) 84 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGSGGGGSGGGGS DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK GLP1 (7-37) with A8G, K34R, and R36G mutations, connectors, and FcKIH-a (LS and STR) 85 H G EGTFTSDVSSYLEGQAAKEFIAWLV R G GGGGSGGGGSGGGGS DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK GLP1 (7-37), connector, and FcKIH-a (LS and STR) with H7Y, A8G, and R36G mutations. 86 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G GGGGSGGGGSGGGGS DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK GLP1 (7-37), connector, FcKIH-a LS and STR with H7Y, A8G, K34R and R36G mutations 87 YG EGTFTSDVSSYLEGQAAKEFIAWLV R G G GGGGSGGGGSGGGGS DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK GIPs (1-42) with A2G mutations, connectors, and FcKIH-b (LS and STR) 88 Y G EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GGGGSGGGGSGGGGS DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK GLP1 (7-37), connector, and FcKIH-a (LS and STR) with H7Y, A8G, and R36G mutations. 173 YG EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGSGGGGS DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK GLP1 (7-37) with A8G and R36G mutations, connectors, and FcKIH-a (LS and STR) 174 H G EGTFTSDVSSYLEGQAAKEFIAWLVKG G G GGGGSGGGGSGGGGS DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

In some embodiments, any of the exemplary incretin agents including the FcKIH-a domain may be combined with an exemplary incretin agent including the FcKIH-b domain. For example, in some embodiments, the incretin agent of SEQ ID NO: 84, 85, 86, 87, 173, or 174 may be combined with the incretin agent of SEQ ID NO: 88. Signal peptide

According to some embodiments, the signal peptide is fused directly or via a linker to the encoded incretin peptide described herein. In some embodiments, the open reading frame of the polynucleotide described herein encodes an incretin having, for example, a signal peptide that functions in mammalian cells.

In some embodiments, the signal peptide is typically characterized by a sequence of about 15 to 30 amino acids in length. In many embodiments, the signal peptide is located at the N-terminus of the incretin, but is not limited thereto. In some embodiments, the signal peptide preferably allows the transport of the incretin encoded by the polynucleotide disclosed herein bound to it to a specific cellular compartment, preferably on the cell surface, in the endoplasmic reticulum (ER), or in the endosome-lysosome compartment.

In some embodiments, the ribonucleic acid sequence encoding the signal peptide allows the incretin encoded by the polynucleotide to be secreted after translation by, for example, cells present in an individual, thus producing a plasma concentration of the bioactive incretin.

In some embodiments, the ribonucleic acid sequence encoding a signal peptide included in the polynucleotide consists of or contains a nucleotide sequence encoding a human signal peptide. In some embodiments, the ribonucleic acid sequence encoding a secretion signal included in the polynucleotide consists of or contains a nucleotide sequence encoding a non-human secretion signal. In some embodiments, the signal peptide may be or contains a viral signal peptide. In some embodiments, such a signal peptide may be or contains the amino acid sequence of MRVLVLLACLAAASNA (SP1-2; SEQ ID NO: 17). In some embodiments, the signal peptide may be or contains the amino acid sequence of MRVMAPRTLILLLSGALALTETWA (husec signal peptide δ GS; SEQ ID NO: 65).

In some embodiments, the signal peptide sequence is selected from those sequences included in Table 10 below, or fragments or variants thereof: Table 10: Exemplary Signal Peptides signal peptide SEQ ID NO: Sequence (amino acids) SP1 16 MRVLVLLACLAAASA SP1-2 17 MRVLVLLACLAAASNA SP1-3 18 MRRVLVLLACLAAASA SP1-4 19 MRRVLVLLACLAAASNA SP1-5 20 MRRRVLVLLACLAAASA SP1-6 twenty one MRRRVLVLLACLAAASNA HSV-1 gD SP twenty two MGGAAARLGAVILFVVIVGLHGVRSKY HSV-2 gD SP twenty three MGRLTSGVGTAALLVVAVGLRVVCA HSV-2 twenty four MGRLTSGVGTAALLVVAVGLRVVCAKYA SARS-CoV-2-S 25 MFVFLVLLPLVSSQCVNLT Human Ig heavy chain signaling peptide 26 MDWIWRILFLVGAATGAHSQM Human Ig heavy chain signaling peptide 27 MDWTWRVFCLLAVAPGAHS HuIgGk signaling peptide 28 METPAQLLFLLLLWLPDTTG IgE heavy chain ε-1 signaling peptide 29 MDWTWILFLVAAATRVHS Japanese encephalitis PRM signaling peptide 30 MLGSNSGQRVVFTILLLLVAPAYS VSVg protein signaling peptide 31 MKCLLYLAFLFIGVNCA Exemplary signal peptides 32 MDWTWILFLVAAATRVHS Exemplary signal peptides 33 ETPAQLLFLLLLWLPDTTG Exemplary signal peptides 34 MLGSNSGQRVVFTILLLLVAPAYS Exemplary signal peptides 35 MKCLLYLAFLFIGVNCA Exemplary signal peptides 36 MWLVSLAIVTACAGA Exemplary signal peptides 37 MFVFLVLLPLVSSQC husec signaling peptide 38 MRVMAPRTLILLLSGALALTETWAGS husec3 signaling peptide 39 MEFGLSWLFLVAILKGVQC Husec signaling peptide (δGS) 65 MRVMAPRTLILLLSGALALTETWA gD1 signaling peptide 66 MGGAAARLGAVILFVVIVGLHGVRG Gaussia luciferase signaling peptide variant 67 MMGVKVLFALICIAVAEA

This disclosure particularly recognizes that the selection of the signal peptide can be important for predicting the cleavage site between the signal peptide and the incretin peptide. In order to enable the delivery and expression of the incretin peptide by polynucleotides, wherein the expressed incretin peptide maintains appropriate function and biological activity, in some embodiments, the signal peptide is selected and included in the incretin preparation to achieve proper cleavage of the incretin into its mature form.

Without being bound by any theory, in the context of the polynucleotides encoding incretins described herein, the cleavage site and type or sequence of the signal peptide can be important for ensuring the correct processing of the N-terminus of the incretin peptide. The signal peptide may contain specific sequences or structures that lead to alternative processing or cleavage sites, ultimately altering the final amino acid sequence of the mature incretin peptide. In relatively small peptides such as GLP1 or GIP (or their variants, and other peptides of similar size/properties), variations in amino acid residues can affect the peptide's biological activity. In some embodiments, signal peptides are selected for inclusion in the incretins described herein to promote proper cleavage of the N-terminus of the incretin peptide, or in other words, to produce a "scarless" N-terminus of the incretin peptide in order to maintain its biological activity. Figures 20 and 21 illustrate the locations of the theoretical cleavage sites of certain exemplary signal peptides and incretins. Figure 20 shows that the A8G mutation promotes the correct N-terminal processing of a GLP1 incretin with a husec signal peptide. Figure 21 shows that the A2G mutation promotes the correct N-terminal processing of a GIP incretin with a husec signal peptide.

The concept and utilization of cleavage of a specific signal peptide to produce a mature peptide with a scarless N-terminus can also be applied to other enteropeptides (e.g., glucagon) and/or other peptides of similar size/properties to GLP1 and GIP described herein. This can be important in the context of delivering incretins (or other similar peptides) as encoding one or more polynucleotides of incretins. Such delivery requires proper translation of intracellular proteins, in addition to post-translational processing (including proper cleavage of the translated peptide). Incretins can be designed and produced comprising one or more incretin peptides fused to another peptide described herein, such that the cleavage of the signal peptide is accurate and does not affect the amino acid sequence of the mature peptide (i.e., producing a "scarless" N-terminus). The scarless N-terminal allowable peptides of intestinal insulin peptides (and other similar peptides, such as other intestinal peptides, such as glucagon) have appropriate functions after being processed into mature peptides.

In some embodiments, the polynucleotide includes a signal peptide coding sequence and one or more incretin peptide coding sequences in the 5' to 3' direction. In some embodiments, the signal peptide coding sequence and one or more incretin peptide coding sequences encode any of the sequences shown in Table 11 below, or sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the sequences shown in Table 11 below. Table 11: Exemplary incretin preparations, wherein examples x2 and x4 include linkers between the repeating units and furin cleavage sites. Incretins Sequence (SP is shown in bold) SEQ ID NO SP1-2, GLP1 (7-37) MRVLVLLACLAAASNA HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 41 SP1-2, GLP1 with K34R mutation (7-37) MRVLVLLACLAAASNA HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG 42 SP1-2, GLP1 (7-36) MRVLVLLACLAAASNA HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR 43 SP1-2, GIP (1-42) MRVLVLLACLAAASNA YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 44 SP1-2, GIP (1-30) MRVLVLLACLAAASNA YAEGTFISDYSIAMDKIHQQDFVNWLLAQK 45 Husec SP, GLP1 (7-37) MRVMAPRTLILLLSGALALTETWA HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 52 Husec SP, GIP (1-42) MRVMAPRTLILLLSGALALTETWA YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 53 husec SP, GIP with A2G mutation (1-42) MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 54 husec SP, GLP1 with A8G mutation (7-37) MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 55 Husec SP, GLP1 with H7Y and A8G (7-37) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 56 gD1 SP, GLP1 (7-37) MGGAAARLGAVILFVVIVGLHGVRG HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 57 gD1 SP, GIP (1-42) MGGAAARLGAVILFVVIVGLHGVRG YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 58 gD1 SP, GIP with A2G mutation (1-42) MGGAAARLGAVILFVVIVGLHGVRG YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 59 gD1 SP, GLP1 with A8G mutation (7-37) MGGAAARLGAVILFVVIVGLHGVRG HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 60 gD1 SP, GLP1 with H7Y and A8G mutations (7-37) MGGAAARLGAVILFVVIVGLHGVRG YGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 61 husec SP, GLP1 (7-37) with A8G mutation, connector MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGGSGGGS 108 husec SP, GIP with A2G mutation (1-30) MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQK 109 SP, GLP1 with A8G mutation (7-37) MMGVKVLFALICIAVAEA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 110 SP, GLP1 (7-37) with A8G mutation, connector MMGVKVLFALICIAVAEA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGSGGGS 111 SP, GIP with A2G mutation (1-42) MMGVKVLFALICIAVAEA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 112 SP, GIP with A2G mutation (1-30) MMGVKVLFALICIAVAEA YGEGTFISDYSIAMDKIHQQDFVNWLLAQK 113 SP, GLP1 (7-37) MMGVKVLFALICIAVAEA HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 114 SP, GLP1 (7-37), connector MMGVKVLFALICIAVAEA HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGSGGGS 115 SP, GIP (1-42) MMGVKVLFALICIAVAEA YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 116 SP, GIP (1-30) MMGVKVLFALICIAVAEA YAEGTFISDYSIAMDKIHQQDFVNWLLAQK 117 gD1 SP, GLP1 with A8G and K34R mutations (7-37) MGGAAARLGAVILFVVIVGLHGVRG HGEGTFTSDVSSYLEGQAAKEFIAWLVRGRG 118 gD1 SP, GLP1 with H7Y, A8G and K34R mutations (7-37) MGGAAARLGAVILFVVIVGLHGVRG YGEGTFTSDVSSYLEGQAAKEFIAWLVRGRG 119 gD1 SP, GIP with A2G mutation (1-30) MGGAAARLGAVILFVVIVGLHGVRG YGEGTFISDYSIAMDKIHQQDFVNWLLAQK 120 Husec SP, GLP1 with A8G and R36G mutations (7-37), connecton, furin protease, GIP (1-42) MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 121 Husec SP, GLP1 with A8G, K34R, and R36G mutations (7-37), connecton, furin protease, GIP (1-42) MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVRGGGGGGGSGGGGSNVRRKRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 122 Husec SP, GLP1 with A8G and R36G mutations (7-37), connecton, furin protease, and GIP with A2G mutation (1-42). MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 123 Husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, connective tissue, furin protease, GIP (1-42) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 124 Husec SP, GLP1 (7-37) with H7Y, A8G, K34R, and R36G mutations, connecton, furin protease, GIP (1-42) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVRGGGGGGGSGGGGSNVRRKRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 125 Husec SP, GLP1 with H7Y, A8G and R36G mutations (7-37), connecton, furin protease, GIP with A2G mutation (1-42) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 126 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin protease, GIP (1-42) with A2G mutation] x2 MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSNVRRKRYGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 127 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G, linker, furin protease, GIP (1-42) with A2G mutation] x4 MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSNV RRKRYGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGG GSNVRRKRYGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGG SGGGGSNVRRKRYGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 128 husec SP, GLP1 (7-37) with A8G and R36G mutations, connector, FcKIH-a (LS and STR) MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGSGGGGSGGGGSDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK 129 husec SP, GLP1 (7-37) with A8G, K34R and R36G mutations, connector, FcKIH-a (LS and STR) MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVRGGGGGGSGGGGSGGGGSDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK 130 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, connector, FcKIH-a (LS and STR) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGSGGGGSGGGGSDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK 131 husec SP, GLP1 (7-37) with H7Y, A8G, K34R and R36G mutations, connector, FcKIH-a (LS and STR) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVRGGGGGGSGGGGSGGGGSDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK 132 husec SP, GIP with A2G mutation (1-42), connector, FcKIH-b (LS and STR) MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK 133 husec SP, GLP1 (7-37) with A8G and R36G mutations, linker, Dula_IgG4 MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 134 husec SP, GLP1 (7-37) with A8G, K34R and R36G mutations, and the linker Dula_IgG4 MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVRGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 135 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, Dula_IgG4 MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 136 husec SP, GIP (1-42), ligands, Dula_IgG4 MRVMAPRTLILLLSGALALTETWA YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 137 husec SP, GIPs with A2G mutation (1-42), linkers, Dula_IgG4 MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 138 Husec SP, GLP1 (7-37) with A8G and R36G mutations, connective tissue, furin, GIP (1-42), connective tissue, Dula IgG4 MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 139 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, connective tissue, furin, GIP (1-42), connective tissue, Dula_IgG4 MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 140 Husec SP, GLP1 (7-37) with A8G and R36G mutations, connective tissue, furin, GIP (1-42) with A2G mutation, connective tissue, Dula_IgG4 MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 141 Husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, connective tissue, furin, GIP (1-42) with A2G mutation, connective tissue, Dula_IgG4 MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 142 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, connector, hAlbumin MRVMAPRTLILLLSGALALTETWA 143 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, connector, aHSA-VHH MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSEVQLLESGGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS 144 husec SP, GIP with A2G mutation (1-42), connector, hAlbumin MRVMAPRTLILLLSGALALTETWA 145 husec SP, GIP with A2G (1-42), connector, aHSA-VHH MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS 146 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin, GIP (1-42), linker, hAlbumin MRVMAPRTLILLLSGALALTETWA 147 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin protease, GIP (1-42), linker, aHSA-VHH MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSEVQLLESGG GLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS 148 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin, GIP (1-42) with A2G mutation, linker, hAlbumin MRVMAPRTLILLLSGALALTETWA 149 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, furin protease, GIP (1-42) with A2G mutation, linker, aHSA-VHH MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSEVQLLESGG GLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIASSGGSTNYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAAVLECRTVVRGYDYWGQGTQVTVSS 150 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations], linker, furin, GIP (1-42) with A2G mutation, x2, linker, hAlbumin MRVMAPRTLILLLSGALALTETWA 151 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations], linker, furin, GIP (1-42) with A2G mutation, x4, linker, hAlbumin MRVMAPRTLILLLSGALALTETWA 152 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, connector, FcKIH-a (LS and STR) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK 175 husec SP, GLP1 (7-37) with A8G and R36G mutations, connector, FcKIH-a (LS and STR) MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSDKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK 176 gD1 SP, GLP1 with K34R mutation (7-37) MGGAAARLGAVILFVVIVGLHGVRG HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG 161 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations, linker, Dula_IgG4 (LS) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG 162 husec SP, GIPs with A2G mutations (1-42), linkers, Dula_IgG4 (LS) MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG 163 Husec SP, GLP1 (7-37) with H7Y, A8G, and R36G mutations, furin, GIP (1-42) with A2G mutation, connecton, and Dula_IgG4 (LS) MRVMAPRTLILLLSGALALTETWA YGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSNVRRKRYGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG 164 Husec SP, GIPs with the A2G mutation (1-42), furin, linker, GLP1 with the H7Y, A8G, and R36G mutations (7-37), linker, Dula_IgG4 (LS) MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQGGGGSGGGGSNVRRKRYGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKSLSLSLG 165 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations], linker, furin, GIP (1-42) with A2G mutation] x4, linker, Dula_IgG4 (LS) MRVMAPRTLILLLSGALALTETWA 166

In some embodiments, the polynucleotide includes, in the 5' to 3' direction, a signal peptide coding sequence; an incretin peptide coding sequence; a linker coding sequence; and a half-life extension coding sequence.

In some embodiments, the polynucleotide includes a signal peptide coding sequence and one or more incretin peptide coding sequences in the 5' to 3' direction, each separated from a linker coding sequence and a protease cleavage site coding sequence, such as a furin protease cleavage site coding sequence. In some such embodiments, a linker coding sequence and a half-life extension coding sequence precede or follow one or more of the incretin peptide coding sequences. Exemplary Polynucleotide Encoding Incretin Agents

In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the sequences shown in Table 12 below. Table 12: Exemplary polynucleotides encoding incretins, wherein examples x2 and x4 include linkers between the repeating units and furin cleavage sites. Description of polynucleotide coding sequences SEQ ID NO sequence Virus SP, GLP1 (7-37) 177 AUGAGAGUGCUGGUGCUGCUGGCUUGUCUGGCCGCUGCCUCUAAUGCUCAUGCCGAGGGCACCUUUACCAGCGACGUGUCCUCUUACCUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCUAAUAG Virus SP, GLP1 with K34R mutation (7-37) 178 AUGAGAGUGCUGGUGCUGCUGGCUUGUCUGGCCGCUGCCUCUAAUGCUCAUGCCGAGGGCACCUUUACCAGCGACGUGUCCUCUUACCUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUCGUCAGAGGCAGAGGAUAAUAG husec SP, GLP1 (7-37)-A8G mutation (codec-optimized variant 1 or "opt1") 179 AUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGGCCCUGGCUCUGACAGAAACAUGGGCUCAUGGCGAGGGCACCUUCACCUCCGAUGUGUCCUCUUACCUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCUAAUAG husec SP, GLP1 (7-37) with A8G mutation, connector (code-optimized variant 1 or "opt1") 180 AUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGGCCCCUGGCUCUGACAGAAACAUGGGCUCAUGGCGAGGGCACCUUCACCUCCGAUGUGUCCUCUUACCUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCGGCGGAGGAAGUGGCCGGAGGAUCUUAAUAG husec SP, GLP1 (7-37)-A8G (code-optimized variant 2 or "optp") 181 AUGAGAGUGAUGGCCCCUCGGACACUGAUUCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCCCACGGCGAGGGCACCUUUACCAGCGAUGUGUCUUCUUAUCUGGAAGGCCAGCCCGCCAAAGAGUUCAUCGCUUGGCUUGUGAAAGGCAGAGGCUAAUAG husec SP, GLP1 (7-37) with A8G mutation, connector (code-optimized variant 2 or "optp") 182 AUGAGAGUGAUGGCCCCUCGGACACUGAUUCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCCCACGGCGAGGGCACCUUUACCAGCGAUGUGUCUUCUUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCUUGGCUUGUGAAAGGCAGAGGUGGUGGCGGAUCUGGCCGGAGGAUCUUAAUAG Virus SP, GIP (1-42) 183 AUGAGAGUGCUGGUGCUGCUGGCUUGUCUGGCCGCUGCCUCUAAUGCCUAUGCCGAGGGCACCUUCAUCAGCGACUACUCUAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAACAUCACCCAGUAAUAG husec SP, GIP with A2G mutation (1-42) (code-optimized variant 1 or "opt1") 184 AUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGGCCCCUGGCUCUGACAGAGACAUGGGCUUAUGGCGAGGGCACCUUCAUCAGCGACUACUCUAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAACAUCACCCAGUAAUAG husec SP, GIP with A2G mutation (1-42) (codec-optimized variant 2 "optp") 185 AUGAGAGUGAUGGCCCCUCGGACACUGAUUCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCCUAUGGCGAGGGCACCUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAACAUCACCCAGUAAUAG husec SP, GIP (1-30) (Variant 1 or "opt1" optimized by the code) 224 AUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGGCCCUGGCUCUGACAGAGACAUGGGCUUAUGGCGAGGGCACCUUCAUCAGCGACUACUCUAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAAUAAUAG husec SP, GIP (1-30) (Variant 1 or "opt1" optimized by the code) 225 AUGAGAGUGAUGGCCCCUCGGACACUGAUUCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCCUAUGGCGAGGGCACCUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAAUAAUAG husec SP, GLP1 (7-37) (a codec-optimized variant 1 or "opt1") 226 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUCACGCCGAGGGCACCUUUACAAGCGACGUGUCCAGCUACCUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGC husec SP, GIP (1-42) (a variant 1 or "opt1" optimized with cryptography) 227 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCCUACGCCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAG husec SP, GLP1 (7-37) with H7Y and A8G mutations (codec-optimized variant 1 or "opt1") 228 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUGAAAGGCAGAGGC gD1 SP, GLP1 (7-37) (Variant 1 or "opt1" optimized by the codeword) 229 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUGGACUGCACGGCGUUAGAGGACACGCCGAGGGAACCUUUACAAGCGACGUGUCCAGCUACCUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGC gD1 SP, GLP1 (7-37) with K34R mutation (codec-optimized variant 1 or "opt1") 230 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUGUGGUCAUCGUUGGACUGCACGGCGUUAGAGGACACGCCGAGGGAACCUUUACAAGCGACGUGUCCAGCUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUCGUCAGAGGCAGAGGA gD1 SP, GLP1 (7-37) with A8G mutation (codec-optimized variant 1 or "opt1") 231 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUGGACUGCACGGCGUUAGAGGACACGGCGAGGGAACCUUUACAAGCGACGUGUCCAGCUACCUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGC gD1 SP, GLP1 (7-37) with H7Y and A8G mutations (codecoin-optimized variant 1 or "opt1") 232 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUGGACUGCACGGCGUCAGAGGAUACGGCGAGGGAACCUUUACAAGCGACGUGUCCAGCUACCUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGC gD1 SP, GIP (1-42) (Variant 1 or "opt1" optimized by the code) 233 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUGGACUGCACGGCGUCAGAGGAUACGCCGAGGGCACCUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAG gD1 SP, GIP (1-42) with A2G mutation (code-optimized variant 1 or "opt1") 234 AUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUUCGUGGUCAUCGUUGGACUGCACGGCGUCAGAGGAUACGGCGAGGGCACCUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAG husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations - linker-furin protease-GIP (1-42) (codon-optimized variant 1 or "opt1") 235 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUG GCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGCCGAGGGAACCUUUAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAG husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation (codon-optimized variant 1 or "opt1") 236 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUG GCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAG husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x2 237 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCG GUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCAC AAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAGGUUCAAACGUUCGCCGCAAAAGAUACGGCGAGGGAACUUUCACCUCUGACGUGUCCUCUUACCUCGAAGGACAGGCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAGGCGGUGGUG GAAGCGGAGGUGGUGGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCUCCGACUACUCCAUUGCUAUGGAUAAGAUUCAUCACAGGAUUUCGUCAACUGGCUCCUCGCUCAGAAAGGGAAGAAAAACGAUUGGAAACAUAACAUCACCCAG husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 (codon-optimized variant 1 or "opt1") 238 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCG GCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGG AAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAGGUUCAAACGUUCGCCGCAAAAGAUACGGCGAGGGAACUUUCACCUCUGACGUGUCCUCUUACCUCGAAGGACAGGCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAG GCGGUGGUGGAAGCGGAGGUGGGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCUCCGACUACUCCAUUGCUAUGGAUAAGAUUCAUCACAGGAUUUCGUCAACUGGCUCCUCGCUCAGAAAGGGAAGAAAAACGAUUGGAAACAU AACAUCACGCAAGGCGGUGGCGGUUCAGGUGGUGGUGGUUCUAACGUCCGGCGGAAACGUUACGGCGAGGGAACCUUUACAUCAGACGUUUCAUCCUACCUUGAGGGGCAAGCUGCAAAAGAGUUUAUUGCCUGGCUCGUGAAAGGUGGUGGCGGUGGCG GAGGUAGCGGAGGCGGCGGAAGCAACGUUCGAAGAAAACGAUACGGCGAGGGGACCUUCAUCUCCGAUUAUAGUAUCGCUAUGGACAAAAUUCAUCAGCAAGACUUUGUUAACUGGUUGCUCGCCCAAAAGGGGAAAAAGAACGAUUGGAAGCACAACAUU ACACAAGGCGGAGGUAGGUGGCGGAGGUGGAAGUAACGUACGACGGAAAAGAUACGGCGAAGGCACCUUCACCUCCGACGUUUCAAGUUACUUGGAGGGACAAGCAGCCAAAGAAUUCAUAGCCUGGCUCGUGAAAGGCGGCGGAGGUGGUGGCGGUA GUGGUGGUGGCGGCUCAAACGUUCGGAGGAAAAGAUACGGCGAGGGCACUUUUAUUAGCGAUUACUCUAUCGCAAUGGAUAAGAUACACCAACAAGACUUUGUCAAUUGGCUCUUGGCACAAAAAGGCAAGAAGAACGAUUGGAAACACAAUAUCACACAG husec SP, GLP1 (7-37)-linker-Dula_IgG4 with H7Y, A8G and R36G mutations (codon-optimized variant 1 or "opt1") 239 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGCUGAAAGCAAAUA CGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCA ACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGUGUCCCUGACCUGCCUGGUCAAGGGC UUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACACCCUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGUAGCGUGAUGCACGAGGCCCUGCACAAUCACUACACCCAGAAGUCCCUGAGCCUGUCUGGGA husec SP, GLP1 (7-37)-linker-Dula_IgG4 (LS) with H7Y, A8G, R36G mutations (codon-optimized variant 1 or "opt1") 240 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGCUGAAAGCAAAUA CGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCA ACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGUGUCCCUGACCUGCCUGGUCAAGGGC UUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACACCCUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUCUGUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUGAGCCUGAGCCUGGGA husec SP, GIP (1-42) with A2G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") 241 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAG GCGGUGGAUCUGCCGAGUCUAAAUACGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCU AGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGUGUCCCUGACCUGCCUGG UCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACCACCUCCUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGUAGCGUGAUGCACGAGGCCCUGCACAAUCACUACACCCAGAAGUCCCUGAGCCUGUCUCUGGGA husec SP, GIP (1-42) with A2G mutation - linker - Dula_IgG4 (LS) (codon-optimized variant 1 or "opt1") 242 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAG GCGGUGGAUCUGCCGAGUCUAAAUACGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCU AGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGUGUCCCUGACCUGCCUGG UCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACCACCUCCUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUCUGUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGA husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - FcKIH-a (codecoin-optimized variant 1 or "opt1") 243 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUGGUGGCGGAGGUUCUGAUAAGACCCACACC UGUCCACCUUGUCCUGCUCCAGAGAGCACAAGAGGCCCUAGCGUGUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUGUCCCACGAAGAUCCCGAAGUGAAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUACAACAGCACC UACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCCUAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCACCUUGCAGAGAAGAAAUGACCAAGAAUCAGGUGUCCCUGUGGUGCCUGGUCAAGGGCUUCUAC CCUAGCGACAUUGCCGUCGAGGGGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAAGCUGACAGUGGACAAGAGCAGGUGGCAGCAGGGCAACGUGUUCAGCUGUUCUGUGCUGCACGAGGCCCUGCACAGCCACUACACACAGAAGUCCCUGAGCCUGUCCUGGCAAG husec SP, GIP with A2G mutation (1-42) - FcKIH-b (code-optimized variant 1 or "opt1") 244 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGA GGCGGUGGAUCUGAUAAGACCCACACCUGUCCACCUUGUCCUGCUCCAGAGAGCACAAGAGGCCCUAGCGUGUUCUUGUUCCCUCCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUGUCCCACGAAGAUCCCGAAGUGAAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAG GAACAGUACAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCCUAUCGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAGCCUCAAGUCUGUACCCUGCCUCCUAGCAGAGAGAAGAAAUGACCAAGAAUCAGGUGUCCCUGAGCUGCGCCGUGAAG GGCUUCUACCCUAGCGAUAUUGCCGUCGAGUGGGAGAGCAACGGCCAGCCUGAGAACAAUUACAAGACCACACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGGUGUCCAAGCUGCAGUGGACAAGAGCAGGUGGCAGCAGGGCAACGUGUUCAGCUGUUCUGUGCUGCACGAGGCCCUGCACAGCCACUACACACAGAAGUCCCUGUCUCUGAGCCCUGGCAAG husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - connector - hAlbumin (code-optimized variant 1 or "opt1") 245 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCA GGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGACGCUCACAAAUCUGAAGUGGCCCACAGAUUCAA GGACCUGGGCGAAGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUG UGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGACAAGCUGUGUACAGUGGCCACACUGAGAGAAACCUACGGCGAGAUGGCCGACUGCUGCGCCAAACA AGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUCCACGACAACGAGGAAACCUUCC UGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCG CUUGCCUGCUGCCUAAACUGGACGAGCUUCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUG CUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCGUGACCGACCUGACAAAGGUGCACACCGAGUGCUGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAG CUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAGGUGGAAAACGACGAGAUG CCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAAUUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCU GACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCCGCCGCUGAUCCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUG GUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUCCACACCU ACACUGGUCGAAGUGUCCAGAAAUCUGGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUUACCUGUCUGGGGCUGAAUCAGCUGUGCGUG CUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCAAAAGAGUUUAAC GCCGAGACCUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAGAGACAGAUCAAGAAACAGACUGCCCUGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCAAAGAACAACUG AAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGUUUCGCCGAAGAAGGCAAGAAACUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUG husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - connector - aHSA-VHH (code-optimized variant 1 or "opt1") 246 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCC UGGCUUGUAAAAGGCGGCGGUGGGCGGAGGAUCUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGAAGUUCAGCUGCUUGAAUCAGGCGGAGGCCUGGUUCAACCUGGCGGAUCUCUGAGACUGAGCUGUGCCGCCUCUGGCU UCACCCUGGAUUAUUACGCCAUCGGCUGGUUCAGACAGGCCCCUGGCAAAGAGAGAGAGGGCGUCAGCUGUAUUGCCAGCAGCGGCGGCUCUACCAAUUACGCCGAUAGCGUGAAGGGCAGAUUCACCAUCAGCAGAGACAACAG CAAGAACACCGUGUACCUCCAGAUGAACAGCCUGAAGCCUGAGGACACCGCCGUGUACUAUUGUGCCGCAGCCGUGCUUGAGUGCAGAACAGUUGUGCGGGGCUACGACUAUUGGGGCCAGGGAACACAAGUGACCGUGUCUUCU husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - furin protease - GIP (1-42) with A2G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") 247 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAG AGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCAC CAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGCCGGAGGAAGCGGAGGCGGCGGAUCUGCUGAAAGCAAAUACGG CCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCC AAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAU UGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGA AAUGACCAAGAAUCAGGGUCCCUGACCUGCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACACCUCCUGUGCUGGACAGCGACG GCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGUAGCGUGAUGCACGAGGCCCUGCACAAUCACUACACCCAGAAGUCCUGAGCCUGUCUCUGGGA husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - furin protease - GIP (1-42) with A2G mutation - linker - Dula_IgG4 (LS) (codon-optimized variant 1 or "opt1") 248 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAG AGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCAC CAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGCCGGAGGAAGCGGAGGCGGCGGAUCUGCUGAAAGCAAAUACGG CCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCC AAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAU UGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGA AAUGACCAAGAAUCAGGGUCCCUGACCUGCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACACCUCCUGUGCUGGACAGCGACG GCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUCUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGA husec SP, GIP (1-42) with A2G mutation - furin protease - GLP1 (7-37) with H7Y, A8G, R36G mutation - linker - Dula_IgG4 (LS) (codon-optimized variant 1 or "opt1") 249 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGG ACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCACA AGCGACGUGUCCAGCUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUUGUGAAAGGCGGUGGUGGUGGCGGAGGAAGCGGUGGCGGAGGUUCAGGUGGCGGUGGAUCUGCCGAGAGCAAAUACGG ACCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUCGGACGUUUCCC AAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAU UGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGA AAUGACCAAGAAUCAGGGUCCCUGACCUGUCUGGUCAAGGGCUUCUACCCUAGCGACAUCGCCGUUGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACCUCCUGUGCUGGACAGCGACG GCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUCUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGA husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 - linker - Dula_IgG4 (LS) (codon-optimized variant 1 or "opt1") 250 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCC GCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCC AUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGCCGGAGGUUCAAACGUUCGC CGCAAAAGAUACGGCGAGGGAACUUUCACCCUCUGACGUGUCCUCUUACCUCGAAGGACAGGCCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAGGCGGUGGUGGAAGCGGAGGUGGU GGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCUCCGACUACUCCAUUGCUAUGGAUAAGAUUCAUCACAGGAUUUCGUCAACUGGCUCCUCGCUCAGAAAGGGAAGAAAAAC GAUUGGAAACAUAACAUCACGCAAGGCGGUGGCGGUUCAGGUGGUGGUGGUUCUAACGUCCGGCGGAAACGUUACGGCGAGGGAACCUUUACAUCAGACGUUUCAUCCUACCUUGAGGGGCAAGCU GCAAAAGAGUUUAUUGCCUGGCUCGAAAGGUGGUGGCGGUGGCGGAGGUAGCGGAGGCGGCGGAAGCAACGUUCGAAGAAAACGAUACGGCGAGGGGACCUUCAUCUCCGAUUAUAGUAUCGCU AUGGACAAAAUUCAUCAGCAAGACUUUGUUAACUGGUUGCUCGCCCAAAAGGGGAAAAAGAACGAUUGGAAGCAACAUACACAAGGCGGAGGUGGUAGUGGGCGGAGGUGGAAGUAACGUACGA CGGAAAAGAUACGGCGAAGGCACCUUCACCUCCGACGUUUCAAGUUACUUGGAGGGACAAGCAGCCAAAGAAUUCAUAGCCUGGCUCGUGAAAGCGGCCGGAGGUGGUGGCGGUAGUGGUGGUGGC GGCUCAAACGUUCGGAGGAAAAGAUACGGCGAGGGCACUUUUAUUAGCGAUUACUCUAUCGCAAUGGAUAAGAUACACCAACAAGACUUUGUCAAUUGGCUCUUGGCACAAAAAGGCAAGAAGAAC GAUUGGAAACACAACAUAACCCAAGGCGGCGGUGGUUCAGGUGGCGGAGGAUCAGGUGGUGGCGGAUCUGCCGAGAGCAAAUACGGACCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGC GGCCCUAGCGUUUUCUUGUUCCCACCUAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUCGUGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUAC GUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUCUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACAAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAAGAAUCAG GUGUCCCUGACCUGUCUGGUCAAGGGCUUCUACCCUAGCGACAUCGCCGUUGAGUGGGAGAGCAACGGACAGCCUGAACAAUUACAAGACCACACCUCCUGUGCUGGACAGCGACGGCUCAUUC UUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUCUGUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGA husec SP, GIP with A2G mutation (1-42) - connector - hAlbumin (code-optimized variant 1 or "opt1") 251 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCC ACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGUGGAUCUGACGCC CACAAAUCUGAAGUGGCCCACAGAUUCAAGGACCUGGGCGAAGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAA CGAAGUGACCGAGUUCGCCAAGACCUGUGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGACAAGCUGUGUACAGUGGCCACACUGAGAGAAACCUACGGCG AGAUGGCCGACUGCUGCGCCAAACAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUC CACGACAACGAGGAAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUG CCAGGCCGCUGAUAAGGCCGCUUGUCUGCUGCCUAAACUGGACGAGCUGCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCU UUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCGUGACCGACCUGACAAAGGUGCACACCGAGUGCUGUCACGGCGACCUGCUUGAG UGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAGGUGG AAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAAUUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCU AGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCCGCCGCUGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUU CAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCU CCACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUUACCUGUCUGGUGCUGAAUCAGCUG UGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCCAAAGAGUU CAACGCCGAGACCUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAGAGACAGAUCAAGAAACAGACUGCCCUGGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCAAAGAACAAC UGAAGGCCGUGAUGGACGACUUCGCCGCCUUUGUCGAGAAGUUGCAAGGCUGACGACAAAGAGACCUGUUUCGCCGAAGAAGGCAAAAAGCUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUG husec SP, GIP (1-42) with A2G mutation - connector - aHSA-VHH (code-optimized variant 1 or "opt1") 252 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCCG GCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGUGGCGGAGGCGGAUCUGAAGUUCAGCUGCUUGAAUCUGGCGGCGGACUGGUUCAACCUGGCGGAUCUCUGAGACUGAGC UGUGCCGCCUCUGGCUUCACCCUGGAUUAUUACGCCAUCGGCUGGUUCAGACAGGCCCCUGGCAAAGAGAGAGGGCGUCAGCUGUAUUGCCAGCAGCGGCGGCUCUACCAAUUACGCCGAUAGCGUGAAGGGCAGAUUCACCAUCAGCAGA GACAACAGCAAGAACACCGUGUACCUCCAGAUGAACAGCCUGAAGCCUGAGGACACCGCCGUGUACUAUUGUGCCGCAGCCGUGCUUGAGUGCAGAACAGUUGUGCGGGGCUACGACUAUUGGGGCCAGGGAACACAAGUGACCGUGUCCUCU husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker-furin protease-GIP (1-42) - linker-hAlbumin (codon-optimized variant 1 or "opt1") 253 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAG AGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGCCGAGGGAACCUUUAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCAC CAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGACGCUCACAAAUCUG AAGUGGCCCACAGAUUCAAGGACCUGGGCGAAGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCC AAGACCUGUGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGACAAGCUGUGUACAGUGGCCACACUGAGAGAAACCUACGGCGAGAUGGCCGACUGCUGCGCCAAACAAG AGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUG UACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCGCUUGCCUGCUGCCUAAACUGGACGA GCUUCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUG CCGAGGUGUCCAAGCUCGUGACCGACCUGACAAAGGUGCACACCGAGUGCUCGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAG CAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAGGUGGAAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGA AUUACGCCGAAGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCC GCCGCUGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCU GCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUCCACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAU UACCUGUCUGGUGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGAC CUACGUGCCAAAAGAGUUUAACGCCGAGACCUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAACGGCAGAUCAAGAAACAGACUGCCCUGGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCA AAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGUUUCGCCGAAGAAGGCAAAAAGCUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUG husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation - linker - hAlbumin (codon-optimized variant 1 or "opt1") 254 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAG AGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCAC CAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGACGCUCACAAAUCUG AAGUGGCCCACAGAUUCAAGGACCUGGGCGAAGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCC AAGACCUGUGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGACAAGCUGUGUACAGUGGCCACACUGAGAGAAACCUACGGCGAGAUGGCCGACUGCUGCGCCAAACAAG AGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUG UACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCGCUUGCCUGCUGCCUAAACUGGACGA GCUUCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUG CCGAGGUGUCCAAGCUCGUGACCGACCUGACAAAGGUGCACACCGAGUGCUCGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAG CAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAGGUGGAAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGA AUUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCC GCCGCUGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCU GCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUCCACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAU UACCUGUCUGGUGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGAC CUACGUGCCAAAAGAGUUUAACGCCGAGACCUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAACGGCAGAUCAAGAAACAGACUGCCCUGGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCA AAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGUUUCGCCGAAGAAGGCAAAAAGCUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUG husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x2 - linker - hAlbumin (codon-optimized variant 1 or "opt1") 255 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUA AAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGC AAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAGGUUCAAACGUUCGCCGCAAAAGAUACGGCGAGGGAACUUUCACCCUGACGUGUCCUCUUACCUCGAAGGACAGGCUGCUAAAGAAUUCAUUGCU UGGCUGGUCAAAGGCGGCGGAGGCGGUGGUGGAAGCGGAGGUGGUGGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCUCCGACUACUCCAUUGCUAUGGAUAAGAUUCAUCAACAGGAUUUCGUCAACUGGCUCCUCGCUC AGAAAGGGAAGAAAAACGAUUGGAAACAUAACAUCACGCAAGGCGGUGGCGGUUCUGGUGGCGGUGGCAGCGGAGGGCGGAUCUGACGCUCACAAAUCUGAAGUGGCCCACAGAUUCAAGGACCUGGGCGAAGAAAAUUUCAAGGCCCUGG UGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUGUGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGACAAGCU GUGUACAGUGGCCACACUGAGAGAAACAUACGGCGAGAUGGCCGACUGCUGCGCCAAACAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUAC AGCCUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCGCU UGCCUGCUGCCUAAACUGGACGAGCUUCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAG UUUGCCGAGGUGUCCAAGCUCGUGACCGACCUGACAAAGGUGCACACCGAGUGCUGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGC UGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAGGUGGAAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAAUUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUG UUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCCGCCGCUGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGG UGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGGCCACAGGUCUCCACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCA AAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUUACCUGUCUGGGUGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAGUGCUGUACCGAGAGCCUCGUGAA UAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCAAAAGAGUUUAACGCCGAGACCUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAACGGCAGAUCAAGAAACAGACUGCCCUGGUGGAACUGGUCAA GCACAAGCCUAAGGCCACCAAAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGUUUUCGCCGAAGAAGGCAAAAAGCUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUG husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 - linker - hAlbumin (codon-optimized variant 1 or "opt1") 256 AUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGA UCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAGGUUCAAACGUUCGCCGCAAAA GAUACGGCGAGGGAACUUUCACCCUCUGACGUGUCCUCUUACCUCGAAGGACAGGCCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAGGCGGUGGUGGAAGCGGAGGUGGUGGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCCCGACUACUCCAUUGCUAUGGAUAAGAU UCAUCAACAGGAUUUCGUCAACUGGCUCCUCGCUCAGAAAGGGAAGAAAAACGAUUGGAAACAUAACAUCACGCAAGGCGGUGGCGGUUCAGGUGGUGGGUUCUAACGUCCCGGCGGAAACGUUACGGCGAGGGAACCUUUACAUCAGACGUUUCAUCCUACCUUGAGGGGCAAGCUGCAAAAGAGUUUAUU GCCUGGCUCGUGAAAGGUGGUGGCGGUGGCGGAGGUAGCGGAGGCGGCGGAAGCAACGUUCGAAGAAAACGAUACGGCGAGGGGACCUUCAUCUCCGAUUAUAGUAUCGCUAUGGACAAAAUUCAUCAGCAAGACUUUGUUAACUGGUUGCUCGCCCAAAAGGGGAAAAAGAACGAUUGGAAGCAACACAUU ACACAAGGCGGAGGUAGGUGGCGGAGGUGGAAGUAACGUACGACGGAAAAGAUACGGCGAAGGCACCUUCACCUCCGACGUUUCAAGUUACUUGGAGGGACAAGCAGCCAAAGAAUUCAUAGCCCUGGCUCGUGAAAGGCGGCGGAGGUGGUGGCGGUAGUGGUGGUGGCGGCUCAAACGUUCGGAGGAAAA GAUACGGCGAGGGCACUUUUAUUAGCGAUUACUCUAUCGCAAUGGAUAAGAUACACCAAGACUUUGUCAAUUGGCUCUUGGCACAAAAAGGCAAGAAGAACGAUUGGAAACACAACAUAACCCAAGGCGGCGGUGGUUCAGGUGGCGGAGGAUCAGGUGGUGGCGGAUCUGACGCCCACAAAUCUGAAGU GGCCCACAGAUUCAAGGACCUCGGCGAGGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUGUGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGC GACAAGCUGUGUACAGUGGCCACACUGAGAGAAACAUACGGCGAAAUGGCCGACUGCUGCGCCAAGCAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUCCACGACAACGAGGAAACCUUCCUG AAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCGCUUGCCUGCUGCCUAAACUGGACGAGCUUCGCGACGAGGGAAAAGCCAGCAGCGCCAAACAGAGACUGAAGU GCGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCGACCGACCUGACAAAGGUGCACACCGAGUGCUGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAU CUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAAGUGGAAAACGACGAGAUGCCUGCCGAUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAAUUACGCCGAGGCCAAAGACGUGUUCCUGGGC AUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCCGCCGCUGAUCCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGC GAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUCCACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUUACCUGUCUGUGGUGC UGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCAAAAGAAUUCAACGCCGAGACCUUUACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAACG GCAGAUCAAGAAACAGACUGCCCUGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCAAAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGCUUUGCCGAAGAAGGCAAAAAGCUGGUGGCCGCCUCUCAAGCUGCUCUGGGACUG Exemplary polynucleotide characteristics

The polynucleotides described herein encode incretins as described herein. Additionally, in some embodiments, the polynucleotides described herein include encoding other elements, such as signal peptides. In some embodiments, the polynucleotides described herein may contain nucleotide sequences encoding a 5' UTR and/or a 3' UTR. In some embodiments, the polynucleotides described herein may contain nucleotide sequences encoding a polyA tail. In some embodiments, the polynucleotides described herein may contain a 5' cap, which may be incorporated during transcription or attached to the polynucleotide post-transcriptionally. 5' Cap

The structural feature of mRNA is a 5' cap structure. Natural eukaryotic mRNA contains a 7-methylguanosine cap (m7GpppN) linked to the mRNA via a 5'-to-5'-triphosphate bridge. In most eukaryotic mRNAs and some viral mRNAs, further modifications can occur at the 2'-hydroxyl group (2'-OH) of the first and subsequent nucleotides (e.g., the 2'-hydroxyl group can be methylated to form 2'-O-Me), producing "cap 1" and "cap 2" pentatonic ends, respectively. Diamond et al., (2014) Cytokine & growth Factor Reviews , 25:543–550, reported that cap 0-mRNA cannot be translated as efficiently as cap 1-mRNA, with the role of 2'-O-Me at the penultimate position of the 5' end of the mRNA being decisive. It has been shown that a lack of 2'-O-Me triggers innate immunity and activates the IFN response. Daffis et al., (2010) Nature , 468:452-456; and Züst et al., (2011) Nature Immunology , 12:137-143.

RNA capping has been well studied and described, for example, in Decroly et al., (2012) Nature Reviews 10: 51-65; and Ramanathan et al., (2016) Nucleic Acids Res; 44(16): 7511–7526, the entire contents of which are hereby incorporated by reference. For example, in some embodiments, the 5' cap structure that may be suitable for the context of this invention is cap 0 (methylation of the first nucleobase, e.g., m7GpppN), cap 1 (extramethylation of the ribose of the adjacent nucleotide of m7GpppN), cap 2 (extramethylation of the ribose of the second nucleotide downstream of m7GpppN), cap 3 (extramethylation of the ribose of the third nucleotide downstream of m7GpppN), cap 4 (extramethylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA ("anti-reverse cap analog"), modified ARCA (e.g., ARCA modified with thiophosphate), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-de-nitro-guanosine, 8-side-oxy-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

As used herein, the term "5'-cap" refers to a structure found at the 5' end of RNA (e.g., mRNA) and generally includes a guanosine nucleotide linked to RNA (e.g., mRNA) via a 5'-to-5'-triphosphate bond (also known as Gppp or G(5')ppp(5')). In some embodiments, the guanosine nucleotide included in the 5' cap may be modified, for example, by methylation at one or more sites (e.g., at the 7 position) on the base (guanine) and/or by methylation at one or more sites on the ribose. In some embodiments, the guanosine nucleotide included in the 5' cap contains a 3'O methylation at the ribose (3'OmeG). In some embodiments, the guanosine nucleotide included in the 5' cap contains a methylation at the 7 position of guanine (m7G). In some embodiments, the guanosine nucleotide included in the 5' cap comprises methylation at the 7-position of guanine and 3'-O methylation at the ribose (m7(3'OmeG)). It will be understood that the notations used in the preceding paragraph, such as "( _m27,3' ^{- O} )G" or "m7(3'OmeG)", are applicable to other structures described herein.

In some embodiments, providing RNA with the 5'-cap disclosed herein can be achieved via in vivo transcription, wherein the 5'-cap is co-transcribed into the RNA strand, or it can be ligated to the RNA post-transcriptionally using a capping enzyme. In some embodiments, co-transcriptional capping using the cap disclosed herein improves RNA capping efficiency compared to co-transcriptional capping using an appropriate reference. In some embodiments, improved capping efficiency can increase RNA transcription efficiency and/or transcription rate, and/or increase the expression of the encoded polypeptide. In some embodiments, changes to polynucleotides produce a non-hydrolyzable cap structure, which can, for example, prevent uncapping and increase RNA half-life.

In some embodiments, the 5' cap used is a cap 0, cap 1, or cap 2 structure. See, for example, Figure 1 by Ramanathan et al. and Figure 1 by Decroly et al., each of which is incorporated herein by reference in its entirety. In some embodiments, the RNA described herein comprises a cap 1 structure. In some embodiments, the RNA described herein comprises a cap 2 structure.

In some embodiments, the RNA described herein includes a cap 0 structure. In some embodiments, the cap 0 structure includes a guanosine nucleotide methylated at the 7-position of guanine (( ^m7 )G). In some embodiments, this cap 0 structure is linked to the RNA via a 5'-to-5'-triphosphate bond and is also referred to herein as ( ^m7 )Gppp. In some embodiments, the cap 0 structure includes a guanosine nucleotide methylated at the 2' position of guanosine ribose. In some embodiments, the cap 0 structure includes a guanosine nucleotide methylated at the 3' position of guanosine ribose. In some embodiments, the guanosine nucleotide included in the 5' cap includes methylation at the 7-position of guanine and at the 2' position of ribose (( _m27,2' ^{- O} )G). In some embodiments, the guanosine nucleotide included in the 5' cap is methylated at the 7' position ^of guanine and at the 2' position of ribose (( _m27,3' ^-O )G).

In some embodiments, the cap 1 structure comprises a guanosine nucleotide methylated at the 7' position of guanine (( ^m7 )G) and, where applicable, at the 2' or 3' position ^of the ribose, and a first nucleotide in RNA methylated at the ^2'O position (( ^{m2' - O} ) _{N1 )} . In some embodiments, ^the cap 1 structure is linked to RNA via 5'-to-5'-triphosphate bonds and is also referred to herein as, for example, (( ^m7 )Gppp( ^{2' -O} ) _N1 ) or ( _m27,3' ^-O )Gppp( ^{2' -O} ) _N1 ), where _N1 is as defined and described herein. In some embodiments, the cap 1 structure contains a second nucleotide _N2 at position 2 and selected from A, G, C, or U, for example, ( ^m7 )Gppp( ^{2' -O} ) _N1pN2 or ( _m27,3' ^-O ) ^{Gppp(2' - O} _{)N1pN2 ,} where each of _N1 and _{N2 is} as defined and described herein.

In some embodiments, the cap 2 structure includes a guanosine nucleotide methylated at the 7-position (( ^m7 )G) and, where applicable, at the 2' or 3' position of the ribose, and a first and second nucleotide of RNA methylated at 2'O (( ^{m2' -O} ) _N1 p( ^{m2' -O} ) _N2 ). In some embodiments, the cap 2 structure includes a guanosine nucleotide methylated at the 7-position (( ^m7 )G) and, where applicable, at the 3' position of the ribose, and a first and second nucleotide of RNA methylated at 2'O. In some embodiments, the cap 2 structure is linked to the RNA via 5'-to-5'-triphosphate bonds and is also referred to herein as, for example, ((m ⁷ )Gppp( ^{2 '-O} )N ₁ p( ^{2 '-O} )N ₂ ) or (m ₂ ^{7 ,3 '-O} )Gppp( ^{2 '-O} )N ₁ p( ^{2 '-O} )N ₂ ), wherein each of N ₁ and N ₂ is as defined and described herein.

In some embodiments, the 5' cap is a dinucleotide cap structure. In some embodiments, the 5' cap is a dinucleotide cap structure containing _N1 , where _N1 is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G* _N1 , where _N1 is as defined above and herein, and G* contains the structure of formula (I): (I) or its salt, wherein each of ^R2 and ^R3 is -OH or _-OCH3 ; and X is O or S.

In some embodiments, ^R2 is -OH. In some embodiments, ^R2 is _-OCH3 . In some embodiments, R3 is -OH. In some embodiments, ^R3 is _-OCH3 . In some embodiments, ^both R2 and ^R3 are -OH. In some embodiments, ^both R2 and ^R3 are _-CH3 . In some embodiments, _both ^R2 and ^R3 are -OH. In some embodiments, ^both R2 ^{and R3} _{are -CH3} .

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

In some embodiments, the 5' cap is a dinucleotide cap O structure (e.g., ( ^m7 ) _GpppN1 , ( _m27,2' ^-O )GpppN1, ( ^{m27,3' -O} _{) GpppN1} , ( _{m7 ) GppSpN1} , ( ^{m27,2' -O} ) _GppSpN1 or ( ^{m27,3' -O )} _GppSpN1 ⁾ , where _N1 is as _defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap O structure (e.g., ( ^m7 ) _GpppN1 , ( _m27,2' ^-O )GpppN1, ( ^{m27,3' -O} _{) GpppN1} , ( _{m7 ) GppSpN1} , ( ^{m27,2' -O )GppSpN1 or (m27,3'} _- ^{O )} _{GppSpN1 )} ^, where _N1 is G. In some embodiments, the 5' cap is a dinucleotide cap O structure (e.g., ( ^m7 ) _GpppN1 , ( _m27,2' ^-O )GpppN1, ( ^{m27,3' -O} ) _GpppN1 , ( _{m7 ) GppSpN1} , ( ^m27,2' _- ^O ) _GppSpN1 or ( ^{m27,3' -O )} _GppSpN1 ), where _N1 is _A , ^U or C. In some embodiments, the 5' cap is a dinucleotide cap 1 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 )} , where _N1 is as defined and described herein. In some embodiments, the 5' cap is selected from the group consisting of: (m ⁷ )GpppG ("Ecap0"), (m ⁷ )Gppp(m ^2'-O )G ("Ecap1"), (m ₂ ^{7,3' -O} )GpppG ("ARCA"), and (m ₂ ^7,2'-O )GppSpG ("β-S-ARCA"). In some embodiments, the 5' cap is (m ⁷ )GpppG ("Ecap0") having the following structure: Or its salt.

In some embodiments, the 5' cap is (m ⁷ )Gppp(m ^{2 '-O} )G ("Ecap1") with the following structure: Or its salt.

In some embodiments, the 5' cap is (m ₂ ^7,3'-O )GpppG ("ARCA") with the following structure: Or its salt.

In some embodiments, the 5' cap is (m ₂ ^{7,2 '-O} )GppSpG ("β-S-ARCA") with the following structure: Or its salt.

In some embodiments, the 5' cap is a trinucleotide cap structure. In some embodiments, the 5' cap is a trinucleotide cap structure containing _{N1 pN2} , wherein _N1 and _N2 are as defined and described herein. In some embodiments, the 5' cap is a dinucleotide cap G* _{N1 pN2} , wherein _N1 and _N2 are as defined above and herein, and G* contains the structure of formula (I): (I) or its salts, wherein ^R2 , ^R3 and X are as defined and described herein.

In some embodiments, the 5' cap is a trinucleotide cap _O structure (e.g., ( ^m7 ) _{GpppN1pN2 ,} ( _m27,2' ^-O ) _{GpppN1pN2 ,} or ( _m27,3' ^{- O )} _GpppN1pN2 ), wherein _N1 and _N2 are as defined and described herein. In some embodiments, the 5' cap is a trinucleotide cap 1 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 cap 2 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 following groups: (m ₂ ^{7,3 '-O} )Gppp(m ^{2 '-O} )ApG ("CleanCap AG", "CC413"), (m ₂ ^{7,3 '-O} )Gppp(m ^{2 '-O} )GpG ("CleanCap GG"), (m ⁷ )Gppp(m ^{2 '-O} )ApG, (m ⁷ )Gppp(m ^{2 '-O} )GpG, (m ₂ ^{7,3 '-O} )Gppp(m ₂ ^{6,2 '-O} )ApG and (m ⁷ )Gppp(m ^{2 '-O} )ApU.

In some embodiments, the 5' cap is a (m ₂ ^{7,3 '-O} )Gppp(m ^{2 '-O} )ApG ("CleanCap AG", "CC413") with the following structure: Or its salt.

In some embodiments, the 5' cap is (m ₂ ^{7,3 '-O} )Gppp(m ^{2 '-O} )GpG ("CleanCap GG") having the following structure: Or its salt.

In some embodiments, the 5' cap is a (m ⁷ )Gppp(m ^{2 '-O} )ApG with the following structure: Or its salt.

In some embodiments, the 5' cap is (m ⁷ )Gppp(m ^{2 '-O} )GpG with the following structure: Or its salt.

In some embodiments, the 5' cap is a (m ₂ ^{7,3' -O} )Gppp(m ₂ ^{6,2' -O} )ApG with the following structure: Or its salt.

In some embodiments, the 5' cap is a (m ⁷ )Gppp(m ^{2 '-O} )ApU with the following structure: Or its salt.

In some embodiments, the 5' cap is a tetranucleotide cap structure. In some embodiments, the 5' cap is a tetranucleotide cap structure comprising _{N1 pN2 pN3} , wherein _N1 , _N2 , and _N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide cap G* _{N1 pN2 pN3} , wherein _N1 , _N2 , and _N3 are as defined above and herein, and G* comprises the structure of formula (I): (I) or its salts, wherein ^R2 , ^R3 and X are as defined and described herein.

In some embodiments, ^{the 5' cap is a tetranucleotide cap O 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 cap 1 structure (e.g., ( ^m7 )Gppp( ^{m2' -O} ) _{N1 pN2 pN3} , ( ^{m27,2' -O} )Gppp( _m2' -O) _{N1 pN2 pN3} , ( ^{m27,3' - O} )Gppp( _m2' ^{- O} ) ^N1 _{pN2 N3 )} , wherein _N1 , _N2 , and _N3 are as defined and described herein. In some embodiments, the 5' cap is a tetranucleotide cap 2 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 _N3 are as defined and _described herein. In some embodiments, the 5' cap is selected from the following groups: (m ₂ ^{7,3 '-O} )Gppp(m ^{2 '-O} )Ap(m ^{2 '-O} )GpG, (m ₂ ^{7,3 '-O} )Gppp(m ^{2 '-O} )Gp(m ^{2 '-O} )GpC, (m ⁷ )Gppp(m ^{2 '-O} )Ap(m ^{2 '-O} )UpA and (m ⁷ )Gppp(m ^{2 '-O} )Ap(m ^{2 '-O} )GpG.

In some embodiments, the 5' cap is (m ₂ ^{7,3 '-O} )Gppp(m ^{2 '-O} )Ap(m ^{2 '-O} )GpG with the following structure: Or its salt.

In some embodiments, the 5' cap is (m ₂ ^{7,3 '-O} )Gppp(m ^{2 '-O} )Gp(m ^{2 '-O} )GpC with the following structure: Or its salt.

In some embodiments, the 5' cap is (m ⁷ )Gppp(m ^{2 '-O} )Ap(m ^{2 '-O} )UpA with the following structure: Or its salt.

In some embodiments, the 5' cap is (m ⁷ )Gppp(m ^{2 '-O} )Ap(m ^{2 '-O} )GpG with the following structure: Or its salt. Cap proximal sequence

In some embodiments, the 5' UTR used according to this disclosure includes a cap proximal sequence, for example, as disclosed herein. In some embodiments, the cap proximal sequence includes a sequence adjacent to the 5' cap. In some embodiments, the cap proximal sequence includes nucleotides at RNA polynucleotide positions +1, +2, +3, +4, and/or +5.

In some embodiments, the cap structure comprises one or more polynucleotides of the proximal cap sequence. In some embodiments, the cap structure comprises an ^m7 guanine cap and nucleotide +1 ( _N1 ) of the RNA polynucleotide. In some embodiments, the cap structure comprises an ^m7 guanine cap and nucleotide +2 ( _N2 ) of the RNA polynucleotide. In some embodiments, the cap structure comprises an ^m7 guanine cap and nucleotides +1 and +2 ( _N1 and _N2 ) of the RNA polynucleotide. In some embodiments, the cap structure comprises an ^m7 guanine cap and nucleotides +1, +2, and +3 ( _N1 , _N2 , and _N3 ) of the RNA polynucleotide.

Those skilled in the art will understand upon reading this disclosure that, in some embodiments, one or more residues of the cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in the RNA because they are already included in the cap body (e.g., the cap 1 structure or the cap 2 structure, etc.); or, in some embodiments, at least some residues in the cap proximal sequence may be added enzymatically (e.g., by polymerases such as T7 polymerase). For example, in some exemplary embodiments of the _m27,3' ^-O Gppp( _m12' ^{- O} )ApG cap, +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).

In some embodiments, the 5' cap is a dinucleotide cap structure, wherein the proximal sequence of the cap contains _N1 of the 5' cap, where _N1 is any nucleotide, such as A, C, G, or U. In some embodiments, the 5' cap is a trinucleotide cap structure (e.g., the trinucleotide cap structure described above and herein), wherein the proximal sequence of the cap contains _N1 and _N2 of the 5' cap, where _N1 and _N2 are independently any nucleotide, such as A, C, G, or U. In some embodiments, the 5' cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structure described above and herein), wherein the proximal sequence of the cap contains _N1 , _N2 , and _N3 of the 5' cap, where _N1 , _N2 , and _N3 are any nucleotide, such as A, C, G, or U.

In some embodiments, for example, where the 5' cap is a dinucleotide cap structure, the proximal cap sequence includes _N1 of the 5' cap, and _N2 , _N3 , _N4 , and _N5 , wherein _N1 to _N5 correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide. In some embodiments, for example, where the 5' cap is a trinucleotide cap structure, the proximal cap sequence includes _N1 and _N2 of the 5' cap, and _N3 , _N4 , and _N5 , wherein _N1 to _N5 correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide. In some embodiments, for example, where the 5' cap is a tetranucleotide cap structure, the proximal sequence of the cap includes _N1 , _N2 , and _N3 of the 5' cap, as well as _N4 and _N5 , where _N1 to _N5 correspond to the positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

In some embodiments, _N1 is A. In some embodiments, _N1 is C. In some embodiments, _N1 is G. In some embodiments, N1 is U. In some embodiments, _N2 is A. In some embodiments, _N2 is C. In some embodiments, _N2 is G. In some embodiments, _N2 is U. In some embodiments, _N3 is A. In some embodiments, _N3 is C. In some embodiments, _N3 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is A. In some embodiments, _N4 is C. In some embodiments, _N4 is G. In some embodiments, _N4 is 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 the embodiments described above and herein (e.g., for _N1 to _N5 ) may be used alone or in combination and/or may be combined with other embodiments of the variables described above and herein (e.g., 5' cap). 5' UTR

In some embodiments, the nucleic acid (e.g., DNA, RNA) used according to this disclosure contains a 5' UTR. In some embodiments, the 5' UTR may contain a plurality of different sequence elements; in some embodiments, such a plurality may be or contain multiple copies of one or more specific sequence elements (e.g., such as those from a specific source or otherwise referred to as functional or characteristic sequence elements). In some embodiments, the 5' UTR contains multiple different sequence elements.

The term "untranslated region" or "UTR" is commonly used in this technique to refer to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide (such as mRNA). Untranslated regions (UTRs) can be located at the 5' (upstream) end of an open reading frame (5' UTR) and/or the 3' (downstream) end of an open reading frame (3' UTR). As used herein, the term "5' UTR" refers to the polynucleotide sequence between the 5' end of a polynucleotide (e.g., the transcription start site) and the start codon of the polynucleotide coding region. In some embodiments, "5' UTR" refers to a polynucleotide sequence that begins at the 5' end of the polynucleotide (e.g., a transcription start site) and terminates one nucleotide (nt) before the start codon (typically AUG) of the polynucleotide coding region, for example, in its natural background. In some embodiments, the 5' UTR contains a Kozak sequence. The 5' UTR is downstream of the 5' cap (if present), for example, directly adjacent to the 5' cap. In some embodiments, the 5' UTR disclosed herein contains, for example, a cap-proximal sequence as defined and described herein. In some embodiments, the cap-proximal sequence contains a sequence adjacent to the 5' cap.

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

In some embodiments, the RNA disclosed herein contains hAg 5' UTR or a fragment thereof.

In some embodiments, the RNA disclosed herein contains 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 with a 5' UTR having the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 49). In some embodiments, the RNA disclosed herein contains the 5' UTR provided in SEQ ID NO: 49. PolyA tail

In some embodiments, the polynucleotides (e.g., DNA, RNA) disclosed herein contain, for example, polyadenylated or poly(A) sequences as described herein. In some embodiments, the poly(A) sequence is located downstream of the 3' UTR, for example, adjacent to the 3' UTR.

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

It has been demonstrated that a poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on RNA levels in transfected eukaryotic cells, as well as on protein levels translated from open reading frames located upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, Vol. 108, pp. 4009-4017, which are incorporated herein by reference).

In some embodiments, the poly(A) sequence according to this disclosure is not limited to a specific length; in some embodiments, the poly(A) sequence is of any length. In some embodiments, the poly(A) sequence comprises at least 20, at least 30, at least 40, at least 80 or at least 100 and at most 500, at most 400, at most 300, at most 200 or at most 150 A nucleotides, and particularly about 120 A nucleotides, substantially consisting of or composed of such number of A nucleotides. In this context, "consistently composed of" means that the majority of nucleotides in the poly(A) sequence, typically at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% by number, are A nucleotides, but the remaining nucleotides may be other nucleotides besides A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "consisting of" means that all nucleotides in the poly(A) sequence, i.e., 100% by number, are A nucleotides. The term "A nucleotide" or "A" refers to adenosine monophosphate.

In some embodiments, based on a DNA template containing repeating dT nucleotides (deoxythymidine) in a strand complementary to the coding strand, a poly(A) sequence is ligated during RNA transcription, such as during the preparation of RNA transcribed in vivo . The DNA sequence (coding strand) encoding the poly(A) sequence is called a poly(A) box.

In some embodiments, the poly(A) box present in the DNA coding strand is essentially composed of dA nucleotides, but interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides. Such a box is disclosed in WO2016/005324, which is hereby incorporated by reference. Any poly(A) box disclosed in WO2016/005324 may be used according to this disclosure. This encompasses a poly(A) box that is essentially composed of dA nucleotides, but interrupted by random sequences of four nucleotides (dA, dC, dG, dT) with an equal distribution and a length of, for example, 5 to 50 nucleotides. At the DNA level, it exhibits stable amplification of plasso DNA in *E. coli* , and at the RNA level, it remains associated with beneficial properties supporting RNA stability and translation efficiency. In some embodiments, the poly(A) sequence contained in the RNA polynucleotide described herein is essentially composed of A nucleotides, but interrupted by random sequences of four nucleotides (A, C, G, U). The length of such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides.

In some embodiments, no nucleotides other than A are located on the 3' flanking side of the poly(A) sequence, that is, the poly(A) sequence is not masked by nucleotides other than A or followed by nucleotides other than A at its 3' end.

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 at most 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly(A) sequence may consist substantially of at least 20, at least 30, at least 40, at least 80, or at least 100 and at most 500, at most 400, at most 300, at most 200, or at most 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 at most 500, at most 400, at most 300, at most 200, or at most 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence contains approximately 120 nucleotides.

In some embodiments, the polyA tail contains 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, about 150, or about 200. In some embodiments, the polyA tail of the string structure may contain 200 or fewer A residues. In some embodiments, the polyA tail of the string structure may contain about 200 A residues. In some embodiments, the polyA tail of the string structure may contain 180 or fewer A residues. In some embodiments, the polyA tail of the string structure may contain about 180 A residues. In some embodiments, the polyA tail may contain 150 or fewer residues.

In some embodiments, the RNA comprises a poly(A) sequence containing the nucleotide sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 50), or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO: 50. In some embodiments, the poly(A) tail comprises the nucleotide sequence according to SEQ ID NO: 50.

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

In some embodiments, the RNA used according to this disclosure contains a 3' UTR. As used herein, the terms "3' non-translated region," "3' non-translated region," or "3' UTR" refer to an mRNA molecule sequence that begins after the stop codon of the open reading frame (OPF) sequence. In some embodiments, the 3' UTR begins immediately after the stop codon of the OPF sequence, for example, in its natural background. In other embodiments, the 3' UTR does not begin immediately after the stop codon of the OPF sequence, for example, in its natural background. The term "3' UTR" preferably does not include the poly(A) sequence. Therefore, the 3' UTR is upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

In some embodiments, the RNA disclosed herein contains a 3' UTR comprising an F element and/or an I element. In some embodiments, the 3' UTR or its proximal sequence contains a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an XhoI site.

In some embodiments, the RNA construct includes an F element. In some embodiments, the F element sequence is the 3' UTR of an amino-terminal cleavage enhancer (AES).

In some embodiments, the RNA disclosed herein contains 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 with the 3’ UTR having the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCUACCCCGAGUCUCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 51). In some embodiments, the RNA disclosed herein contains the 3' UTR provided in SEQ ID NO: 51.

In some embodiments, the 3'UTR is a FI element as described in WO2017/060314, which is incorporated herein by reference in its entirety. RNA Format

At least three different formats have been developed for use with RNA compounds (e.g., pharmaceutical compounds): unmodified uridine-containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplifying mRNA (saRNA). Each of these platforms exhibits unique characteristics. Generally, in all three formats, the RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTRs), and has a polyA tail at the 3' end. The ORFs of uRNA and modRNA vectors encode incretins. saRNA has multiple ORFs.

In some embodiments, the RNA described herein may have modified nucleosides. In some embodiments, the RNA contains modified nucleosides that replace at least one (e.g., each) uridine.

As used in this article, the term "uracil" describes a substance that can be found in one of the nucleobases of RNA nucleic acids. The structure of uracil is: .

As used in this article, the term "uridine" describes one of the nucleosides that can be found in RNA. The structure of uridine is: .

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

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

"Pseudouridine" is an example of a modified nucleoside, which is an isomer of uridine in which uracil is linked to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methyl-pseuuridine (m1Ψ), which has the following structure: .

N1-Methyl-pseudo-UTP has the following structure: .

Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the following structure: .

In some embodiments, one or more uridines in the RNA described herein are replaced by modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

In some embodiments, the RNA comprises a modified nucleoside replacing at least one uridine. In some embodiments, the RNA comprises a modified nucleoside replacing each uridine.

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 includes pseudouridine (ψ). In some embodiments, the modified nucleoside includes N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleoside includes 5-methyl-uridine (m5U). In some embodiments, the RNA may contain more than one type of modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside includes pseudouridine (ψ) and N1-methyl-pseudouridine (m1ψ). In some embodiments, the modified nucleosides include pseudouridine (ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleosides include N1-methyl-pseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleosides include pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyluridine (m5U).

In some embodiments, the modified nucleotides of one or more (e.g., all) uridines in the substitute RNA may be one or more of the following: 3-methyluridine (m3U), 5-methoxyuridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseuuridine, 2-thio-pseuuridine, 5-hydroxyuridine (ho5U), 5-aminoallyluridine, 5-halogen-uridine (e.g., 5-iodouridine or 5-bromouridine), 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 (mnm5s) e2U), 5-aminomethyluridine (ncm5U), 5-carboxymethylaminomethyluridine (cmnm5U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), 5-propynyluridine, 1-propynyl-pseudouridine, 5-tauronic acid methyluridine (τm5U), 1-tauronic acid methyl-pseudouridine, 5-tauronic acid methyl-2-thiouridine (τm5s2U), 1-tauronic acid methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl- Pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-de-nitro-pseudouridine, 2-thio-1-methyl-1-de-nitro-pseudouridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydrouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 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-aminomethylmethyl-2'-O-methyl-uridine (nc) m5Um), 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-arasu-uridine, 2'-F-uridine, 2'-OH-arasu-uridine, 5-(2-carbonmethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

In some embodiments, the RNA contains other modified nucleosides or further modified nucleosides, such as modified cytidine. For example, in some embodiments, 5-methylcytidine partially or completely, preferably completely, replaces cytidine in the RNA. In some embodiments, the RNA contains 5-methylcytidine and one or more of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the RNA contains 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA contains 5-methylcytidine replacing each cytidine and N1-methyl-pseudouridine (m1ψ) replacing each uridine.

In some embodiments disclosed herein, RNA is referred to as "replicant RNA" or simply "replicant," particularly "self-replicating RNA" or "self-amplifying RNA." In a particularly preferred embodiment, the replicant or self-replicating RNA is derived from or contains elements derived from a single-stranded (ss) RNA virus (particularly a positive-stranded ss RNA virus, such as Alfa virus). Alfa virus is a typical example of a positive-stranded RNA virus. Alfa virus replicates in the cytoplasm of infected cells (for a review of the Alfa virus life cycle, see José et al., Future Microbiol. , 2009, Vol. 4, pp. 837–856, which are incorporated herein by reference in their entirety). The total genome length of many alfa viruses typically ranges from 11,000 to 12,000 nucleotides, and the genome RNA usually has a 5' cap and a 3' poly(A) tail. The alfa virus genome encodes non-structural proteins (involved in viral RNA transcription, modification, and replication, as well as protein modification) and structural proteins (forming viral particles). Two open reading frames (ORFs) are usually present in the genome. The four non-structural proteins (nsP1-nsP4) are usually encoded together by a first ORF located near the 5' end of the genome, while the alfa virus structural proteins are encoded together by a second ORF located downstream of the first ORF and extending near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with Alfa virus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., Vol. 87, pp. 111–124). Post-infection, that is, in the early stages of the viral life cycle, the (+)-stranded genomic RNA is used directly, like messenger RNA, to translate the open reading frame encoding the non-structural multiprotein (nsP1234).

Alfa virus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In a simplified approach, a first ORF encodes an Alfa virus-derived RNA-dependent RNA polymerase (replicaase), which mediates the self-amplification of RNA after translation. A second ORF, encoding an Alfa virus structural protein, is replaced by an open reading frame encoding a protein of interest (e.g., an incretin). The Alfa virus-based trans-replication system relies on Alfa virus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicaase, and the other nucleic acid molecule can be trans-replicated by this replicaase (hence the name trans-replication system). Trans-replication requires the presence of two such nucleic acid molecules in a given host cell. Nucleic acid molecules that can be trans-replicated by a replicase must contain certain Alphavirus sequence elements that allow the Alphavirus replicase to recognize and synthesize RNA.

Characteristics of an unmodified uridine platform may include, for example, one or more inherent adjuvant effects and good tolerability and safety. Characteristics of a modified uridine (e.g., pseudouridine) platform may include reduced adjuvant effects, dulled activation of innate immune receptors, and therefore good tolerability and safety. Characteristics of a self-amplifying platform may include, for example, long duration of protein expression, good tolerability and safety, and the potential for higher efficacy at very low vaccine doses.

This disclosure provides optimized RNA constructs for improving manufacturability, encapsulation, performance levels (and/or timing), etc. Certain components are discussed below, and some preferred embodiments are illustrated herein. Codon Optimization and GC Enrichment

As used herein, the term "codon optimization" refers to altering the codons in the coding region of a nucleic acid molecule (e.g., a polynucleotide) to reflect typical codon usage by the host organism (e.g., an individual receiving the polynucleotide), preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, in some embodiments, coding region codon optimization is used to achieve optimal performance of the RNA molecules described herein in the individual seeking treatment. In some embodiments, codon optimization may be performed such that inserting codons that yield frequently occurring tRNAs replaces "rare codons." In some embodiments, codon optimization may include increasing the G/C content of the RNA coding region described herein compared to the guanosine/cytosine (G/C) content of the corresponding coding sequence of wild-type RNA, wherein the amino acid sequence encoded by the RNA is better unmodified compared to the amino acid sequence.

In some embodiments, the coding sequence (also referred to as the "coding region") is codon-optimized for expression in individuals (e.g., humans) who intend to administer the compound (e.g., a pharmaceutical compound). Therefore, in some embodiments, the sequence in such a polynucleotide (e.g., a polynucleotide) may differ from the wild-type sequence encoding the relevant incretin, even when the amino acid sequence of the incretin is wild-type.

In some embodiments, code optimization strategies are used to represent behavior in relevant individuals (e.g., humans) and even, in some cases, to represent behavior in specific cells or tissues.

Different species exhibit specific preferences for certain codons of particular amino acids. Without being bound by any single theory, codon preference (differences in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), which in turn is considered to depend particularly on the nature of the translated codons and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell generally reflects the most frequently used codons in peptide synthesis. Therefore, codon optimization can be used to tailor genes for optimal gene expression in a given organism. Codon usage tables are available, for example, at the "Codon Usage Database" at www.kazusa.orjp/codon/, and these tables can be adjusted in various ways. Computer algorithms for codon optimization of specific sequences to achieve performance in specific individuals or cells are also available, such as Gene Forge (Aptagen; Jacobus, PA).

In some embodiments, the polynucleotides (e.g., polynucleotides) disclosed herein are codon-optimized, wherein the codons in the polynucleotides (e.g., polynucleotides) are adapted for use with human codons (referred to herein as "human codon-optimized polynucleotides"). Codons encoding the same amino acid occur at different frequencies in individuals (e.g., humans). Therefore, in some embodiments, the encoding sequences of the polynucleotides disclosed herein are modified such that the frequencies of codons encoding the same amino acid correspond to the naturally occurring frequencies of that codon used according to human codons, for example, as shown in Table 13. For example, in the case of amino acid Ala, the wild-type coding sequence is preferably adjusted as follows: codon "GCC" is used at a frequency of 0.40, codon "GCT" at a frequency of 0.28, codon "GCA" at a frequency of 0.22, and codon "GCG" at a frequency of 0.10, etc. (see Table 13). Therefore, in some embodiments, this procedure (as illustrated for Ala) is applied to each amino acid encoded by a polynucleotide coding sequence to obtain a sequence suitable for human codon use. Table 13: Human codon usage table with frequencies indicated for each amino acid. amino acids password frequency amino acids password frequency 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 0 42 Lou CTC 0.20 Tyr TAC* 0.58 Met ATG* 1 Stop TGA* 0 61 Asn AAT 0.44 Stop TAG 0.17 Asn AAC* 0.56 Stop TAA 0.22

Certain strategies for codon optimization and/or G/C enrichment for human performance are described in WO2002/098443, which is incorporated herein by reference in its entirety. In some embodiments, multi-parameter optimization strategies may be used to optimize the coding sequence. In some embodiments, optimization parameters may include parameters that affect protein performance, such as transcriptional levels, mRNA levels, and/or translational levels. In some embodiments, exemplary optimization parameters include, but are not limited to, transcriptional level parameters (including, for example, GC content, common splice sites, recessive splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); mRNA level parameters (including, for example, RNA instability motifs, ribosome entry sites, repetitive sequences, and combinations thereof); translational level parameters (including, for example, codon usage, premature poly(A) sites, ribosome entry sites, secondary structures, and combinations thereof); or combinations thereof. In some embodiments, the encoded sequence can be optimized using the GeneOptimizer algorithm as described in the following literature: 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 contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the encoded sequence can be optimized by the Eurofins adaptation and optimization algorithm "GENEius" compared to other optimization algorithms, as described in Eurofins' Application Notes: Eurofins' adaptation and optimization software "GENEius", the entire contents of which are incorporated herein by reference for the purposes described herein.

In some embodiments, the coding sequence used according to this disclosure has an increased G/C content compared to the wild-type coding sequence of the relevant incretin. In some embodiments, the guanosine/cytidine (G/C) content of the coding region is modified relative to the wild-type coding sequence of the relevant incretin, but the amino acid sequence encoded by the polynucleotide is not modified.

Without being bound by any particular theory, it is proposed that GC enrichment can improve the translation of rewarded sequences. Generally, sequences with increased G (guanosine)/C (cytidine) content are more stable than sequences with increased A (adenosine)/U (uridine) content. The fact that several codons encode the same amino acid (so-called genetic code merging) allows for the identification of the most favorable codons for stability (so-called alternative codon usage). Based on the amino acid encoded by a polynucleotide, there are various possibilities for modifying ribonucleic acid sequences compared to their wild-type sequences. In particular, codons containing A and/or U nucleotides can be modified by replacing them with other codons encoding the same amino acid but lacking A and/or U, or containing lower A and/or U nucleotide content.

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

In some embodiments, the stability and transduction efficiency of the polynucleotide may be incorporated into one or more elements established to contribute to the stability and/or transduction efficiency of the polynucleotide; exemplary such elements are described, for example, in WO2007/036366, which is incorporated herein by reference. In some embodiments, to enhance the performance of the polynucleotide used according to this disclosure, the polynucleotide may be modified within its coding region (i.e., the sequence encoding the peptide or protein to be expressed) without altering the sequence of the expressed peptide or protein, for example, to increase GC content to increase mRNA stability and/or to perform codon optimization, and thus enhance transduction in cells. Exemplary Polynucleotide Encoding Incretin Agents

In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to any of the sequences shown in Table 14 below. In addition to sequences encoding incretins and signal peptides, the polynucleotide includes the exemplary polynucleotide features described herein, including the proximal cap sequence (AAUA), the 5' UTR sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 49), the polyA tail sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAA ... UTR sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC (SEQ ID NO: 51). Table 14: Exemplary polynucleotides encoding incretins, where examples x2 and x4 include linkers between repeating units and furin cleavage sites. SEQ ID NO Incretins sequence 257 GLP1 (7-37) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGCUGGUGCUGCUGGCUUGUCUGGCCGCUGCCUCUAAUGCUCAUGCCGAGGGCACCUUUACCAGCGACGUGUCCUCUUACCUGGAAGGCCAG GCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCC ACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCC CAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 258 GLP1 with K34R mutation (7-37) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGCUGGUGCUGCUGGCUUGUCUGGCCGCUGCCUCUAAUGCUCAUGCCGAGGGCACCUUUACCAGCGACGUGUCCUCUUACCUGGAAGGCCAG GCCGCCAAAGAGUUUAUCGCCUGGCUCGUCAGAGGCAGAGGAUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCC ACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCC CAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 259 GIP (1-42) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGCUGGUGCUGCUGGCUUGUCUGGCCGCUGCCUCUAAUGCCUAUGCCGAGGGCACCUUCAUCAGCGACUACUCUAUCGCCAUGGACAAGAUCCACCAGCAG GACUUCGUGAACUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAACAUCACCCAGUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAG GUAUGCUCCCACCUCCACCUGCCCCACACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAU ACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 260 GLP1 with A2G mutation (7-37) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCUCAUGGCGAGGGCACCUUCACCUCCGAUGUGUCC UCUUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAU GCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 261 GLP1 (7-37) connector with A2G mutation GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCUCAUGGCGAGGGCACCUUCACCUCCGAUGUGUCCUCUUAC CUGGAAGGCCAGGCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCGGCGGAGGAAGUGGCGGAGGAUCUUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCG GGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAG CUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 262 GIPs with A2G mutations (1-42) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAGACAUGGGCUUAUGGCGAGGGCACCUUCAUCAGCGACUACUCUAUCGCCAUG GACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAACAUCACCCAGUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGAC CUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUA AGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 263 husec SP, GIP (1-30) (Variant 1 or "opt1" optimized by the code) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUCGGACACUGAUCCUGCUGCUUUCUGGUGCCCUGGCUCUGACAGAGACAUGGGCUUAUGGCGAGGGCACCUUCAUCAGCGACUACUCU AUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAAUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGC UCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACU AACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 264 husec SP, GLP1 (7-37) with A2G mutation GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUCGGACACUGAUUCUGCCGCUGUGGUGCCCUGGCUCUGACAGAAACAUGGGCCCACGGCGAGGGCACCUUUACCAGCGAUGUGUCU UCUUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCUUGGCUUGUGAAAGGCAGAGGCUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAU GCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 265 husec SP, GLP1 (7-37) connector with A2G mutation GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUCGGACACUGAUUCUGCCGCUGUGGUGCCCUGGCUCUGACAGAAACAUGGGCCCACGGCGAGGGCACCUUUACCAGCGAUGUGUCUUCUUAU CUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCUUGGCUUGUGAAAGGCAGAGGUGGUGGCGGAUCUGGCGGAGGAUCUUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCG GGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAG CUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 266 husec SP, GIP with A2G mutation (1-42) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUCGGACACUGAUUCUGCCGCUGUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCCUAUGGCGAGGGCACCUUCAUCAGCGACUACAGCAUCGCCAUG GACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAACAUCACCCAGUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGAC CUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUA AGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 267 husec SP, GIP (1-30) (Variant 1 or "opt1" optimized by the code) GAAUAAACUAGUAUUCUUCUGCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUCGGACACUGAUUCUGCCGCUGUCUGGUGCCCUGGCUCUGACAGAAACAUGGGCCUAUGGCGAGGGCACCUUCAUCAGCGACUACAGC AUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAACUGGCUGCUGGCCCAGAAAUAAUAGGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGC UCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACU AACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 268 husec SP, GLP1 (7-37) (a codec-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUCACGCCGAGGGCACCUUUACAAGCGACGUGUCC AGCUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAU GCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 269 husec SP, GIP (1-42) (a variant 1 or "opt1" optimized with cryptography) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCCUACGCCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUG GACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGAC CUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUA AGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 270 husec SP, GLP1 (7-37) with H7Y and A8G mutations (codec-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCC AGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUGAAAGGCAGAGGCUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAU GCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 271 gD1 SP, GLP1 (7-37) (Variant 1 or "opt1" optimized by the codeword) GAAUAAACUAGUAUUCUUCGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUGUGGUCAUCGUUGGACUGCACGGCGUUAGAGGACACGCCGAGGGAACCUUUACAAGCGACGUGU CCAGCUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUA UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUA CUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 272 gD1 SP, GLP1 (7-37) with K34R mutation (codec-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUCGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUGUGGUCAUCGUUGGACUGCACGGCGUUAGAGGACACGCCGAGGGAACCUUUACAAGCGACGUGU CCAGCUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUCGUCAGAGGCAGAGGAUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUA UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUA CUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 273 gD1 SP, GLP1 (7-37) with A8G mutation (codec-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUGUGGUCAUCGUUGGACUGCACGGCGUUAGAGGACACGGCGAGGGAACCUUUACAAGCGACGUGU CCAGCUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUA UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUA CUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 274 gD1 SP, GLP1 (7-37) with H7Y and A8G mutations (codecoin-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUCGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUGUGGUCAUCGUUGGACUGCACGGCGUCAGAGGAUACGGCGAGGGAACCUUUACAAGCGACGUGU CCAGCUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUGGUCAAAGGCAGAGGCUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUA UGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUA CUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 275 gD1 SP, GIP (1-42) (Variant 1 or "opt1" optimized by the code) GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUGUGGUCAUCGUUGGACUGCACGGCGUCAGAGGAUACGCCGAGGGCACCUUCAUCAGCGACUACAGCAUCGCC AUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCG ACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACU AAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 276 gD1 SP, GIP (1-42) with A2G (a cryptographically optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUCGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGGCGGAGCUGCUGCUAGACUGGGAGCCGUGAUUCUGUGUGGUCAUCGUUGGACUGCACGGCGUCAGAGGAUACGGCGAGGGCACCUUCAUCAGCGACUACAGCAUCGCC AUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCG ACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACU AAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 277 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations - linker-furin protease-GIP (1-42) (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCU UGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGCCGAGGGAACCUUUAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAGUAAUGAGGAUC CGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCA GUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 278 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCU UGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAGUAAUGAGGAUC CGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCA GUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 279 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x2 GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCU UCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACA UUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGCCGGAGGUUC AAACGUUCGCCGCAAAAGAUACGGCGAGGGAACUUUCACCUCCUGACGUGUCCUCUUACCUCGAAGGACAGGCCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAGGCGGUGGUGGAAGCGGAGGUGGUGGCA GUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCUCCGACUACUCCAUUGCUAUGGAUAAGAUUCAUCACAGGAUUUCGUCAACUGGCUCCUCGCUCAGAAAGGGAAGAAAAAACGAUUGGAAACAUAACAUC ACCCAGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGC UAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCA AUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 280 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUG GUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGCCGGAGGUUCAAACGUUCGCCGCAAAA GAUACGGCGAGGGAACUUUCACCUCCUGACGUGUCCUCUUACCUCGAAGGACAGCCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAGGCGGUGGGAAGCGGAGGUGGUGGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCCCGACUACUCCAUUGCUAUGGAUAAGAUUCAUCACAGGAUUUCGUCAACUGGC UCCUCGCUCAGAAAGGGAAGAAAAACGAUUGGAAACAUAACAUCACGCAAGGCGGUGGCGGUUCAGGUGGUGGGUUCUAACGUCCGGCGGAAACGUUACGGCGAGGGAACCUUUACAUCAGACGUUUCAUCCUACCUUGAGGGGCAAGCUGCAAAAGAGUUUAUUGCCUGGCUCGAAAGGUGGUGGCGGUGGCGGGAGGUAGCGGAGGCGGCGGAA GCAACGUUCGAAGAAAACGAUACGGCGAGGGGACCUUCAUCUCCGAUUAUAGUAUCGCUAUGGACAAAAUUCAUCAGCAAGACUUUGUUAACUGGUUGCUCGCCCAAAAGGGGAAAAAGAACGAUUGGAAGCACAACAUUACACAAGGCGGAGGUGGUAGUGGCGGAGGUGGAAGUAACGUACGACGGAAAAGAUACGGCGAAGGCACCUUCACCUCC GACGUUUCAAGUUACUUGGAGGGACAAGCAGCCAAAGAAUUCAUAGCCUGGCUCGUGAAAGCGGCGGGAGGUGGUGGCGGUAGUGGUGGUGGCGGCUCAAACGUUCGGAGGAAAAGAUACGGCGAGGGCACUUUUAUUAGCGAUUACUCUAUCGCAAUGGAUAAGAUACACCAACAAGACUUUGUCAAUUGGCUCUUGGCACAAAAAGGCAAGAAGAAC GAUUGGAAACACAAUAUCACACAGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACAC CCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 281 husec SP, GLP1 (7-37)-linker-Dula_IgG4 with H7Y, A8G and R36G mutations (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAG GCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGCUGAAAGCAAAUACGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUC CCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUA CAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGA AAUGACCAAGAAUCAGGUGUCCCUGACCUGCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACCUCCUGGUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACA AGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGUAGCGUGAUGCACGAGGCCCUGCACAAUCACUACACCCAGAAGUCCCUGAGCCUGUCUGGGAUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUC UCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUU AACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 282 husec SP, GLP1 (7-37)-linker-Dula_IgG4 with H7Y, A8G, R36G mutations (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAG GCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGCUGAAAGCAAAUACGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUC CCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUA CAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGA AAUGACCAAGAAUCAGGUGUCCCUGACCUGCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACCUCCUGGUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACA AGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUCUGUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGAUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUC UCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUU AACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 283 husec SP, GIP (1-42) with A2G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAU CCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGUGGAUCUGCCGAGUCUAAAUACGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCU GGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAG GAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGC CUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGGUCCCUGACCUGCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGGGAGAGCAACGGACAGCCUGAACAAUUACAAGACCACACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGAC UGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGUAGCGUGAUGCACGAGGCCCUGCACAAUCACUACACCCAGAAGUCCCUGAGCCUGUCUCUGGGAUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUAC CCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAA GUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 284 husec SP, GIP (1-42) with A2G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAU CCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGUGGAUCUGCCGAGUCUAAAUACGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCU GGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAG GAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCUGCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGC CUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGGUCCCUGACCUGCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGGGAGAGCAACGGACAGCCUGAACAAUUACAAGACCACACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGAC UGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUCUGUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGAUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUAC CCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAA GUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 285 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - connector - FcKIH-a (code-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAA GGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGAGGCGGAGGAAGCGGUGGCGGCGGAUCUGGUGGCGGAGGUUCUGAUAAGACCCACCUGUCCACCUUGUCCUGCUCCAGAGAGCACAAGAGGCCCUAGCGUGUUCUUGUUCCCUCCAAAGC CUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUGUCCCACGAAGAUCCCGAAGUGAAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUACAACAGCACCUACAGAGUGGU GUCCGUGCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCCUAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCACCUUGCAGAGAAGAAAUGACCA AGAAUCAGGUGUCCCUGUGGGCCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAAGCUGACAGUGGACAAGAGCAG GUGGCAGCAGGGCAACGUGUUCAGCUGUUCUGUGCUGCACGAGGCCCUGCACAGCCACUACACACAGAAGUCCCUGAGCCUGUCCUGGCAAGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUC CCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUA ACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 286 husec SP, GIP (1-42) with A2G mutation - connector - FcKIH-b (code-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGA UCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGUGGAUCUGAUAAGACCCACCUGUCCACCUUGUCCUGCUCCAGAGAGCACAAGAGGCC CUAGCGUGUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGGACGUGUCCCACGAAGAUCCCGAAGUGAAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGU ACAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGCCCUGCCUGCUCCUAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAGCCUCAAGUCUGUACCCUGCCUCCUA GCAGAGAAGAAAUGACCAAGAAUCAGGGUCCCUGAGCUGCGCCGUGAAGGGCUUCUACCCUAGCGAUAUUGCCGUCGAGUGGGAGAGCAACGGCCAGCCUGAGAACAAUUACAAGACCACACCUCCUGCUGGACAGCGACGGCUCAUUCUUCCUGGUGUCCAAGCUGACA GUGGACAAGAGCAGGUGGCAGCAGGGCAACGUGUUCAGCUGUUCUGUGCUGCACGAGGCCCUGCACAGCCACUACACAGAAGUCCCUGUCUCUGAGCCCUGGCAAGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACC CCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAG UUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 287 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - connector - hAlbumin (code-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGAC GUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGCGGAGGAAGCGGAGGCGGCGGAUCUGACGCUCACAAAUCUGAAGUGGCCCACAGAUUCAAGGACCU GGGCGAAGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUGUGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGA GCCUGCACACCCUGUUCGGCGACAAGCUGUGUACAGUGGCCACACUGAGAGAAACCUACGGCGAGAUGGCCGACUGCUGCGCCAAACAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUC GUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCAC CGAGUGUUGUCAGGCCGCUGAUAAGGCCGCUUGCCUGCUGCCUAAACUGGACGAGCUUCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUG CUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCGUGACCGACCUGACAAAGGUGCACACCGAGUGCUGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAU CAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAGGUGGAAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAA UUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCCGCCGCUGAUCCUCACGAGU GUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUCC ACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUUACCUGUCUGGGGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCUGUGUC CGAUAGAGUGACCAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCAAAAGAGUUUAACGCCGAGACCUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAGA GACAGAUCAAGAAACAGACUGCCCUGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCAAAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGUUUCGCCGAAGAA GGCAAGAAACUGGUGGCCGCUUCUCAGGCUGCUGGGACUGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCC ACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCC CAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 288 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - connector - aHSA-VHH (code-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUCGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGC GAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGCGGAGGAAGCGGAGGCGGCGGAUC UGAAGUUCAGCUGCUUGAAUCAGGCGGAGGCCUGGUUCAACCUGGCGGAUCUCUGAGACUGAGCUGUGCCGCCUCUGGCUUCACCCUGGAUUAUUACGCCAUCGGCUGGUUCAGACAGGCCCCUGGCAAA GAGAGAGGGCGUCAGCUGUAUUGCCAGCAGCGGCGGCUCUACCAAUUACGCCGAUAGCGUGAAGGGCAGAUUCACCAUCAGCAGAGACAACAGCAAGAACACCGUGUACCUCCAGAUGAACAGCCUGAA GCCUGAGGACACCGCCGUGUACUAUUGUGCCGCAGCCGUGCUUGAGUGCAGAACAGUUGUGCCGGGGCUACGACUAUUGGGGCCAGGGAACACAAGUGACCGUGUCUUCUUAAUGAGGAUCCGAUCUGGUA CUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCC AAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 289 husec SP, GLP-1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAU CGCCUGGCUUGUAAAAGGCGGCGGUGGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAU CACCCAAGGUGGCGGUGGAAGUGGCGCGGGAGGAAGCGGAGGCGGCGGAUCUGCUGAAAGCAAAUACGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGU GGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCU GCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGUGUCCCUGACCUGCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAA GACCACACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGUAGCGUGAUGCACGAGGCCCUGCACAAUCACUACACCCAGAAGUCCCUGAGCCUGUCUCGGAUAAUGAGGAUCCGAUCUGGUACUGCAUGC ACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAAC CUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 290 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAU CGCCUGGCUUGUAAAAGGCGGCGGUGGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAU CACCCAAGGUGGCGGUGGAAGUGGCGCGGGAGGAAGCGGAGGCGGCGGAUCUGCUGAAAGCAAAUACGGCCCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUGGU GGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCU GCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGUGUCCCUGACCUGCCUGGUCAAGGGCUUCUACCCUAGCGACAUUGCCGUCGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAA GACCACACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGAUAAUGAGGAUCCGAUCUGGUACUGCAUGC ACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAAC CUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 291 husec SP, GIP (1-42) with A2G mutation - linker - furin protease - GLP1 with H7Y, A8G, R36G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGU GAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCACAAGCGACGUGUCCAGCUACCUGGAAGGCCAGCCCGCCAAAGAGUUUAUCGCCUGGCUUGUGAAAGG CGGUGGUGGUGGCGGAGGAAGCGGUGCGGGAGGUUCAGGUGGCGGUGGAUCUGCCGAGAGCAAAUACGGACCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCUCCAAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUCGU GGACGUUUCCCAAGAGGACCCUGAGGUGCAGUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACAAGGGCCU GCCUAGCAGCAUCGAGAAGACCAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGUGUCCCUGACCUGUCUGGUCAAGGGCUUCUACCCUAGCGACAUCGCCGUUGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAA GACCACACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUGCUGCACGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGAUAAUGAGGAUCCGAUCUGGUACUGCAUGC ACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAAC CUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 292 husec SP, [GLP1-linker-furin protease-GIP (1-42)- with H7Y, A8G, R36G mutations] x4-linker-Dula_IgG4 (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUCGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGU CCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGAC AAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAGGUUCAAACGUUCGCCGCAAAAGAUACGGCGAGGGAACUUUCACCUCUGA CGUGUCCUCUUACCUCGAAGGACAGGCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAGGCGGUGGGAAGCGGAGGUGGGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCUCCGACUACUCCAUUGCUA UGGAUAAGAUUCAUCAACAGGAUUUCGUCAACUGGCUCCUCGCUCAGAAAGGGAAGAAAAACGAUUGGAAACAUAACAUCACGCAAGGCGGUGGCGGUUCAGGUGGUGGGGUUCUAACGUCCGGCGGAAACGUUACGGCGAGGGAACCUUUACA UCAGACGUUUCAUCCUACCUUGAGGGGCAAGCUGCAAAAGAGUUUAUUGCCUGGCUCGAAAGGUGGUGGCGGUGGCGGAGGUAGCGGAGGCGGCGGAAGCAACGUUCGAAGAAAACGAUACGGCGAGGGGACCUUCAUCUCCGAUUAUAGUAU CGCUAUGGACAAAAUUCAUCAGCAAGACUUUGUUAACUGGUUGCUCGCCCAAAAGGGGAAAAAGAACGAUUGGAAGCAACAUACACAAGGCGGAGGUGGUAGUGGGCGGAGGUGGAAGUAACGUACGACGGAAAAGAUACGGCGAAGGCACCU UCACCUCCGACGUUUCAAGUUACUUGGAGGGACAAGCAGCCAAAGAAUUCAUAGCCUGGCUCGUGAAAGGCGGCGGAGGUGGUGGCGGUAGUGGUGGGGCGGCUCAAACGUUCGGAGGAAAAGAUACGGCGAGGGCACUUUUAUUAGCGAUUACU CUAUCGCAAUGGAUAAGAUACACCAACAAGACUUUGUCAAUUGGCUCUUGGCACAAAAAGGCAAGAAGAACGAUUGGAAACACAACAUAACCCAAGGCGGCGGUGGUUCAGGUGGCGGAGGAUCAGGUGGUGGCGGAUCUGCCGAGAGCAAAUAC GGACCUCCUUGUCCACCUUGUCCUGCUCCAGAAGCUGCUGGCGGCCCUAGCGUUUUCUUGUUCCCACCUAAGCCUAAGGACACCCUGAUGAUCAGCAGAACCCCUGAAGUGACCUGCGUGGUCGUGGACGUUUCCCAAGAGGACCCUGAGGUGCA GUUCAAUUGGUACGUGGACGGCGUGGAAGUGCACAACGCCAAGACCAAGCCUAGAGAGGAACAGUUCAACAGCACCUACAGAGUGGUGUCCGUGCCUGACCGUGCUGCACCAGGAUUGGCUGAACGGCAAAGAGUACAAGUGCAAGGUGUCCAACA AGGGCCUGCCUAGCAGCAUCGAGAAGACAAUCAGCAAGGCCAAGGGCCAGCCAAGAGAACCUCAAGUGUACACCCUGCCUCCAAGCCAAGAGGAAAUGACCAAGAAUCAGGUGUCCCUGACCUGUCUGGUCAAGGGCUUCUACCCUAGCGACAUC GCCGUUGAGUGGGAGAGCAACGGACAGCCUGAGAACAAUUACAAGACCACCUCCUGUGCUGGACAGCGACGGCUCAUUCUUCCUGUACAGCAGACUGACCGUGGACAAGAGCAGGUGGCAAGAGGGCAACGUGUUCAGCUGCUCUGUGCUGCA CGAAGCCCUGCACAGCCACUACACCCAGAAGUCUCUGAGCCUGAGCCUGGGAUAAUGAGGAUCCGAUCUGGUACUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCC CACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUA ACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 293 husec SP, GIP with A2G mutation (1-42) - connector - hAlbumin (code-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCAUCAGCGAUUAC AGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCGGAGGUGGAAGCGGAGGCGGUGGAUCUGACGCCCACAAAU CUGAAGUGGCCCACAGAUUCAAGGACCUGGGCGAAGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUGUGUGGCCGA CGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGACAAGCUGUGUACAGUGGCCACACUGAGAGAAACCUACGGCGAGAUGGCCGACUGCUGCGCCAAACAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGAC GACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCA AGAGAUACAAGGCCGCCUUCACCGAGUGUUGCCAGGCCGCUGAUAAGGCCGCUUGUCUGCUGCCUAAACUGGACGAGCUGCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCGCCAGCCUCCAGAAGUUUGGCGAGAGAGC CUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCGUGACCGACCUGACAAAGGUGCACACCGAGUGCUGUCACGGCGACCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCC AAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAGGUGGAAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCA AGGACGUGUGCAAGAAUUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCCGCCGC UGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUG CCACAGGUCUCCACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUUACCUGUCUGUGGUGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGA CCCCUGUGUCCGAUAGAGUGACCAAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCCAAAGAGUUCAACGCCGAGACCUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGA GAAAGAGAGACAGAUCAAGAAACAGACUGCCCUGGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCAAAGAACAACUGAAGGCCGUGAUGGACGACUUCGCCGCCUUUGUCGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGUUUCGCC GAAGAAGGCAAAAAGCUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCAC CUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAAC CCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 294 husec SP, GIP (1-42) with A2G mutation - connector - aHSA-VHH (code-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUCGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGG GCACCUUCAUCAGCGAUUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGCGGCGGAGGAUCUGGCG GAGGUGGAAGUGGCGGAGGCGGAUCUGAAGUUCAGCUGCUUGAAUCUGGCGGCGGACUGGUUCAACCUGGCGGAUCUCUGAGACUGAGCUGUGCCGCCUCUGGCUUCACCCUGGAUUAUUACGCCAUCGGCUGGU UCAGACAGGCCCCUGGCAAAGAGAGAGGGCGUCAGCUGUAUUGCCAGCAGCGGCGGCUCUACCAAUUACGCCGAUAGCGUGAAGGGCAGAUUCACCAUCAGCAGAGACAACAGCAAGAACACCGUGUACCUCC AGAUGAACAGCCUGAAGCCUGAGGACACCGCCGUGUACUAUUGUGCCGCAGCCGUGCUUGAGUGCAGAACAGUUGUGCGGGGCUACGACUAUUGGGGCCAGGGAACACAAGUGACCGUGUCCUCUUAAUGAGGA UCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGA CACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 295 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker-furin protease-GIP (1-42) - linker-hAlbumin (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUA UCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGCCGAGGGAACCUUUAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAG GACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAAGAAGCGGAGGGCGGAUCUGACGCUCACAAAUCUGAAGUGGCCCACAGAUUCAAGGACCUGGGCGAAGAAA AUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUGUGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGA CAAGCUGUGUACAGUGGCCACACUGAGAGAAACCUACGGCGAGAUGGCCGACUGCUGCGCCAAACAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGC CUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCGCUUGCCUGCUGCCU AAACUGGACGAGCUUCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCG UGACCGACCUGACAAAGGUGCACACCGAGUGCUGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUG CAUCGCCGAGGUGGAAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAAUUACGCCGAAGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGU GGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCCGCCGCUGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAG CUGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUCCACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUU ACCUGUGUGGUGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCAAAAGAGUUUAACGCCGAGAC CUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAACGGCAGAUCAAGAAACAGACUGCCCUGGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCAAAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAG GCUGACGACAAAGAGACCUGUUUCGCCGAAGAAGGCAAAAAGCUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGG UCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 296 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation - linker - hAlbumin (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUA UCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAG GACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAAGAAGCGGAGGGCGGAUCUGACGCUCACAAAUCUGAAGUGGCCCACAGAUUCAAGGACCUGGGCGAAGAAA AUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUGUGUGGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGA CAAGCUGUGUACAGUGGCCACACUGAGAGAAACCUACGGCGAGAUGGCCGACUGCUGCGCCAAACAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGC CUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCGCUUGCCUGCUGCCU AAACUGGACGAGCUUCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCG UGACCGACCUGACAAAGGUGCACACCGAGUGCUGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUG CAUCGCCGAGGUGGAAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAAUUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGU GGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAAAAGUGCUGUGCCGCCGCUGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAG CUGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUCCACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUU ACCUGUGUGGUGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCAAAAGAGUUUAACGCCGAGAC CUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAACGGCAGAUCAAGAAACAGACUGCCCUGGGGGAACUGGUCAAGCACAAGCCUAAGGCCACCAAAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAG GCUGACGACAAAGAGACCUGUUUCGCCGAAGAAGGCAAAAAGCUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGG UCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 297 husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x2 - linker - hAlbumin (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCA AAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUGGCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGA ACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGGCGGAGGUUCAAACGUUCGCCGCAAAAGAUACGGCGAGGGAACUUUCACCCUGACGUGUCCUCUUACCUCGAAGGACAGGCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAGGCGGUGGUGGAAGC GGAGGUGUGGGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCUCCGACUACUCCAUUGCUAUGGAUAAGAUUCAUCACAGGAUUUCGUCAACUGGCUCCUCGCUCAGAAAGGGAAGAAAAACGAUUGGAAACAUAACAUCACGCAAGGCGGUGGCGGUUCUGGUGGCGGU GGCAGCGGAGGCGGCGGAUCUGACGCUCACAAAUCUGAAGUGGCCCACAGAUUCAAGGACCUGGGCGAAGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUGUGU GGCCGACGAGAGCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGACAAGCUGUGUACAGUGGCCACACUGAGAGAAACAUACGGCGAGAUGGCCGACUGCUGCCAAACAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACU CGUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCG CUUGCCUGCUGCCUAAACUGGACGAGCUUCGCGACGAGGGAAAAGCCAGCUCUGCCAAGCAGAGACUGAAGUGCCAGCCUCCAGAAGUUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCGUGACCG ACCUGACAAAGGUGCACACCGAGUGCUGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGCGAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAGGUGGAAAACGACGAG AUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAAUUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUACGAGACCACACUGGAA AAGUGCUGUGCCGCCGCUGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUC CACACCUACACUGGUCGAAGUGUCCAGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUUACCUGUCUGUGGGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAGUGCUGUACCGAGAG CCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCAAAAGAGUUUAACGCCGAGACCUUCACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAACGGCAGAUCAAGAAACAGACUGCCCUGGUGGAACUGGUCAAGCACAAGCCUAAGGCCACCA AAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGUUUCGCCGAAGAAGGCAAAAAGCUGGUGGCCGCUUCUCAGGCUGCUCUGGGACUGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCU UUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAU AAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 298 husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 - linker - hAlbumin (codon-optimized variant 1 or "opt1") GAAUAAACUAGUAUUCUUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAGAGUGAUGGCCCCUAGAACACUGAUCCUGCUGCUGUCUGGUGCCCUGGCUCUGACAGAAACUUGGGCUUACGGCGAGGGCACCUUCACAAGCGACGUGUCCAGCUAUCUGGAAGGCCAGGCCGCCAAAGAGUUCAUCGCCUGGCUUGUAAAAGGCGGCGGUGGUG GCGGAGGAUCUGGCGGAGGCGGAUCUAACGUGCGGAGAAAGAGAUACGGCGAAGGGACAUUCAUCAGCGACUACAGCAUCGCCAUGGACAAGAUCCACCAGCAGGACUUCGUGAAUUGGCUGCUGGCCCAGAAGGGCAAGAAGAACGACUGGAAGCACAAUAUCACCCAAGGUGGCGGUGGAAGUGGCGCCGGAGGUUCAAACGUUCGCCGCAAAAGAUACG GCGAGGAACUUUCACCUCCUGACGUGUCCUUACCUCGAAGGACAGGCUGCUAAAGAAUUCAUUGCUUGGCUGGUCAAAGGCGGCGGAGGCGGUGGUGGAAGCGGAGGUGGUGGCAGUAACGUUCGUAGGAAACGCUACGGCGAAGGCACUUUUAUCCCGACUCCAUUGCUAUGGAUAAGAUUCAUCAACAGGAUUUCGUCAACUGGCUCCUCGCUC AGAAAGGGAAGAAAACGAUUGGAAACAUAACAUCACGCAAGCGGUGGCGGUUCAGGUGGUGGGGUUCUAACGUCCCGGCGGAAACGUUACGGCGAGGGAACCUUUACAUCAGACGUUUCAUCCUACCUUGAGGGGCAAGCUGCAAAAGAGUUUAUUGCCUGGCUCGAAAGGUGGUGGCGGUGGCGGAGGUAGCGGAGGCGGCGGAAGCAACGUUCGAA GAAAACGAUACGGCGAGGGGACCUUCAUCUCCGAUUAUAGUAUCGCUAUGGACAAAAUUCAUCAGCAAGACUUUGUUAACUGGUUGCUCGCCCAAAAGGGGAAAAAGAACGAUUGGAAGCACAACAUACACAAGGCGGAGGUGGUAGUGGCGGAGGUGGAAGUAACGUACGACGGAAAAGAUACGGCGAAGGCACCUUCACCUCCGACGUUUCAAGUUACU UGGAGGGACAAGCAGCCAAAGAAUUCAUAGCCUGGCUCGUGAAAGGCGGCGGAGGUGGUGGCGGUAGUGGUGGUGGCGGCUCAAACGUUCGGAGGAAAAGAUACGGCGAGGGCACUUUAUUAGCGAUUACUCUAUCGCAAUGGAUAAGAUACACCAACAAGACUUUGUCAAUUGGCUCUUGGCACAAAAAGGCAAGAAGAACGAUUGGAAACACAACAUAA CCCAAGGCGGCGGUGGUUCAGGUGGCGGGAGGAUCAGGUGGUGGCGGAUCUGACGCCCACAAAUCUGAAGUGGCCCACAGAUUCAAGGACCUCGGCGAGGAAAAUUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAACAGUGCCCUUUCGAGGACCACGUGAAGCUGGUCAACGAAGUGACCGAGUUCGCCAAGACCUGUGUGGCCGACGAGA GCGCCGAGAAUUGCGAUAAGAGCCUGCACACCCUGUUCGGCGACAAGCUGUGUACAGUGGCCACACUGAGAGAAACAUACGGCGAAAUGGCCGACUGCUGCGCCAAGCAAGAGCCCGAGAGAAACGAGUGCUUCCUCCAGCACAAGGACGACAAUCCUAAUCUGCCUAGACUCGUGCGGCCUGAGGUGGACGUGAUGUGUACAGCCUUCCACGACAACGAGG AAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCCAGAAGACACCCUUACUUCUACGCCCCUGAGCUGCUGUUCUUUGCCAAGAGAUACAAGGCCGCCUUCACCGAGUGUUGUCAGGCCGCUGAUAAGGCCGCUUGCCUGCUGCCUAAACUGGACGAGCUUCGCGACGAGGGAAAAGCCAGCAGCGCCAAACAGAGACUGAAGUGCGCCAGCCUCCAGAAG UUUGGCGAGAGAGCCUUUAAGGCCUGGGCCGUUGCUAGACUGAGCCAGAGAUUCCCUAAGGCCGAGUUUGCCGAGGUGUCCAAGCUCGUGACCGACCUGACAAAGGUGCACACCGAGUGCUGCCACGGCGAUCUGCUUGAGUGUGCCGACGACAGAGCUGACCUGGCCAAGUACAUCUGCGAGAAUCAGGACAGCAUCAGCAGCAAGCUGAAAGAGUGCUGC GAGAAGCCUCUGCUGGAAAAGAGCCACUGCAUCGCCGAAGUGGAAAACGACGAGAUGCCUGCCGAUCUGCCUUCUCUGGCCGCCGAUUUCGUGGAAAGCAAGGACGUGUGCAAGAAUUACGCCGAGGCCAAAGACGUGUUCCUGGGCAUGUUCCUGUACGAAUACGCUAGACGGCACCCUGACUACAGCGUGGUGCUGCUGCUGAGACUGGCCAAAACCUAC GAGACCACACUGGAAAAGUGCUGUGCCGCCGCUGAUCCUCACGAGUGUUACGCCAAAGUGUUCGACGAGUUCAAGCCACUGGUGGAAGAACCUCAGAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUCCAGAACGCCCUGCUCGUGCGGUACACCAAGAAGGUGCCACAGGUCUCCACACCUACACUGGUCGAAGUGUCC AGAAAUCUGGGCAAAGUGGGCAGCAAGUGCUGCAAGCACCCUGAGGCCAAGAGAAUGCCUUGCGCCGAGGAUUACCUGUCUGUGGGCUGAAUCAGCUGCGUGCUGCACGAGAAGACCCCUGUGUCCGAUAGAGUGACCAAAGUGCUGUACCGAGAGCCUCGUGAAUAGAAGACCUUGCUUCAGCGCCCUGGAAGUGGACGAGACCUACGUGCCAAAAGAA UUCAACGCCGAGACCUUUACCUUCCACGCCGACAUCUGUACCCUGAGCGAGAAAGAACGGCAGAUCAAGAAACAGACUGCCCUGGUGGAACUGGUCAAGCACAAGCCUAAGGCCACCAAAGAACAACUGAAGGCCGUGAUGGACGAUUUCGCCGCUUUCGUCGAGAAGUGUUGCAAGGCUGACGACAAAGAGACCUGCUUUGCCGAAGAAGGCAAAAAGCUG GUGGCCGCCUCUCAAGCUGCUCUGGGACUGUAAUGAGGAUCCGAUCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCA CACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUCGAGCUAGCAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA RNA delivery technology

The provided polynucleotides can be delivered for the therapeutic applications described herein using any suitable method known in this art, including, for example, delivery as naked RNA, or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, etc.) and/or peptide-based vectors. See, for example, Wadhwa et al. , “Opportunities and Challenges in the Delivery of mRNA-Based Vaccines”, Pharmaceutics (2020) 102 (27 pages), the contents of which are incorporated herein by reference for information on various methods that can be used to deliver the polynucleotides described herein.

In some embodiments, one or more polynucleotides may be formulated together with lipid nanoparticles for delivery (e.g., dosing).

In some embodiments, lipid nanoparticles may be designed to protect polynucleotides from extracellular RNases and/or engineered for the systematic delivery of RNA to target cells (e.g., liver, intestinal, or pancreatic cells). In some embodiments, these lipid nanoparticles are particularly suitable for delivering polynucleotides when administered intraperitoneally, intravenously, or intramuscularly. Particles for delivering at least one polynucleotide

The polynucleotides provided herein can be delivered via particles. In the context of this disclosure, the term "particle" refers to a structured entity formed by molecules or molecular complexes. In some embodiments, the term "particle" refers to a micrometer-sized or nanometer-sized structure, such as a micrometer-sized or nanometer-sized compact structure dispersed in a medium. In some embodiments, the particle is a particle containing nucleic acids, such as a particle containing polynucleotides.

Electrostatic interactions between positively charged molecules (such as polymers and lipids) and negatively charged nucleic acids (e.g., polynucleotides) participate in particle formation. This leads to the complexation and spontaneous formation of nucleic acid particles (e.g., ribonucleic acid particles). In some embodiments, the nucleic acid particles (e.g., ribonucleic acid particles) are nanoparticles.

"Nucleic acid particles" (e.g., ribonucleic acid particles) are particles that encompass or contain nucleic acids and are used to deliver nucleic acids (e.g., polynucleotides) to target sites of interest (e.g., cells, tissues, organs, and similar sites). Nucleic acid particles (e.g., ribonucleic acid particles) can be formed from: (i) at least one cationic or cationically ionizable lipid or lipid-like material, (ii) at least one cationic polymer (such as protamine), or a mixture of (i) and (ii), and (iii) nucleic acids (e.g., polynucleotides). Nucleic acid particles (e.g., ribonucleic acid particles) include multiple lipid nanoparticles (single lipid nanoparticles) and lipid complexes (LPX).

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

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

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

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

The nucleic acid particles (e.g., ribonucleic acid particles) described herein can be prepared using a variety of methods, which 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.

As used herein, the term "colloid" refers to a class of homogeneous mixtures in which the dispersed particles do not settle. The insoluble particles in the mixture can be microscopic, ranging in size from 1 to 1000 nanometers. The mixture may be called a colloid or a colloidal suspension. Sometimes the term "colloid" refers only to the particles in the mixture rather than the entire suspension.

The term "average diameter" or "mean diameter" refers to the average hydrodynamic diameter of a particle as measured by dynamic laser light scattering (DLS), where data analysis is performed using a so-called cumulant algorithm, the results of which provide the so-called Z-mean with a long dimension and a dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321, which is incorporated herein by reference). In this document, the terms "average diameter," "mean diameter," "diameter," or "size" are used synonymously with this value of the Z-mean.

The "polydispersity index" is better calculated based on dynamic light scattering measurements using the so-called cumulative analysis mentioned in the definition of "mean diameter". Under certain prerequisites, it can be regarded as a measure of the size distribution of an assembly of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).

Different types of nucleic acid particles have previously been described as suitable for delivering nucleic acids in microparticle form (e.g., Kaczmarek et al., 2017, Genome Medicine 9, 60, which is incorporated herein by reference). For non-viral nucleic acid delivery media, the encapsulation of nucleic acids in nanoparticles physically protects the nucleic acids from degradation and, depending on specific chemical properties, can facilitate cellular uptake and endosome escape.

This disclosure describes particles comprising nucleic acids (e.g., polynucleotides), at least one cationic or cationically ionizable lipid or lipid-like material, and/or at least one cationic polymer bound to the nucleic acid (e.g., polynucleotides) 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 acids (e.g., polynucleotides) complexed with the particles in various forms through non-covalent interactions. The particles described herein are not viral particles, and in particular, they are not infectious viral particles, i.e., they cannot infect cells.

Some of the embodiments described herein involve compositions, methods and uses that involve more than one, such as 2, 3, 4, 5, 6 or even more nucleic acid species (e.g., polynucleotide species).

In nucleic acid particle formulations (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles), it is possible to separately formulate each nucleic acid species (e.g., polynucleotide species) into individual nucleic acid particle formulations. In that case, each individual nucleic acid particle formulation (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) will contain one nucleic acid species (e.g., polynucleotide species). Individual nucleic acid particle formulations (e.g., ribonucleic acid particles, e.g., ribonucleic acid nanoparticles) can exist as separate entities, for example, in separate containers. Such formulations can be obtained by separately providing each nucleic acid species (e.g., polynucleotide species) (usually in the form of solutions containing nucleic acids) and a particle-forming agent, thereby allowing particle formation. The corresponding particles will exclusively contain the specific nucleic acid species (e.g., polynucleotide species) provided during particle formation (individual particle formulations).

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

Alternatively, different nucleic acid species (e.g., polynucleotide species) can be formulated together as "combined microparticle formulations". Such formulations are obtained by providing a combination of different nucleic acid species (e.g., polynucleotide species) and a particle-forming agent (typically a combination solution), thereby allowing particle formation. In contrast to "mixed microparticle formulations", "combined microparticle formulations" will typically contain particles comprising more than one nucleic acid species (e.g., polynucleotide species). In combined microparticle compositions, different nucleic acid species (e.g., polynucleotide species) are typically present together within a single particle.

In some embodiments, when present in the provided nucleic acid particles (e.g., ribonucleic acid particles, such as lipid nanoparticles), the nucleic acids (e.g., polynucleotides) are resistant to degradation by nucleases in aqueous solutions.

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

Cationic polymers have been recognized for use in the development of particulate delivery media, as reported in PCT Application Publication No. WO2021/001417, the entire contents of which are incorporated herein by reference. As used herein, the term "polymer" means an assembly comprising one or more molecules, such molecules comprising repeating units of one or more monomers. As used herein, "polymer," "polymer material," and "polymer assembly" are used interchangeably and, unless otherwise stated, refer to an assembly of polymer molecules. Those skilled in the art will understand that a polymer assembly comprises polymer molecules of different lengths (e.g., comprising different amounts of monomers). The polymer assemblies described herein are characterized by one or more of the following: normalized molecular weight (Mn), weight-average molecular weight (Mw), and/or polydispersity index (PDI). In some embodiments, these repeating units may be entirely identical (“homogene”); or, in some cases, more than one type of repeating unit may be present within the polymer material (“hybrid” or “copolymer”). In some cases, the polymer is biologically derived, such as biopolymers, like proteins. In some cases, additional portions may also be present in the polymer material, such as targeting portions, as described herein.

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

In some embodiments, the polymeric materials used according to this disclosure are biocompatible. In some embodiments, the biocompatible materials are biodegradable, for example, capable of chemical and/or biological degradation in physiological environments (such as in vivo).

In some embodiments, the polymer material may be or contain protamine or polyalkylimide.

As those familiar with this technique will recognize, the term "protamine" is generally used to refer to any of a variety of relatively low molecular weight, strongly alkaline proteins rich in arginine, which are found to be particularly associated with DNA, replacing somatic histones in sperm cells of various animals (such as fish). Specifically, the term "protamine" is generally used to refer to a protein found in fish sperm that is strongly alkaline, water-soluble, does not coagulate upon heat, and primarily produces arginine upon hydrolysis. In purified form, protamine is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

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

In some embodiments, the polyalkylene imide comprises polyethyleneimine (PEI) and/or polypropylene imide. In some embodiments, the preferred polyalkylene imide is polyethyleneimine (PEI). In some embodiments, the average molecular weight of PEI is preferably 0.75∙10² to 10⁷ Da, more preferably 1000 to 10⁵ Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, and even more preferably 20000 to 25000 Da.

The cationic materials (e.g., polymeric materials, including polycationic polymers) considered herein include those capable of electrostatically binding nucleic acids. In some embodiments, the cationic polymeric materials considered herein include any cationic polymeric material to which nucleic acids can bind (e.g., by forming complexes with nucleic acids or forming vesicles therewith that encapsulate or encapsulate nucleic acids).

In some embodiments, the particles described herein may comprise polymers other than cationic polymers, such as nonionic polymer materials and/or anionic polymer materials. In summary, anionic and neutral polymer materials are referred to herein as nonionic polymer materials. Lipid compositions: Lipids and lipid-like materials

Lipids and lipid-like materials have also been considered for use in the development of particle delivery media. The terms "lipid" and "lipid-like material" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, where appropriate, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are also commonly referred to as amphiphiles. Lipids are generally poorly soluble in water. In an aqueous environment, their amphiphilic nature allows molecules to self-assemble into organized structures and different phases. One of these phases consists of a lipid bilayer, as it exists in vesicles, multilayer/monolayer lipid bodies, or membranes in an aqueous environment. Hydrophobicity can be imparted by including nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic groups. Hydrophilic groups may include polar and/or charged groups and include carbohydrate, phosphate, carboxyl, sulfate, amino, thiohydrogen, nitro, hydroxyl, and other similar groups.

Typically, amphiphilic compounds have a polar head connected to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the nonpolar portion is insoluble in water. Additionally, the polar portion may carry a formal positive charge or a formal negative charge. Alternatively, the polar portion may carry both a formal positive and negative charge and be an amphoteric ion or an inner salt. For the purposes of this disclosure, the amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

"Lipid-like materials" are substances that are structurally and/or functionally related to lipids but may not be considered lipids in the strict sense. For example, the term includes compounds that can form an amphiphilic layer when present in an aqueous environment as vesicles, multilayer/monolayer liposomes, or membranes, and includes surfactants or compounds synthesized with both hydrophilic and hydrophobic portions. Generally, the term refers to molecules containing hydrophilic and hydrophobic portions with different structural structures, which may be similar to or dissimilar to the structure of lipids.

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

Generally, lipids can be classified into eight categories: fatty acids, glycerol lipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketone subunits), sterols, and isoprenol lipids (derived from the condensation of isoprenol subunits). Although the term "lipid" is sometimes used synonymously with fat, fat is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

Fatty acids are a group of different molecules composed of hydrocarbon chains terminated by carboxylic acid groups; this arrangement gives the molecule a polar hydrophilic end and a water-insoluble nonpolar hydrophobic end. The carbon chain, typically between four and 24 carbons in length, can be saturated or unsaturated and can be linked to functional groups containing oxygen, halogen, nitrogen, and sulfur. If fatty acids contain double bonds, cis or trans geometric isomers may exist, significantly affecting the molecular configuration. Cis double bonds cause the fatty acid chain to bend, an effect of complexation with more double bonds in the chain. Other major lipid classes within the fatty acid family include fatty esters and fatty amides.

Glycerides are composed of mono-, di-, and tri-substituted glycerols, the most well-known being the fatty acid triesters of glycerol, known as triglycerides. The term "triglycerides" is sometimes used synonymously with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are typically esterified from different fatty acids. Other subclasses of glycerides are represented by glycosylglycerols, characterized by the presence of one or more glycosidic residues linked to glycerol via glycosidic bonds.

Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions), consisting of a glycerol core linked to two fatty acid-derived "tails" by ester bonds and a "head" group linked to a phosphate ester bond. Examples of glycerophospholipids commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid) include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

Neurosphingolipids are members of a complex family of compounds that share a common structural feature: a sphingosine-based backbone. The main sphingosine-based backbone in mammals is generally referred to as neursphingosine. Neurosphingosine (N-amide-sphingosine-based) is the main subclass of sphingosine-based derivatives of fatty acids linked by amide. These fatty acids are typically saturated or monounsaturated, with chain lengths of 16 to 26 carbon atoms. The main sphingomyelin in mammals is sphingomyelin (neuraminic phosphocholine), while insects primarily contain neuraminic phosphoethanolamine, and fungi possess phytoneuraminic phosphoinositol and a mannose-containing head group. Glycosphingolipids are a family of different molecules consisting of one or more glycosidic residues linked by glycosidic bonds to sphingosine bases. Examples of such substances include simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

Sterols, such as cholesterol and its derivatives, or tocopherol and its derivatives, together with glycerophospholipids and sphingomyelin, are important components of membrane lipids.

Glycolipids are compounds in which fatty acids are directly linked to the glycosidic backbone, forming a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone present in glycerol lipids and glycerophospholipids. The most familiar glycolipid is the acetylated glucosamine precursor of the lipid A component of lipopolysaccharides in Gram-negative bacteria. The typical lipid A molecule is a disaccharide of glucosamine, which has derived up to seven fatty acid acetylated chains. The smallest lipopolysaccharide required for growth in Escherichia coli is Kdo2-lipopolysaccharide, which is a hexacetylated disaccharide of glucosamine glycosylated by two 3-deoxy-D-manno-octyl ketone (Kdo) residues.

Polyketides are synthesized by polymerizing acetyl and aliphatic subunits using classical enzymes and iterative and multimodal enzymes that share mechanical features with fatty acid synthases. Polyketides comprise a vast array of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, exhibiting immense structural diversity. Many polyketides are cyclic molecules, and their backbones are typically further modified through glycosylation, methylation, hydroxylation, oxidation, or other processes.

Lipids and lipid-like materials can be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist as uncharged or neutral zwitterionic forms at a selected pH.

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

In some embodiments, the cationic or cationically ionizable lipids or lipid-like materials considered for use herein include any cationic or cationically ionizable lipids or lipid-like materials capable of electrostatically binding nucleic acids. In one embodiment, the cationic or cationically ionizable lipids or lipid-like materials considered for use herein may be coupled to nucleic acids, for example, by forming complexes with nucleic acids or forming vesicles therein that encapsulate or encapsulate nucleic acids.

Cationic lipids or lipid-like materials are characterized by their net positive charge (e.g., at the relevant pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acids via electrostatic interactions. Generally, cationic lipids have lipophilic moieties, such as sterols, acetyl chains, diacetyl groups, or multiple acetyl chains, and the head group of the lipid typically carries a positive charge.

In some embodiments, cationic lipids or lipid-like materials possess a net positive charge only at certain pH levels, particularly acidic pH levels, and preferably no net positive charge, or even neutral charge, at different, preferably higher pH levels, such as physiological pH levels. This ionization behavior is considered to enhance efficacy by facilitating internal escape and reducing toxicity compared to particles that remain cationic at physiological pH levels.

In some embodiments, the cation or cation-ionizable lipid or lipid-like material includes a head group comprising at least one nitrogen atom (N) that is positively charged or capable of being protonated.

Examples of cationic lipids include, but are not limited to, 1,2-dioleyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and 3-(N-(N′,N′-dimethylaminoethane)-aminomethyl)cholesterol (DC-C). 1,2-Dioxoyl-3-dimethylammonium chloride (DODAP); 1,2-dioxo-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; 1,2-dioxo-3-dimethylammonium chloride (DODAC); 1,2-distearateoxy-N,N-dimethyl-3-aminopropane (DS) DMA), 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylazonium (DMRIE), 1,2-dimyristyl-sn-glycerol-3-ethylcholesterol phosphate (DMEPC), 1,2-dimyristyl-3-trimethylammonium propane (DMTAP), 1,2-dioleoxypropyl-3-dimethyl-hydroxyethylammonium bromide (DOR) IE) and trifluoroacetic acid 2,3-dioleoxy-N-[2-(sperminemethamine)ethyl]-N,N-dimethyl-1-propane (DOSPA), 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleoxy-N,N-dimethylaminopropane (DLenDMA), bis(octadecylaminoglycyl) Spermine (DOGS), 3-dimethylamino-2-(cholesterol-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′-(cholesterol-5-en-3-β-oxy)-3′-oxopaloxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N′-dioleoylaminomethyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinyl... Aminomethyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinylaminomethyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinyl-4-dimethylaminomethyl-[1,3]-dioxane (DLin-K-DMA), 2,2-dilinyl-4-dimethylaminoethyl-[1,3]-dioxane (DLin-K-XTC2-DMA), 2,2-dilinyl-4-(2-dimethylaminoethyl)-[1,3]-dioxane (DLin-KC2-DMA), heptadecyl-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA), N-(2- Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane ammonium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecyloxy)-1-propane ammonium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2, 3-Bis(dodecyloxy)-1-propane ammonium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane ammonium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propane ammonium bromide (βA) E-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oxylatedoxy)prop-1-ammonium (DOBAQ), 2-({8-[(3β)-cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]prop-1-amine (octyl) 1,2-Dimyristyl-3-dimethylammonium-propane (DMDAP), 1,2-dimyristyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylmethylamino)ethyl]-3,4-di[oleoxy] -Benzylamine (MVL5), 1,2-dioleoyl-sn-glycerol-3-ethylcholesterol phosphate (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropyl-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propyl-1-ammonium bromide (DMORIE), 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanyl)dioctanoic acid di((Z)-non-2-en-1-yl) ester (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propyl-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propyl-1-amine (DMDM) A) Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylaminomethyl-ethyl)-{2-[(2-dodecylaminomethyl-ethyl)-2-{(2-dodecylaminomethyl-ethyl)-[2-(2-dodecylaminomethyl-ethylamino)-ethyl]amino}-ethylamino)propionic acid (lipid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]pyrazin-1-yl]ethyl]amino]dodecyl-2-ol (lipid C12-200), LIPOFECTIN® (Commercially available cationic liposomes containing DOTMA and 1,2-dioleyl-sn-3-phosphate ethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminemethoxy)ethyl)-N,N-dimethyltrifluoroacetate ammonium (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic liposomes containing bis(octadecylaminoglycyl)carboxyspermine (DOGS) in ethanol, from Promega Corp., Madison, Wis.), or any combination thereof. Other suitable cationic lipids used in this 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. Other suitable cationic lipids used in this disclosure include those described in WO2010/053572. (Including C12-200 as described in paragraph [00225]) and the cationic lipids described in WO2012/170930, which are incorporated herein by reference for the purposes described herein. Additional suitable cationic lipids used in this disclosure include HGT4003, HGT5000, HGTS001, HGT5001, and HGT5002 (see US2015/0140070, which is incorporated herein by reference in its entirety).

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

In some embodiments, the amino or cationic lipids disclosed herein may have at least one protonable or deprotonable group, such that the lipids are positively charged at pH values equal to or lower than physiological pH (e.g., pH 7.4) and neutral at a second pH value, preferably equal to or higher than physiological pH. It will be understood that the addition or removal of protons varies with pH as an equilibrium process, and the reference to charged or neutral lipids refers to the property of the dominant species, and does not require all lipids to exist in a charged or neutral form. Lipids having more than one protonable or deprotonable group or being zwitterionic are not excluded and are equally applicable in the context of this invention.

In some embodiments, the protonable lipids have pKa of protonable groups in the range of about 4 to about 11, for example, about 5 to about 7.

In some embodiments, the cationic lipids may comprise 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 lipids present in the lipid composition utilized according to this disclosure. Additional lipids or lipid-like materials

In some embodiments, the formulations used according to this disclosure may contain 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). In summary, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. In some embodiments, formulations that optimize nucleic acid particles by adding other hydrophobic components (such as cholesterol and lipids) in addition to ionizable/cationic lipids or lipid-like substances may, for example, enhance particle stability and nucleic acid delivery efficiency.

In some embodiments, lipids or lipid-like materials that may or may not affect the overall charge of the particles may be incorporated. In some embodiments, such lipids or lipid-like materials are non-cationic lipids or lipid-like materials.

In some embodiments, non-cationic lipids may include, for example, one or more anionic lipids and/or neutral lipids. "Anionic lipids" are negatively charged (e.g., at a selected pH).

"Co-lipids" exist in an uncharged, neutral zwitterionic form (e.g., at a chosen pH), or in some embodiments, in a cationic or positively charged form at physiological pH. In some embodiments, the formulation comprises one of the following co-lipid components: (1) phospholipids, (2) cholesterol or a derivative thereof; or (3) a mixture of phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholesterol ketones, cholesterol ketones, serotonols, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol, and their derivatives and mixtures thereof.

Specific exemplary phospholipids that may be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. In particular, these phospholipids include diacetylphosphatidylcholine, such as distearate phosphatidylcholine (DSPC), dioleophosphatidylcholine (DOPC), dimyristophosphatidylcholine (DMPC), pentacarbamate phosphatidylcholine, dilaurate phosphatidylcholine, dipalmitophosphatidylcholine (DPPC), diarachidonicylphosphatidylcholine (DAPC), diarachidonicylphosphatidylcholine (DBPC), and triacylphosphatidylcholine. Choline (DTPC), bis(2-tetrafluoromethylphospholipid)choline (DLPC), palmitic oleyl-phospholipid (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), 1-oleyl-2-cholestylosyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16) Lyso PC and phosphatidylethanolamines, particularly diacetylated phosphatidylethanolamines, such as dioleylphosphatidylethanolamine (DOPE), distearylylphosphatidylethanolamine (DSPE), dipalmitylphosphatidylethanolamine (DPPE), dimyristylphosphatidylethanolamine (DMPE), dilaurylphosphatidylethanolamine (DLPE), diphyranylphosphatidylethanolamine (DPyPE), and other phosphatidylethanolamine lipids with different hydrophobic chains.

In some embodiments, the formulations used according to this disclosure include DSPC or DSPC and cholesterol.

In some embodiments, the formulations used according to this disclosure contain ionizable or cationic auxiliary lipids.

In some embodiments, the formulations used according to this disclosure include both cationic lipids and additional (non-cationic) lipids.

In some embodiments, the formulations described herein include polymer-coupled lipids, such as polyethylene glycol-coupled lipids. "PEG-coupled lipids" or "PEG-coupled lipids" comprise both a lipid portion and a polyethylene glycol portion. PEG-coupled lipids are known in this art.

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

In some embodiments disclosed herein, the RNA described herein may be present in RNA-lipid complex particles.

"RNA-liposome complex particles" contain lipids (particularly cationic lipids) and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA lead to the complexation and spontaneous formation of RNA-liposome complex particles. Positively charged liposomes can generally be synthesized using cationic lipids (such as DOTMA) and additional lipids (such as DOPE). In one embodiment, the RNA-liposome complex particles are nanoparticles.

In some embodiments, the RNA-lipoprotein complex particles comprise both cationic lipids and additional lipids. In one exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

In some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In 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 at least one cationic lipid to at least one additional lipid is about 2:1.

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

The RNA-liposome complex particles and compositions comprising RNA-liposome complex particles described herein can be used to deliver RNA to target tissues after non-enteral administration, particularly after intravenous administration. The RNA-liposome complex particles can be prepared using liposomes, which can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In another embodiment, the aqueous phase contains, for example, about 5 mM of acetic acid. Liposomes can be used to prepare RNA-liposome complex particles by mixing liposomes with RNA. In one embodiment, the liposomes and RNA-liposome complex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleyl-3-trimethylammonium-propane (DOTAP). In one embodiment, at least one additional lipid comprises 1,2-di-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol, and/or 1,2-dioleyl-sn-glycerol-3-phosphate choline (DOPC). In one embodiment, at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and at least one additional lipid comprises 1,2-di-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine (DOPE). In one embodiment, liposomes and RNA-liposome complex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine (DOPE).

RNA-lipoprotein complex particles targeting the spleen are described in WO2013/143683, which is incorporated herein by reference. It has been found that RNA-lipoprotein complex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Therefore, RNA accumulation and/or RNA expression occur in the spleen after administration of the RNA-lipoprotein complex particles. Therefore, the RNA-lipoprotein complex particles disclosed herein can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lungs and/or liver after administration of the RNA-lipoprotein complex particles. In one embodiment, RNA accumulation and/or RNA expression occur in antigen-presenting cells (such as professional antigen-presenting cells in the spleen) after administration of RNA-lipoprotein complex particles. Therefore, the RNA-lipoprotein complex particles disclosed herein can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and/or macrophages. Lipoprotein Nanoparticles (LNPs)

In some embodiments, the nucleic acids (such as RNA) described herein are delivered in the form of lipid nanoparticles (LNPs). In some embodiments, the LNP may comprise any lipid capable of forming a particle, with one or more nucleic acid molecules linked to the particle, or encapsulating one or more nucleic acid molecules therein.

In some embodiments, the LNP comprises one or more cationic lipids and one or more stable lipids. Stable lipids include neutral lipids, auxiliary lipids, and polyethylene glycol-modified lipids.

In some embodiments, LNPs comprise cationic lipids, co-lipids, sterols, polymer-coupled lipids; and RNA encapsulated within or coupled to lipid nanoparticles.

In some embodiments, the co-lipids are phosphatidylinosylcholine, such as 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitinyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristinyl-sn-glycerol-3-phosphate choline (DMPC), 1-palmitinyl-2-oleyl-sn-glycerol-3-phosphate choline (POPC), 1,2-dioleyl-sn-glycerol-3-phosphate choline (DOPC), phosphatidylinosylethanolamines such as 1,2-dioleyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and sphingomyelin (SM). In some embodiments, the auxiliary lipid is 1,2-dioleyl-3-trimethylammonium-propane (DOTAP), 1-α-phosphatidylserine (PS), or DOPE. In some embodiments, the auxiliary 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 auxiliary lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOTAP, PS, and SM. In some embodiments, the auxiliary lipid is DSPC. In some embodiments, the auxiliary lipid is DOTAP. In some embodiments, the auxiliary lipid is DOPE. In some embodiments, the auxiliary lipid is PS.

In some implementations, the sterol is cholesterol.

In some embodiments, the polymer-coupled lipid is a polyethylene glycol-modified lipid (PEG lipid). In some embodiments, the PEG lipid is selected from polyethylene glycol-modified diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG) ( e.g., 1,2-dimyristyl-rac-glycerol-3-methoxypolyethylene glycol-2000). (PEG2000-DMG); polyethylene glycolated phosphatidylethanolamine (PEG-PE); PEG diacylglycerol succinate (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); polyethylene glycolated ceramide (PEG-cer); or PEG diekoxy Propylcarbamates, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecoxy)propyl)carbamate and 2,3-di(tetradecoxy)propyl-1-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the PEGylated ester is 1,2-dimyristyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (C14-PEG2000). In some embodiments, the PEGylated ester has the following structure: Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

^R12 and ^R13 are each independently a straight-chain or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60. In some embodiments, ^R12 and ^R13 are each independently a straight-chain saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 40 to 55. In some embodiments, the average w is about 45. In some embodiments, ^R12 and ^R13 are each independently a straight-chain saturated alkyl chain containing about 14 carbon atoms, and w has an average value of about 45.

In some embodiments, the polyethylene glycol ester is DMG-PEG 2000, for example having the following structure: .

In some embodiments, the polyethylene glycol-modified lipid is or contains 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide having the following chemical structure: Or a pharmaceutically acceptable salt thereof, where n' is an integer from 45 to 50.

In some embodiments, the cationic lipid component of LNP has the structure of formula (III): (III) or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein: one of ^L1 or ^L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x- , -SS-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a- , NR ^a C(=O)NR ^a- , -OC(=O)NR ^a- , or -NR ^a C(=O)O-, and the other of ^L1 or ^L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) _x- , -SS-, -C(=O)S-, SC(=O)-, -NR ^a C(=O)-, -C(=O)NR ^a -, NR ^a C(=O)NR ^a -, -OC(=O)NR ^a - or -NR ^a C(=O)O- or direct bond; ^G1 and ^G2 are each independently unsubstituted _C1 - _C12 alkyl or _C1 - _C12 alkenyl; ^G3 is _C1 - _C24 alkyl, _C1 - _C24 alkenyl, _C3 - _C8 cycloalkyl, _C3 - _C8 cycloalkyl; ^Ra is H or _C1 - _C12 alkyl; ^R1 and ^R2 are each independently _C6 - _C24 alkyl or _C6 - _C24 alkenyl; ^R3 is H, ^OR5 , CN, -C(=O) ^OR4 , -OC(=O) ^R4 or ^-NR5 C(=O) ^R4 ; ^R4 is _C1 - _C12 alkyl; R ⁵ is H or _C1 - _C6 alkyl; and x is 0, 1 or 2.

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB): or Wherein: A is a 3 to 8-membered cycloalkyl or extended cycloalkyl ring; ^R6 is independently H, OH or _C1 - _C24 alkyl each time it appears; n is an integer in the range of 1 to 15.

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

In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID): or Where y and z are each an integer in the range of 1 to 12.

In any of the foregoing embodiments of equation (III), one of ^L1 or ^L2 is -O(C=O)-. For example, in some embodiments, each of ^L1 and ^L2 is -O(C=O)-. In some different embodiments of either of the foregoing, ^L1 and ^L2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, each of ^L1 and ^L2 is -(C=O)O-.

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

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

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

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

In some of the foregoing embodiments of formula (III), ^R6 is H. In other foregoing embodiments, ^R6 is a _C1 - _C24 alkyl group. In other embodiments, ^R6 is OH.

In some embodiments of formula (III), ^G3 is unsubstituted. In other embodiments, G3 is substituted. In various embodiments, ^G3 is a straight-chain _C1 - _C24 alkyl or a straight-chain _C1 - _C24 alkenyl.

In some other aforementioned embodiments of formula (III), ^R1 or ^R2 , or both, are _C6 - _C24 alkenyl groups. For example, in some embodiments, ^R1 and ^R2 each independently have the following structures: Wherein: ^R7a and ^R7b are independently H or _C1 - _C12 alkyl groups each time they appear; and a is an integer from 2 to 12.

^R7a , ^R7b , and a are each chosen such that ^R1 and ^R2 each independently contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5 to 9 or 8 to 12.

In some of the foregoing embodiments of formula (III), ^R7a occurring at least once is H. For example, in some embodiments, ^R7a is H every time it occurs. In other different foregoing embodiments, ^R7b occurring at least once is a _C1 - _C8 alkyl group. For example, in some embodiments, the _C1 - _C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tributyl, n-hexyl, or n-octyl.

In different embodiments of equation (III), ^R1 or ^R2 or both have one of the following structures: ; ; ; ; ; ; ; ; ; .

In some of the foregoing embodiments of formula (III), ^R3 is OH, CN, -C(=O) ^OR4 , -OC(=O) ^R4 or -NHC(=O) ^R4 . In some embodiments, ^R4 is methyl or ethyl.

In various embodiments, the cationic lipids of formula (III) have one of the structures listed in Table 15 below. Table 15: Exemplary cationic lipid structures of formula (III) Number Structure III-1 III-2 III-3 III-4 III-5 III-6 III-7 III-8 III-9 III-10 III-11 III-12 III-13 III-14 III-15 III-16 III-17 III-18 III-19 III-20 III-21 III-22 III-23 III-24 III-25 III-26 III-27 III-28 III-29 III-30 III-31 III-32 III-33 III-34 III-35 III-36

In various embodiments, the cationic lipids possess one of the structures listed in Table 16 below. Table 16: Exemplary cationic lipid structures of formula AF Number Structure A B C D E F

In some embodiments, the LNP comprises cationic lipids as ionizable lipid-like materials (lipids). In some embodiments, the cationic lipids have one of the following structures as described in Melamed et al., Science Advances , 2023, 9, eade1444 (the entire contents of which are incorporated herein by reference): X-1 X-2 X-3 X-4

In some embodiments, the lipid nanoparticles may have an average size (e.g., average diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, the lipid nanoparticles according to this disclosure may have an average size (e.g., average diameter) of about 50 nm to about 100 nm. In some embodiments, the lipid nanoparticles may have an average size (e.g., average diameter) of about 50 nm to about 150 nm. In some embodiments, the lipid nanoparticles may have an average size (e.g., average diameter) of about 60 nm to about 120 nm. In some embodiments, the lipid nanoparticles according to this disclosure may have an average size (e.g., average diameter) of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm or 150 nm. The term "average diameter" or "mean diameter" refers to the average hydrodynamic diameter of a particle as measured by dynamic laser light scattering (DLS), where data analysis is performed using a so-called cumulant algorithm, the results of which provide the so-called Z-mean with a long dimension and a dimensionless polydispersity index (PI) (Koppel, Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321, which is incorporated herein by reference). Here, the terms "average diameter,""meandiameter,""diameter," or "size" of a particle are used synonymously with this value of the Z-mean.

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

The lipid nanoparticles described herein are characterized by their "N/P ratio," which is the molar ratio of cation (nitrogen) groups ("N" in N/P) in cationized polymers to anion (phosphate) groups ("P" in N/P) in RNA. It should be understood that cation groups are groups in cation form ( e.g., N ⁺ ) or groups that can be ionized to become cations. Using a single number in the N/P ratio ( e.g., approximately 5 N/P ratio) is intended to mean that the number is greater than 1; for example, approximately 5 N/P ratio means 5:1. In some embodiments, the lipid nanoparticles described herein have an N/P ratio greater than or equal to 5. In some embodiments, the lipid nanoparticles described herein have an N/P ratio of about 5, 6, 7, 8, 9, or 10. In some embodiments, the N/P ratio of the lipid nanoparticles described herein is from about 10 to about 50. In some embodiments, the N/P ratio of the lipid nanoparticles described herein is from about 10 to about 70. In some embodiments, the N/P ratio of the lipid nanoparticles described herein is from about 10 to about 120.

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

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

In some embodiments, the formulation used for IP delivery comprises lipid nanoparticles, wherein the lipid nanoparticles comprise approximately 35 mol% cationic lipids; approximately 40 mol% cofactor lipids; approximately 22.5 mol% cholesterol; and approximately 2.5 mol% PEG-conjugated lipids. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% cationic lipids X-2, X-3, or X-4; approximately 40 mol% DOTAP, DOPE, or PS; approximately 22.5 mol% cholesterol; and approximately 2.5 mol% C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-2; approximately 40 mol% of DOTAP; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-3; approximately 40 mol% of DOTAP; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-4; approximately 40 mol% of DOTAP; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-2; approximately 40 mol% of DOPE; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-3; approximately 40 mol% of DOPE; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-4; approximately 40 mol% of DOPE; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-2; approximately 40 mol% of PS; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-3; approximately 40 mol% of PS; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. In some embodiments, the lipid nanoparticles comprise approximately 35 mol% of cationic lipid X-4; approximately 40 mol% of PS; approximately 22.5 mol% of cholesterol; and approximately 2.5 mol% of C14-PEG2000. Not wishing to be limited by any theory, it is anticipated that these formulations will lead to enhanced delivery of lipid nanoparticles to pancreatic β-cells and enhanced in vivo expression of encoded incretins within pancreatic β-cells, particularly when administered via intraperitoneal (ip) delivery. An exemplary method for manufacturing lipid nanoparticles is provided.

Lipids containing nucleic acids and lipid nanoparticles and methods for preparing them are known in this art, including, for example, U.S. Patent Nos. 8,569,256, 5,965,542, and U.S. Patent Publications Nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, and 2013/032. No. 3269, No. 2013/0245107, No. 2013/0195920, No. 2013/0123338, No. 2013/0022649, No. 2013/0017223, No. 2012/0295832, No. 2012/0183581, No. 2012/0172411, No. 2012/0027803, No. 2012/0058188, No. 2011/0311583, No. 2011/0311582, No. 2011/0262527, No. 2011/0216622, No. 2011/011712 No. 5, No. 2011/0091525, No. 2011/0076335, No. 2011/0060032, No. 2010/0130588, No. 2007/0042031, No. 2006/0240093, No. 2006/0083780, No. 2006/0008910, No. 2005/0175682, No. 2005/017054, No. 2005/0118253, No. 2005/0064595, No. 2004/0142025, No. 2007/0042031, No. 1999/009076 and PC The entire disclosure of each of the patents described in T Publication Nos. WO99/39741, WO2018/081480, WO2017/004143, WO2017/075531, WO2015/199952, WO2014/008334, WO2013/086373, WO2013/086322, WO2013/016058, WO2013/086373, WO2011/141705 and WO2001/07548 is incorporated herein by reference for the purposes described herein.

For example, in some embodiments, cationic lipids, co-lipids (e.g., DSPC and/or cholesterol), and polymer-coupled lipids can be dissolved in ethanol at a predetermined molar ratio (e.g., the molar ratio described herein). In some embodiments, multiple lipid nanoparticles (single lipid nanoparticles) are prepared at a total lipid to polynucleotide weight ratio of approximately 10:1 to 30:1. In some embodiments, such polynucleotides can be diluted to 0.2 mg/mL in an acetate buffer.

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

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

In some embodiments, solutions containing RNA-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.

In some embodiments, RNA-encapsulated lipid nanoparticles can be processed by filtration.

In some embodiments, the particle size and/or internal structure of lipid nanoparticles (with or without RNA) can be monitored using appropriate techniques such as small-angle X-ray scattering (SAXS) and/or cryo-transmission electron microscopy (CryoTEM). Pharmaceutical compositions

This disclosure provides compositions comprising one or more of the polynucleotides described herein, such as pharmaceutical compositions. Pharmaceutical formulations may additionally comprise pharmaceutically acceptable excipients, as used herein, including any and all solvents, dispersion media, diluents or other liquid media, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, and analogues, as appropriate for a desired specific dosage form. Remington's *The Science and Practice of Pharmacy*, 21st edition, Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any known adjuvant medium is incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or by interacting with any other component of the pharmaceutical composition in a harmful manner, its use is considered within the scope of this disclosure.

In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration (FDA). In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), the European Pharmacopeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulators, surfactants and/or emulsifiers, disintegrants, binders, preservatives, stabilizers, lubricants and/or oils. Such excipients may be included in the pharmaceutical formulation as appropriate. Excipients (such as cocoa butter and suppository waxes, colorants, coating agents, sweeteners, flavorings and/or aromatizers) may be present in the composition at the discretion of the formulation engineer.

For general considerations in the preparation and/or manufacture of pharmaceutical preparations, see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference).

In some embodiments, the pharmaceutical compositions provided herein may be formulated in accordance with the techniques described in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005 (which are incorporated herein by reference) with one or more pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients.

The pharmaceutical compositions described herein can be administered using appropriate methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration may depend on a variety of factors, including, but not limited to, the stability and/or pharmacokinetics and/or pharmacodynamics of the pharmaceutical compositions described herein.

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

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

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

Therapeutic compounds must typically be sterile and stable under manufacturing and storage conditions. Compounds can be formulated as solutions, microemulsions, lipid nanoparticles, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and similar alcohols), or suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using surfactants. In many cases, it is preferable to include isotonic agents in the compound, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. In some embodiments, prolonged absorption of injectable compounds can be achieved by including agents that delay absorption (e.g., monostearate and gelatin) in the compound.

Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or more of the ingredients listed above (as needed) into a suitable solvent, followed by sterilization and/or microfiltration. In some embodiments, pharmaceutical compositions can be prepared by methods described herein and/or known in the art.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of antimicrobial agents can be ensured by sterilization processes and by including various antibacterial and antifungal agents, such as para-hydroxybenzoic acid esters, chlorobutanol, phenolic sorbic acid, and their analogues. It is also desirable to include isotonic agents (such as sugars, sodium chloride, and their analogues) in the pharmaceutical compositions described herein. Furthermore, prolonged absorption in injectable pharmaceutical forms can be achieved by including agents that delay absorption (such as aluminum monostearate and gelatin).

The formulations of the pharmaceutical compositions described herein can be prepared by any method known in or subsequently developed in pharmacological techniques. Generally, such preparation methods include the steps of mixing an active agent with a diluent or another excipient and/or one or more other excipients, and then, if necessary and/or desired, shaping and/or packaging the product into desired single or multiple dosage units.

The pharmaceutical compositions disclosed herein 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 a discrete amount of a pharmaceutical composition containing at least one pre-quantitative RNA product produced using the systems and/or methods described herein.

The relative amounts of polynucleotides in any additional components encapsulated in lipid nanoparticles, pharmaceutically acceptable excipients, and/or pharmaceutical compositions may vary depending on the individual seeking treatment, the target cells, the disease, or the condition, and may also depend on the route of administration of the composition.

In some embodiments, the pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms using methods known to those skilled in the art. The actual dosage level of the active ingredient (e.g., polynucleotides encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and mode of administration, without toxicity to the patient. The chosen dosage level will depend on various pharmacokinetic factors, including the activity of the specific combination disclosed herein, the route of administration, the timing of administration, the excretion rate of the specific compound, the duration of treatment, other drugs, compounds and/or materials used in combination with the specific combination, the age, sex, weight, disease, general health condition and medical history of the patient being treated, and similar factors known in medical technology.

Physicians familiar with this technique can easily determine and prescribe the effective dosage of the required drug combination. For example, a physician can start with a dosage of the active ingredient used in the drug combination (e.g., polynucleotides encapsulated in lipid nanoparticles) at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

In some embodiments, the pharmaceutical composition described herein is formulated (e.g., but not limited to intravenous, intramuscular, or subcutaneous administration) to deliver an active dose that imparts to a plasma concentration of an incretin encoded by at least one polynucleotide (e.g., those described herein), the polynucleotide mediating its pharmacological activity via its primary mode of action (an agonist of GLP1 and/or GIP).

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

In some embodiments, the pharmaceutical composition described herein may further comprise one or more active agents other than at least one polynucleotide encoding an incretin agent. For example, in some embodiments, such other active agents may be or comprise another known treatment for obesity or obesity-related disorders or diseases. In some embodiments, exemplary treatments may be those included in Table 1 herein.

This disclosure provides the following understanding: incretins can be combined with the polynucleotides and/or combinations provided herein, for example, for the treatment or prevention of obesity and obesity-related diseases or conditions. Exemplary incretins that can be used with the combinations described herein include, but are not limited to, the incretins, fragments thereof, or combinations thereof provided in Table 1.

This disclosure further provides the following insight: incretins, combinations of incretins, and incretin mimics can be encoded in polynucleotides. Delivery of one or more incretin agents via polynucleotide delivery can achieve the same or substantially the same efficacy as known incretins and incretin mimics (which are peptide-based products (e.g., those in Table 1)), but with a lower injection volume per dose.

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

Although the descriptions of pharmaceutical compositions provided herein primarily pertain to those suitable for human administration, those skilled in the art will understand that such compositions are generally suitable for administration to a wide range of animals. Modifications to pharmaceutical compositions suitable for human administration are well known to make them suitable for administration to a wide range of animals, and such modifications can be designed and/or performed by a competent veterinarian through routine experiments (if applicable). Patient Population

The techniques provided herein can be used to treat and/or prevent obesity or obesity-related diseases or conditions. As described herein, the techniques include polynucleotides encoding incretin agents, their immunoglobulin chains, or fragments thereof. Therefore, this disclosure provides pharmaceutical compositions for the treatment and/or prevention of obesity and obesity-related diseases or conditions (e.g., type 2 diabetes (T2D), early T1D (e.g., within 3 months of T1D diagnosis), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease, or kidney disease). In some embodiments, the pharmaceutical compositions comprise polynucleotides as described herein.

In some embodiments, an individual is an individual who has and/or is susceptible to obesity or an obesity-related disease or condition. In some embodiments, an individual may be defined by one or more criteria such as age group, sex, genetic background, pre-existing clinical disorder and/or previous exposure to therapy.

In some embodiments, an individual may be identified as requiring the pharmaceutical combination described herein based on screening tools used for obesity and obesity-related diseases and conditions. For example, in some embodiments, an individual may be identified as requiring the pharmaceutical combination described herein based on results obtained from enzyme immunoassay (EIA), Western ink spot and/or PCR testing, and/or weight, and/or waist circumference, and/or body mass index.

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

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

In some implementations, an individual is not currently considered obese. In some implementations, an individual is at risk of developing obesity.

In some implementations, individuals have and/or are at increased risk of obesity, prediabetes, type 2 diabetes (T2D, and its complications), early T1D, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease (e.g., characterized by major cardiovascular events (MACE), including CV death, non-fatal myocardial infarction, non-fatal stroke, heart failure with preserved ejection fraction (HFpEF)), kidney disease, or premature death.

In some embodiments, individuals have and/or are susceptible to additional comorbidities related to or unrelated to obesity, including any of the following: prediabetes, type 2 diabetes, early type 1 diabetes, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease (e.g., characterized by major cardiovascular events (MACE), including CV death, non-fatal myocardial infarction, non-fatal stroke, heart failure with preserved ejection fraction (HFpEF)), kidney disease, and an increased risk of premature death.

In some implementations, the individual has not previously received treatment for obesity or obesity-related diseases.

In some embodiments, individuals with and/or predisposition to obesity or obesity-related disorders may have received or are currently receiving other therapies for obesity. In some embodiments, individuals are currently receiving or have received one or more of the treatments listed in Table 1. In some embodiments, individuals with and/or predisposition to obesity or obesity-related disorders may have received or are currently receiving lifestyle interventions, such as reducing calorie intake and/or increasing physical activity.

In some embodiments, the individual has received one or more of the treatments listed in Table 1 for more than 1 week, more than 2 weeks, more than 3 weeks, more than 4 weeks, more than 5 weeks, more than 6 weeks, more than 7 weeks, more than 8 weeks, more than 12 weeks, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, or more than 1 year. In some embodiments, the individual responded to another treatment when given the polynucleotides, combinations, or pharmaceutical combinations described herein. In some embodiments, the individual did not respond to another treatment when given the polynucleotides, combinations, or pharmaceutical combinations described herein.

In some embodiments, the individual has previously received one or more treatments for obesity or obesity-related conditions (e.g., one or more treatments listed in Table 1). In some embodiments, the individual has received prior treatment for obesity for more than 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 1 year. In some embodiments, the individual responded to the prior treatment. In some embodiments, the individual did not respond to the prior treatment.

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

In some implementations, the individual did not receive any other treatment for obesity or obesity-related conditions in the previous month. In some implementations, the individual did not receive any other treatment for obesity or obesity-related conditions in the past year. In some implementations, the individual did not receive any other treatment for obesity or obesity-related conditions in the past two years. Treatment Methods

In some embodiments, the pharmaceutical composition described herein can be taken up by cells to produce a encoded incretin agent at a therapeutically relevant serum concentration. Therefore, this disclosure provides methods for using the pharmaceutical composition described herein. For example, in some embodiments, the methods provided herein involve administering the pharmaceutical composition described herein to an individual.

As used herein, the terms "administering" or "administration" generally refer to the administration of a composition to an individual to achieve delivery as an agent (e.g., at least one polynucleotide encoding an incretin agent as described herein) to a target or therapeutic site. Those generally skilled in this art will recognize that administration can be performed on individuals (e.g., humans) via various pathways in appropriate settings. Administration may be via, for example, the bronchus (e.g., via bronchial infusion), cheek, percutaneous (which may include one or more of the following: topical dermal, intradermal, interdermal, percutaneous, etc.), enteroenteric, intraarterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrasheath, intravenous, intraventricular, intra-organ (e.g., intrahepatic), mucosa, nasal, oral, rectal, subcutaneous, sublingual, topical, trachea (e.g., via tracheal infusion), vagina, vitreous body, etc. In a preferred embodiment, administration may be intramuscular, intraperitoneal, intravenous, or subcutaneous.

In some embodiments, administration of the pharmaceutical composition results in the delivery of one or more polynucleotides (e.g., encoding incretins) as described herein to an individual. In some embodiments, administration of the pharmaceutical composition to an individual results in the expression in the individual of an incretin encoded by the administered polynucleotide. In some embodiments, administration of the pharmaceutical composition to an individual results in the expression in the individual of an incretin encoded by the administered polynucleotide.

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

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

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

Those familiar with this technique recognize that the therapy can be administered in cycles. In some implementations, the pharmaceutical combination described herein is administered in one or more cycles.

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

In some implementations, a dosing cycle may involve multiple doses, for example, depending on the pattern, such as daily dosing during the dosing cycle, or dosing every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 2 weeks, every month, or every 2 months within a cycle.

In some embodiments, multiple administration cycles may be administered. For example, in some embodiments, at least two administration cycles may be administered (including, for example, at least three administration cycles, at least four administration cycles, at least five administration cycles, at least six administration cycles, at least seven administration cycles, at least eight administration cycles, at least nine administration cycles, at least ten administration cycles, or more). In some embodiments, the desired number of administration cycles may vary depending on the type of treatment (e.g., monotherapy versus combination therapy). In some embodiments, at least three to eight administration cycles may be administered.

In some embodiments, there may be a "rest period" between drug administration cycles; in other embodiments, there may be no rest period between drug administration cycles. In some embodiments, there may be a rest period between drug administration cycles and there may be no rest period.

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

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

In some embodiments, the dosing cycle involves, for example, administering a cumulative dose over a specific time period and, as appropriate, through multiple doses, which may be administered, for example, at set intervals and/or according to a set pattern. In some embodiments, the cumulative dose may be administered through multiple doses at set intervals, such that there is at least some temporal overlap in the bio and/or pharmacokinetic effects of such multiple doses on the target cells or the treated individual. In some embodiments, the cumulative dose may be administered through multiple doses at set intervals, such that the bio and/or pharmacokinetic effects of such multiple doses on the target cells or the treated individual can be additive. For example, in some embodiments, the cumulative dose of X mg can be administered by two doses, each of X/2 mg, which are administered sufficiently close in time to allow the bio and/or pharmacokinetic effects of the X/2 mg dose on the target cells or the treated individual to be additive.

In some embodiments, dosing may be adjusted based on the individual's response to the therapy. For example, in some embodiments, if one or more parameters used in a safety pharmacology assessment indicate that a previous dose may not meet the physician's medical safety requirements, dosing may involve administering a higher dose followed by a lower dose. In some embodiments, dose escalation may be performed at one or more levels. Not wishing to be bound by any particular theory, this disclosure specifically provides the following insight: the pharmaceutically guided dose escalation (PGDE) approach can be applied to determine the appropriate dose of the pharmaceutical combination described herein.

In some implementations, the pharmaceutical combination described herein may be administered to an individual as a single therapy.

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

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

In some embodiments, the combination therapy may include administration of a pharmaceutical composition comprising at least one polynucleotide encoding an incretin and administration of a pharmaceutical composition comprising a dipeptidyl peptidase-4 (DPP-4) inhibitor. Without being bound by any theory, administration of a pharmaceutical composition comprising at least one polynucleotide encoding an incretin and combined with the pharmaceutical composition comprising a DPP-4 inhibitor may increase the efficacy of the pharmaceutical composition by prolonging the activity of the incretin. In some embodiments, the pharmaceutical composition comprising at least one polynucleotide encoding an incretin is administered co-administered with another pharmaceutical composition comprising one or more DPP-4 inhibitors.

The administration of a DPP-4 inhibitor or a pharmaceutical composition containing a DPP-4 inhibitor may be, for example, by oral administration. In some embodiments, the DPP-4 inhibitor comprises sitagliptin, vildagliptin, saxagliptin, linagliptin, giglitazone, alagliptin, ticagliptin, alogliptin, trogliptin, octagliptin, elegliptin, gogliptin, dugliptin, neogliptin, repagliptin, degliptin, cogliptin, folagliptin, prolagliptin, berberine, or any combination thereof.

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

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

In some embodiments, a dosing cycle may involve, for example, the simultaneous or sequential administration of a pharmaceutical composition comprising one or more DPP-4 inhibitors and a pharmaceutical composition comprising at least one polynucleotide encoding incretin. In some embodiments, multiple dosing cycles may be administered. For example, in some embodiments, at least two dosing cycles may be administered (including, for example, at least three dosing cycles, at least four dosing cycles, at least five dosing cycles, at least six dosing cycles, at least seven dosing cycles, at least eight dosing cycles, at least nine dosing cycles, at least ten dosing cycles, or more). In some embodiments, at least three to eight dosing cycles may be administered.

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

In some embodiments, the efficacy of the administered treatment can be evaluated periodically in the dosing regimen by monitoring individuals receiving the combination (e.g., a pharmaceutical combination) provided herein. For example, in some embodiments, the efficacy of the administered treatment can be evaluated periodically, such as weekly, bi-weekly, 4-weekly, 5-weekly, 6-weekly, 7-weekly, 8-weekly, or longer. Manufacturing Method

Individual polynucleotides can be produced by methods known in this art. For example, in some embodiments, polynucleotides can be produced by in vivo extracellular transcription, such as using a DNA template. The plastid DNA used as an in vivo extracellular transcription template to produce the polynucleotides described herein is also within the scope of this disclosure.

In the presence of a suitable RNA polymerase (e.g., a recombinant RNA polymerase, such as T7 RNA polymerase) containing ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP), a DNA template can be used for in vitro RNA synthesis. In some embodiments, polynucleotides (e.g., those described herein) can be synthesized in the presence of modified ribonucleotide triphosphates. For example, 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).

Those familiar with this technique will know that during in vivo transcription, RNA polymerase (e.g., as described herein and/or utilized) typically passes through at least a portion of a single-stranded DNA template in the 3'→5' direction to produce single-stranded complementary RNA in the 5'→3' direction.

In some embodiments of polynucleotides containing a polyA tail, as will be appreciated by those skilled in the art, such a polyA tail can be encoded in a DNA template, for example, by using a suitable tailing PCR primer, or the polyA tail can be added to the polynucleotide after in vivo transcription, for example, by enzymatic treatment (e.g., using a poly(A) polymerase, such as E. coli poly(A) polymerase). Suitable poly(A) tails are described above. In some embodiments, the poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, the linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 40).

In some embodiments, those skilled in the art will understand that adding a 5' cap to RNA (e.g., mRNA) promotes RNA recognition and ligation to ribosomes to initiate transcription and enhances transcription efficiency. Those skilled in the art will also understand that the 5' cap protects RNA products from 5' exonuclease-mediated degradation, thereby increasing half-life. Methods for capping are known in the art; those generally skilled in the art will understand that in some embodiments, capping can be performed after in vivo transcription in the presence of a capping system (e.g., an enzyme-based capping system, such as the capping enzyme of vaccinia virus). In some embodiments, the cap and multiple ribonucleotide triphosphates can be introduced during in vivo transcription, such that the cap is incorporated into the polynucleotide during transcription (also known as co-transcriptional capping). In some embodiments, a batching program that adds GTP multiple times during the reaction can be used to maintain a low concentration of GTP for efficient capping of RNA. A suitable 5' cap is described above. For example, in some embodiments, the 5' cap contains m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

After RNA transcription, the DNA template is digested. In some embodiments, digestion can be achieved using DNase I under appropriate conditions.

In some embodiments, the polynucleotides transcribed in vivo may be provided in a buffer solution, such as HEPES, phosphate buffer, citrate buffer, or acetate buffer; in some embodiments, such solutions may be buffered to a pH in the range of, for example, about 6.5 to about 7.5; in some embodiments, about 7.0. In some embodiments, the production of polynucleotides may further include one or more of the following steps: purification, mixing, filtration, and/or filling.

In some embodiments, polynucleotides may be purified (e.g., in some embodiments following in vitro transcription reactions), for example, to remove components utilized or formed during the generation process, such as proteins, DNA fragments, and/or nucleotides. Various nucleic acid purification techniques known in this art can be used according to this disclosure. Certain purification steps may be or include one or more of the following: precipitation, column chromatography (including, for example, but not limited to, anion, cation, hydrophobic interaction chromatography (HIC)), and solid-based purification (e.g., magnetic bead-based purification). In some embodiments, magnetic bead-based purification may be used to purify polynucleotides, and in some embodiments it may be or include magnetic bead-based chromatography. In some embodiments, polynucleotides may be purified using hydrophobic interaction chromatography (HIC) and/or filtration. In some embodiments, polynucleotides may be purified using HIC followed by filtration.

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

In some embodiments, a batch of polynucleotides may be further processed by one or more filtration and/or concentration steps. For example, in some embodiments, after the polynucleotides have been decontaminated with dsRNA, they may be further filtered (e.g., by tangential flow filtration in some embodiments) to adjust the concentration of the polynucleotides to the desired RNA concentration and/or to replace the buffer with a drug substance buffer.

In some embodiments, the polynucleotides may be processed by 0.2 μm filtration before being filled into a suitable container.

In some embodiments, polynucleotides and their compositions may be manufactured according to processes as described herein or otherwise known in the art.

In some embodiments, polynucleotides and their combinations can be manufactured on a large scale. For example, in some embodiments, a batch of polynucleotides can be manufactured on 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.

In some embodiments, RNA quality control may be performed and/or monitored at any time during the production of polynucleotides and/or compositions thereof. For example, in some embodiments, RNA quality control parameters, including RNA consistency (e.g., sequence, length, and/or RNA properties), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and/or monitored after one or more steps of the polynucleotide production process (e.g., after in vivo transcription) and/or after each purification step.

In some embodiments, the stability of polynucleotides (e.g., produced by in vivo transcription) and/or compositions comprising one or more RNAs can be evaluated under various test storage conditions (e.g., room temperature versus refrigerator or sub-zero temperatures) for 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, polynucleotides (e.g., the polynucleotides described herein) and/or compositions thereof can be stably stored at refrigerator temperatures (e.g., from 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, polynucleotides (e.g., the polynucleotides described herein) and/or combinations thereof can be stored stably at sub-zero temperatures (e.g., -20°C or lower) for at least one month or longer, including at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months or longer. In some embodiments, polynucleotides (e.g., the polynucleotides described herein) and/or combinations thereof can be stored stably at room temperature (e.g., at about 25°C) for at least one month or longer.

In some embodiments, one or more evaluations (e.g., as release tests) may be used during the manufacture, or other preparation or use of the polynucleotide.

In some embodiments, one or more quality control parameters may be evaluated to determine whether the polynucleotides described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for allocation). 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, those skilled in the art will recognize that, in some embodiments, one or more analytical tests may be used for RNA quality assessment. Examples of such analytical tests may include, but are not limited to, gel electrophoresis, UV absorbance, and/or PCR assays.

In some embodiments, a batch of polynucleotides may be evaluated against one or more of the characteristics described herein to determine the next action step. For example, if RNA quality assessment indicates that a batch of polynucleotides meets or exceeds relevant acceptance criteria, that batch of polynucleotides may be designated for use in the manufacture and/or formulation and/or dispensing of one or more further steps. Otherwise, if a batch of polynucleotides does not meet or exceeds the acceptance criteria, alternative actions may be taken (e.g., discarding the batch).

In some embodiments, a batch of polynucleotides that meets the evaluation results can be used to manufacture and/or formulate and/or dispense one or more further steps. DNA architecture

This disclosure provides, in particular, DNA constructs that can encode one or more incretins or components thereof as described herein. In some embodiments, the DNA constructs provided by and/or utilized according to this disclosure are contained in a carrier.

Non-limiting examples of vectors include plasmid vectors, granule vectors, phage vectors such as λ phage, viral vectors (such as retrotransmitters, adenoviruses, or baculoviruses), or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC)). In some embodiments, the vector is a performance vector. In some embodiments, the vector is a selection vector. Generally, a vector is a nucleic acid construct (e.g., a construct that is or encodes a payload or endows it with a specific function) that can receive or otherwise link to a nucleic acid element of interest.

The expression vector can be a plasmid, virus, or other vector, and typically includes a expressible sequence of interest (e.g., an encoded sequence) functionally linked to 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, the system is ex vivo (e.g., an in vivo transcription system); in other embodiments, the system is in vivo (e.g., bacteria, yeast, plants, insects, fish, vertebrates, mammalian cells or tissues, etc.).

Selection vectors are generally used for modification, engineering, and/or replication (e.g., by in vivo replication, such as in simple systems like bacteria or yeast, or in vitro, such as by amplification processes like polymerase chain reactions or other amplification procedures). In some embodiments, selection vectors may lack phenotype signals.

In many embodiments, the vector may include replication elements, such as primer-binding sites and/or start sites for replication. In many embodiments, the vector may include insertion or modification sites, such as restriction endonuclease recognition sites and/or guided RNA binding sites.

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

Those skilled in this art will recognize that various techniques can be used to produce 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, and other techniques are well-established and useful tools and techniques. Alternatively or additionally, certain nucleic acids can be prepared or assembled by chemical and/or enzymatic synthesis. In some embodiments, combinations of known methods are used to prepare recombinant polynucleotides.

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

In some embodiments, the expression vector comprises a polynucleotide of the protein and/or polypeptide disclosed herein, the polynucleotide being operatively linked to one or more sequences controlling expression (e.g., promoters, initiation signals, termination signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, one or more sequences controlling expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence controlling expression (e.g., a promoter) is used. In some embodiments, more than one sequence controlling expression (e.g., a promoter) is used to achieve a desired level of expression for multiple polynucleotides encoding multiple proteins and/or polypeptides. In some embodiments, multiple recombinant proteins and/or polypeptides are expressed from the same vector (e.g., a bicistronic vector, a tricistronic vector, a polycistronic vector). In some embodiments, multiple polypeptides are expressed, each expressed on a separate carrier.

In some embodiments, the expression vector containing the polynucleotides disclosed herein is used to produce RNA and/or proteins and/or peptides in host cells. In some embodiments, the host cells may be extracellular (e.g., cell lines), such as cells or cell lines suitable for producing the polynucleotides disclosed herein and proteins and/or peptides encoded by such polynucleotides (e.g., human embryonic kidney (HEK cells), Chinese hamster ovary cells, etc.).

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

Various methods for introducing a performance vector into host cells are known in this art. In some embodiments, transfection can be used to introduce the vector into host cells. In some embodiments, transfection is performed, for example, using calcium phosphate transfection, lipid transfection, or polyethyleneimine-mediated transfection. In some embodiments, the vector can be introduced into host cells using transduction.

In some embodiments, after the vector is introduced into the host cells, the transformed host cells are cultured to allow the expression of the recombinant polynucleotides. In some embodiments, the transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours, or longer. The transformed host cells are cultured under growth conditions (e.g., temperature, carbon dioxide level, growth medium) depending on the requirements of the selected host cells. Those skilled in the art will recognize that the culture conditions for the selected host cells are well known in the art. Example 1: Generation of polynucleotides encoding exemplary incretins

This example describes a method for generating polynucleotide sequences encoding incretins. This example further describes the design of polynucleotides that enable transient in vivo incretin production following ip/iv/im/sc administration.

The method of this example includes: (1) selecting a DNA fragment encoding an incretin (e.g., GLP1, GIP, or a variant thereof) into a DNA plasmid suitable for RNA expression. A suitable 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 typically also includes restriction sites that enable the selection of a DNA fragment encoding an incretin downstream of the region encoding the 5' UTR and Kozak sequence and upstream of the region encoding the 3' UTR and polyA tail sequence. Examples of suitable DNA plasmids can also be found in WO2021/214204, which is hereby incorporated herein by reference in its entirety. (2) Verification of the selected strains by control digestion and conditional sequencing. (3) Linearization of DNA plastids encoding incretins. (4) Synthesis of polynucleotides encoding incretins. (5) Biochemical characterization of polynucleotides encoding incretins. (6) Transfection of polynucleotides encoding incretins into HEK cells and quantification of incretin levels. Codon optimization .

To best represent exemplary incretins, DNA sequences were generated based on the amino acid sequences of GLP1 (7-37), GIP (1-42), and their truncated or mutant variants fused to exemplary signal peptides (SP), including sequences (SP1-2, MRVLVLLACLAAASNA) and SPs shown in SEQ ID NO: 65 and 66, as shown in Table 11 above.

The amino acid sequence is translated into a DNA nucleotide sequence. If any of Eam1104I (GAAGAG), BamHI (GGATCC), PstI (CTGCAG), SbfI (CCTGCAGG), XhoI (CTCGAG), SpeI (ACTAGT), BspEI (TCCGGA), SacI (GAGCTC), Ear1 (CTCTTCN^NNN), and NheI (GCTAGC) (or other enzymes) are used for linearization or for selecting ploids, restriction sites targeting these enzymes are eliminated as appropriate after optimization. The presence of regions showing high homology to the T7 RNA polymerase termination signal sequence "ATCTGTT" and subsequent multiple "T" residues is also examined in the sequence.

Optimization is performed using GeneOptimizer® software provided by Life Technologies GmbH GeneArt®. This software adjusts codon usage by using the most frequently used codons and adjusts the GC content of the uploaded sequence for the selected phenotype (in this case, Homo sapiens ). Simultaneously, GeneOptimizer® removes sequence duplications, introns, hidden 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 stabilizing RNA secondary structures and unwanted sequences such as restriction sites. The output sequence is then used to sequence the DNA fragment string. Those familiar with this technique will recognize that alternative methods for codon optimization are available. In addition, this article provides extra information on cryptographic optimization methods. (Selection/breeding)

Each intestinal insulin sequence was selected and colonized into DNA plasmids (e.g., pST5). This can be accomplished, for example, by in vivo assembly. Garcia-Nafria, "IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly," Scientific Reports , Vol. 6, Article No. 27459 (2016), which is incorporated herein by reference in its entirety. Plasmid DNA Preparation

Plasmid DNA is prepared by selecting strains for inoculation into the culture medium. The selected strains are verified, as needed, by control digestion and sequencing. After cell harvesting, the culture is purified according to the manufacturer's instructions, such as using the QIAGEN Plasmid Plus Maxi kit. DNA concentration is determined by UV spectroscopy. The DNA is stored in certified RNase-free and DNase-free reaction tubes. Linearization and DNA purification are then performed.

Linearization of plastid DNA was 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 was measured by UV spectroscopy, control digestion, and sequencing as needed. In vivo extracellular transcription.

RNA was then generated, for example, following the process described in Kreiter et al., Cancer Immunol. Immunother . 2007, 56, 1577–87 and WO2021/214204, with capped RNA as appropriate. All of these references are incorporated herein by reference in their entirety. Methylpseudouridine was used in the in vivo transcription reaction and incorporated into the generated RNA. The resulting RNA was purified by cellulose to separate single-stranded RNA, followed by concentration determination by UV spectroscopy. RNA integrity was determined by microfluidic-based electrophoresis. Further biochemical characterization of the resulting RNA was performed as needed. Transfection and Expression

RNA encoding an incretin was transfected into HEK cells, for example, via electroporation, and the resulting incretin levels were quantified. HEK cells, such as HEK293T cells, were washed with chilled medium. Electroporation was performed in pre-chilled cuvettes. Cells and RNA in each sample were at typical concentrations for RNA electroporation. After electroporation, cells were incubated on ice.

The cells were then transferred to a expression medium (e.g., Expi293) and counted. The cells were seeded at the typical concentration used for expression and incubated at 37°C for, for example, 48 hours. The supernatant was then harvested by centrifugation, carefully aspirated to avoid disturbing the cell sediment, and stored at 4°C.

For example, the performance of incretin agents can be quantified using ELISA or Western blot analysis of cell culture supernatant. Example 2: Generating reporter cell lines to monitor incretin activity.

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

The method of this example includes: (1) selecting DNA fragments encoding incretin receptors (e.g., GLP1R and/or GIPR) into DNA plasmids (e.g., pT2). (2) stably transfecting HEK293 cells with DNA plasmids encoding incretin receptors and mRNA transposases. (3) sorting cells by FACS for high, medium, and low incretin receptor expression (e.g., GLP1R and/or GIPR expression), performing mass sorting as needed, followed by single-cell sorting. (4) confirming stable expression of incretin receptors (e.g., GLP1R and/or GIPR). (5) generating a master cell bank .

Exemplary DNA sequences encoding incretin receptors are shown in Table 15. These DNA sequences were selected and implanted into DNA plasmids (e.g., pT2). Slightly different variants of GLP1R, GLP1R_mutR and GLP1R_mutL, are encoded (GLP1R_mutR encodes the GLP1R with the L260F mutation as opposed to the GLP1R encoded by GLP1R_mutL). This can be accomplished, for example, by in vivo assembly. Garcia-Nafria, "IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly," Scientific Reports , Vol. 6, Article No. 27459 (2016), which is incorporated herein by reference in its entirety. Table 17: Sequences encoding exemplary incretin receptors. receptor sequence SEQ ID NO GLP1R_mutL atggccggcgcccccggcccgctgcgccttgcgctgctgctgctcgggatggtgggcagggccggcccccgcccccagggtgccactgtgtccctctggggagacggtgcagaaatggcgagaataccgacgccagtgccagcgctccctgactgaggatccacctcctgccacagact tgttctgcaaccggaccttcgatgaatacgcctgctggccagatggggagccaggctcgttcgtgaatgtcagctgcccctggtacctgccctgggccagcagtgtgccgcagggccacgtgtaccggttctgcacagctgaaggcctctggctgcagaaggacaactccagcctgcc ctggagggacttgtcggagtgcgaggagtccaagcgaggggagagaagctccccggaggagcagctcctgttcctctacatcatctacacggtgggctacgcactctccttctctgctctggttatcgcctctgcgatcctcctcggcttcagacacctgcactgcacccggaactac atccacctgaacctgtttgcatccttcatcctgcgagcattgtccgtcttcatcaaggacgcagccctgaagtggatgtatagcacagccgcccagcagcaccagtgggatgggctcctctcctaccaggactctctgagctgccgcctggtgtttctgctcatgcagtactgtgtggc ggccaattactactggctcttggtggagggcgtgtacctgtacacactgctggccttctcggtcttAtctgagcaatggatcttcaggctctacgtgagcataggctggggtgttcccctgctgtttgttgtcccctggggcattgtcaagtacctctatgaggacgagggctgctgg accaggaactccaacatgaactactggctcattatccggctgcccattctctttgccattggggtgaacttcctcatctttgttcgggtcatctgcatcgtggtatccaaactgaaggccaatctcatgtgcaagacagacatcaaatgcagacttgccaagtccacgctgacactcat ccccctgctggggactcatgaggtcatctttgcctttgtgatggacgagcacgcccgggggaccctgcgcttcatcaagctgtttacagagctctccttcacctccttccaggggctgatggtggccatctttatactgctttgtcaacaatgaggtccagctggaatttcggaagagc tgggagcgctggcggcttgagcacttgcacatccagagggacagcagcatgaagcccctcaagtgtcccaccagcagcctgagcagtggagccacggcgggcagcagcatgtacacagccacttgccaggcctcctgcagctgatgaTGgcgacctaggcatatgtgtacaacgcgtga 46 GLP1R_mutF atggccggcgcccccggcccgctgcgccttgcgctgctgctgctcgggatggtgggcagggccggcccccgcccccagggtgccactgtgtccctctggggagacggtgcagaaatggcgagaataccgacgccagtgccagcgctccctgactgaggatccacctcctgccacagact tgttctgcaaccggaccttcgatgaatacgcctgctggccagatggggagccaggctcgttcgtgaatgtcagctgcccctggtacctgccctgggccagcagtgtgccgcagggccacgtgtaccggttctgcacagctgaaggcctctggctgcagaaggacaactccagcctgcc ctggagggacttgtcggagtgcgaggagtccaagcgaggggagagaagctccccggaggagcagctcctgttcctctacatcatctacacggtgggctacgcactctccttctctgctctggttatcgcctctgcgatcctcctcggcttcagacacctgcactgcacccggaactac atccacctgaacctgtttgcatccttcatcctgcgagcattgtccgtcttcatcaaggacgcagccctgaagtggatgtatagcacagccgcccagcagcaccagtgggatgggctcctctcctaccaggactctctgagctgccgcctggtgtttctgctcatgcagtactgtgtggc ggccaattactactggctcttggtggagggcgtgtacctgtacacactgctggccttctcggtcttctctgagcaatggatcttcaggctctacgtgagcataggctggggtgttcccctgctgtttgttgtcccctggggcattgtcaagtacctctatgaggacgagggctgctgg accaggaactccaacatgaactactggctcattatccggctgcccattctctttgccattggggtgaacttcctcatctttgttcgggtcatctgcatcgtggtatccaaactgaaggccaatctcatgtgcaagacagacatcaaatgcagacttgccaagtccacgctgacactcat ccccctgctggggactcatgaggtcatctttgcctttgtgatggacgagcacgcccgggggaccctgcgcttcatcaagctgtttacagagctctccttcacctccttccaggggctgatggtggccatctttatactgctttgtcaacaatgaggtccagctggaatttcggaagagc tgggagcgctggcggcttgagcacttgcacatccagagggacagcagcatgaagcccctcaagtgtcccaccagcagcctgagcagtggagccacggcgggcagcagcatgtacacagccacttgccaggcctcctgcagctgatgaTGgcgacctaggcatatgtgtacaacgcgtga 47 GIPR atggatgactacctctccgatcctgcagctgctgctgcggctctcactgtgcgggctgctgctccagagggcggagacaggctctaaggggcagacggcgggggagctgtaccagcgctgggaacggtaccgcagggagtgccaggagaccttggcagccgcggaaccgccttcaggcctcgcctgta acgggtccttcgatatgtacgtctgctgggactatgctgcacccaatgccactgcccgtgcgtcctgcccctggtacctgccctggcaccaccatgtggctgcaggtttcgtcctccgccagtgtggcagtgatggccaatggggactttggagagaccatacacaatgtgagaacccagagaagaat gaggcctttctggaccaaaggctcatcttggagcggttgcaggtcatgtacactgtcggctactccctgtctctcgccacactgctgctagccctgctcatcttgagtttgttcaggcggctacattgcactagaaactatatccacatcaacctgttcacgtctttcatgctgcgagctgcggccat tctcagccgagaccgtctgctacctcgacctggcccctaccttggggaccaggcccttgcgctgtggaaccaggccctcgctgcctgccgcacg gcccagatcgtgacccagtactgcgtgggtgccaactacacgtggctgctggtggagggcgtctacctgcacagtctcctggtgctcgtggggagg ctccgaggagggccacttccgctactacctgctcctcggctggggggcccccgcgcttttcgtcattccctgggtgatcgtcaggtacctgtacgagaacacgcagtgctgggagcgcaacgaagtcaaggccatttggtggattatacggaccccccatcctcatgaccatcttgattaatttcctca tttttatccgcattcttggcattctcctgtccaagctgaggacacggcaaatgcgctgccgggattaccggctgaggctggctcgctccacgct gacgctggtgcccctgctgggtgtccacgaggtggtgtttgctcccgtgacagaggaacaggcccggggcgccctgcgcttcgccaagctcggc tttgagatcttcctcagctccttccagggcttcctggtcagcgtcctctactgcttcatcaacaaggaggtgcagtcggagatccgccgtggct ggcaccactgccgcctgcgccgcagcctgggcgaggagcaacgccagctcccggagcgcgccttccgggccctgccctccggctccggcccggg cgaggtccccaccagccgcggcttgtcctcggggaccctcccagggcctgggaatgaggccagccgggagttggaaagttactgcttgccaactttcttgtacaaagttggcattataagaaagcattgcttatcaatttgttgcaacgaatgatgaTGgcgacctaggcatatgtgtacaacgcgtga 48 Plastid DNA Preparation

Plasmid DNA was prepared by selecting strains for inoculation into the culture medium. The selected strains were verified, if necessary, by control digestion and, if necessary, sequencing. After cell harvesting, the culture was purified according to the manufacturer's instructions, such as using the QIAGEN Plasmid Plus Maxi kit. DNA concentration was determined by UV spectroscopy. The DNA was stored in certified RNase-free and DNase-free reaction tubes. Transfection

Plassomal DNA encoding incretin receptors and RNA encoding transposases (e.g., SB100X transposase) were transfected into HEK cells, for example, via electroporation. HEK cells, such as HEK293 cells, were washed with chilled medium. Electroporation was performed in pre-chilled cuvettes. Cells, plassomal DNA, and RNA in each sample were at typical concentrations for electroporation. After electroporation, cells were incubated on ice. Cell sorting was then performed.

Cells are then sorted using FACS (e.g., using the BD FACSMelody™ Cell Sorter) for high, medium, and low intestinal insulin receptor expression (e.g., GLP1R and/or GIPR expression), with mass sorting performed as needed, followed by single-cell sorting. Stable expression of intestinal insulin receptors in the selected strains is confirmed, and a master cell bank is generated. The resulting cell lines may include one or more of the following: ^high HEK293_GLP1R, ^medium HEK293_GLP1R, ^low HEK293_GLP1R, ^high HEK293_GIPR, ^medium HEK293_GIPR, ^low HEK293_GIPR, ^high HEK293_GLP1R/ ^high GIPR, ^medium HEK293_GLP1R/ ^medium GIPR, and ^low HEK293_GLP1R/ ^low GIPR. Example 3: Evaluation of the functionality of polynucleotide-encoded incretins.

This example describes a method for evaluating the functionality of incretins encoded by polynucleotides.

The method in this example includes: (1) Transfecting HEK293_GLP1R/GIPR reporter cells from Example 2 with GLP1 or GIP RNA from Example 1. The reporter cells can be incubated, for example, for 24 hours post-transfection before collecting the supernatant. (2) Quantifying downstream signal induction by measuring, for example, cAMP release in the supernatant, using a cAMP-Glo™ assay or ELISA.

The method of this example also includes: (1) transfecting wild-type HEK293 cells with GLP1 or GIP RNA from Example 1. (2) collecting the supernatant containing GLP1 and GIP. (3) incubating HEK293_GLP1R/GIPR reporter cells from Example 2 with the supernatant. The reporter cells may be incubated for, for example, 24 hours before collecting the supernatant. (4) quantifying downstream signal induction by measuring, for example, cAMP release in the supernatant, such as using the cAMP-Glo™ assay or ELISA. Example 4: In vitro functionalization of polynucleotides encoding incretins

This example demonstrates that polynucleotides encoding incretins, as described herein, can induce the production of incretins.

Methods: In this example, 6 × ^10⁴ HEK293t17 cells were seeded per well in three different 48-well plates and grown overnight at 37°C in a 5% CO₂ incubator. Cells were transfected using a Lipofectamine Messenger MAX kit (ThermoFisher Scientific, catalog number LMRNA003) with 0.6 μg of polynucleotide candidates (encoding GLP1 (7-37), GLP1 (7-37)-(K34R), GIP (1-30), and GIP (1-42) polynucleotides). Cells were then incubated. Supernatants were collected at 3, 6, 24, 48, and 72 hours post-transfection. At 3 and 6 hours post-transfection, the supernatant was collected and frozen at -80°C. At 24 hours post-transfection, collect the supernatant and replace the medium in the wells with fresh medium. Incubate the wells further until 48 and 72 hours post-transfection. Collect the supernatant at 24, 48, and 72 hours post-transfection and store at -80°C until further analysis.

The concentrations of GIP and GLP1 in the supernatant were then quantified using ELISA (Human GIP (Total) ELISA Kit and GLP1 (7-36) Active ELISA Kit, Merck Millipore). Statistical analysis was performed by one-way ANOVA followed by a post-hoc Tukey test.

Results: ELISA results showed that the polynucleotides encoding the incretins GLP1 (7-37), GLP1 (7-37)-(K34R), and GIP (1-42) were translated into proteins, reaching peak concentrations after 24 hours (see Figures 10, 11, and 12, respectively). Surprisingly, the results showed that GLP1 (7-37)-(K34R) was translated more efficiently than GLP1 (7-37), as indicated by a significant increase in GLP1 (7-37)-(K34R) concentration over 6 hours. Example 5: Production of polynucleotides encoding exemplary incretins

This example describes the production of polynucleotides encoding various incretins. This example further describes the design of polynucleotides that enable transient in vivo production of incretins after ip/iv/im/sc administration.

The method of this example includes: (1) Selecting a DNA fragment encoding an incretin (e.g., GLP1, GIP, or a variant thereof) into a DNA plasmid suitable for RNA expression. A suitable 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 typically also includes restriction sites that allow selection of a DNA fragment encoding an incretin downstream of the region encoding the 5' UTR and Kozak sequence and upstream of the region encoding the 3' UTR and polyA tail sequence. Examples of suitable DNA plasmids can also be found in WO2021/214204, which is hereby incorporated herein by reference in its entirety. (2) Verification of the selected strains by control digestion and conditional sequencing. (3) Linearization of DNA plastids encoding incretins. (4) Synthesis of polynucleotides encoding incretins. (5) Biochemical characterization of polynucleotides encoding incretins. (6) Transfection of polynucleotides encoding incretins into HEK cells and quantification of incretin levels.

Design of Exemplary Incretin Agents: To best express exemplary incretins, DNA sequences were generated based on the amino acid sequences of GLP1 (7-37), GIP (1-42), and their truncated or mutant variants fused to exemplary signal peptides (SP). These signal peptides include the viral signal peptides SP1-2 (“viral SP”) of SEQ ID NO: 17 and the husec (δ GS) signal peptide (“husec”) of SEQ ID NO: 65, as shown in Tables 10 and 11 above. For incretin agents designed to include the husec signal peptide, two different codon optimization methods were tested to determine whether the codon optimization methods affected the translation efficiency and functionality of the final incretin peptide. In addition, some incretins are designed to observe whether the linkers fused to the incretin peptides affect the performance and functionality of the incretins. Exemplary incretins resulting from this example are shown in Table 18 below. Table 18: Exemplary Incretins describe SEQ ID NO: Sequence (signal peptides are shown in bold) Virus SP, GLP1 (7-37) 41 MRVLVLLACLAAASNA HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG Virus SP, GLP1 with K34R mutation (7-37) 42 MRVLVLLACLAAASNA HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG husec SP, GLP-1 (7-37) with A8G (code-optimized variant 1 or "opt1") 55 MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG husec SP, GLP-1 (7-37) with A8G mutation, connector (code-optimized variant 1 or "opt1") 108 MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGGSGGGS husec SP, GLP-1 (7-37) with A8G mutation (codec-optimized variant 2 or "optp") 55 MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG husec SP, GLP-1 (7-37) with A8G mutation, connector (code-optimized variant 2 or "optp") 108 MRVMAPRTLILLLSGALALTETWA HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGGGGSGGGS Virus SP, GIP (1-42) 44 MRVLVLLACLAAASNA YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ husec SP, GIP with A2G mutation (1-42) (code-optimized variant 1 or "opt1") 54 MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ husec SP, GIP with A2G mutation (1-42) (code-optimized variant 2 or "optp") 54 MRVMAPRTLILLLSGALALTETWA YGEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Password optimization

The amino acid sequence is translated into a DNA nucleotide sequence. If any of Eam1104I (GAAGAG), BamHI (GGATCC), PstI (CTGCAG), SbfI (CCTGCAGG), XhoI (CTCGAG), SpeI (ACTAGT), BspEI (TCCGGA), SacI (GAGCTC), Ear1 (CTCTTCN^NNN), and NheI (GCTAGC) (or other enzymes) are used for linearization or for selecting ploids, restriction sites targeting these enzymes are eliminated as appropriate after optimization. The presence of regions showing high homology to the T7 RNA polymerase termination signal sequence "ATCTGTT" and subsequent multiple "T" residues is also examined in the sequence.

Optimization was performed using GeneOptimizer® software provided by Life Technologies GmbH's GeneArt®, employing two strategies. Both strategies generally optimize codon usage by using the most frequently used codons and adjusting the GC content of the uploaded sequence for the chosen phenotype (in this case, Homo sapiens ). Simultaneously, codon optimization removes sequence duplications, introns, hidden 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 stabilizing RNA secondary structures and unwanted sequences such as restriction sites. The output sequence is then used for sequencing the DNA fragment string. Those skilled in this technique will recognize that alternative methods for codon optimization are available. In addition, this article provides extra information on cryptographic optimization methods. (Selection/breeding)

Each intestinal insulin sequence was selected and colonized into a DNA plasmid (e.g., pST5). This can be accomplished, for example, by in vivo assembly. Garcia-Nafria, "IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly," Scientific Reports , Vol. 6, Article No. 27459 (2016).

The nucleotide sequences of the incretins generated from the plasmids produced in this example are shown in Table 19 below. Table 19: Exemplary Polynucleotides Encoding Incretins describe SEQ ID NO: sequence Virus SP, GLP1 (7-37) 177 ATGAGAGTGCTGGTGCTGCTGGCTTGTCTGGCCGCTGCCTCTAATGCTCATGCCGAGGGCACCTTTACCAGCGACGTGTCCTCTTACCTGGAAGGCCAGGCCGCCAAAGAGTTTATCGCCTGGCTGGTCAAAGGCAGAGGCTAATAG Virus SP, GLP1 with K34R mutation (7-37) 178 ATGAGAGTGCTGGTGCTGCTGGCTTGTCTGGCCGCTGCCTCTAATGCTCATGCCGAGGGCACCTTTACCAGCGACGTGTCCTCTTACCTGGAAGGCCAGGCCGCCAAAGAGTTTATCGCCTGGCTCGTCAGAGGCAGAGGATAATAG husec SP, GLP1 (7-37) with A8G mutation (codec-optimized variant 1 or "opt1") 179 ATGAGAGTGATGGCCCCTCGGACACTGATCCTGCTGCTTTCTGGTGCCCTGGCTCTGACAGAAACATGGGCTCATGGCGAGGGCACCTTCACCTCCGATGTGTCCTCTTACCTGGAAGGCCAGGCCGCCAAAGAGTTTATCGCCTGGCTGGTCAAAGGCAGAGGCTAATAG husec SP, GLP1 (7-37) with A8G mutation, connector (code-optimized variant 1 or "opt1") 180 ATGAGAGTGATGGCCCCTCGGACACTGATCCTGCTGCTTTCTGGTGCCCTGGCTCTGACAGAAACATGGGCTCATGGCGAGGGCACCTTCACCTCCGATGTGTCCTCTTACCTGGAAGGCCAGGCCGCCAAAGAGTTTATCGCCTGGCTGGTCAAAGGCAGAGGCGGCGGAGGAAGTGGCGGAGGATCTTAATAG husec SP, GLP1 (7-37) with A8G mutation (codec-optimized variant 2 or "optp") 181 ATGAGAGTGATGGCCCCTCGGACACTGATTCTGCTGCTGTCTGGTGCCCTGGCTCTGACAGAAACATGGGCCCACGGCGAGGGCACCTTTACCAGCGATGTGTCTTCTTATCTGGAAGGCCAGGCCGCCAAAGAGTTCATCGCTTGGCTTGTGAAAGGCAGAGGCTAATAG husec SP, GLP1 (7-37) with A8G mutation, connector (code-optimized variant 2 or "optp") 182 ATGAGAGTGATGGCCCCTCGGACACTGATTCTGCTGCTGTCTGGTGCCCTGGCTCTGACAGAAACATGGGCCCACGGCGAGGGCACCTTTACCAGCGATGTGTCTTCTTATCTGGAAGGCCAGGCCGCCAAAGAGTTCATCGCTTGGCTTGTGAAAGGCAGAGGTGGTGGCGGATCTGGCGGAGGATCTTAATAG Virus SP, GIP (1-42) 183 ATGAGAGTGCTGGTGCTGCTGGCTTGTCTGGCCGCTGCCTCTAATGCCTATGCCGAGGGCACCTTCATCAGCGACTACTCTATCGCCATGGACAAGATCCACCAGCAGGACTTCGTGAACTGGCTGCTGGCCCAGAAGGGCAAGAAGAACGACTGGAAGCACAACATCACCCAGTAATAG husec SP, GIP with A2G mutation (1-42) (code-optimized variant 1 or "opt1") 184 ATGAGAGTGATGGCCCCTCGGACACTGATCCTGCTGCTTTCTGGTGCCCTGGCTCTGACAGAGACATGGGCTTATGGCGAGGGCACCTTCATCAGCGACTACTCTATCGCCATGGACAAGATCCACCAGCAGGACTTCGTGAACTGGCTGCTGGCCCAGAAGGGCAAGAAGAACGACTGGAAGCACAACATCACCCAGTAATAG husec SP, GIP with A2G mutation (1-42) (code-optimized variant 2 or "optp") 185 ATGAGAGTGATGGCCCCTCGGACACTGATTCTGCTGCTGTCTGGTGCCCTGGCTCTGACAGAAACATGGGCCTATGGCGAGGGCACCTTCATCAGCGACTACAGCATCGCCATGGACAAGATCCACCAGCAGGACTTCGTGAACTGGCTGCTGGCCCAGAAGGGCAAGAAGAACGACTGGAAGCACAACATCACCCAGTAATAG Plastid DNA Preparation

Plasmid DNA is prepared by selecting a strain to be inoculated into the culture medium. The selected strain is verified, if necessary, by control digestion and sequencing. After cell harvesting, the culture is purified according to the manufacturer's instructions, for example, using the QIAGEN Plasmid Plus Maxi kit. DNA concentration is determined by UV spectroscopy. The DNA is stored in certified RNase-free and DNase-free reaction tubes. Linearization and DNA purification are then performed.

Linearization of plastid DNA was 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 was measured by UV spectroscopy, control digestion, and sequencing as needed. In vivo extracellular transcription.

RNA was then generated, for example, following the process described in Kreiter et al., Cancer Immunol. Immunother . 2007, 56, 1577–87 and WO2021/214204, with capped RNA as appropriate. All of these references are incorporated herein by reference in their entirety. Methylpseudouridine was used in the in vivo transcription reaction and incorporated into the generated RNA. The resulting RNA was purified by cellulose to separate single-stranded RNA, followed by concentration determination by UV spectroscopy. RNA integrity was determined by microfluidic-based electrophoresis. Further biochemical characterization of the resulting RNA was performed as needed. Transfection and Expression

RNA encoding an incretin was transfected into HEK cells, for example, via electroporation, and the resulting incretin levels were quantified. HEK cells, such as HEK293T cells, were washed with chilled medium. Electroporation was performed in pre-chilled cuvettes. Cells and RNA in each sample were at typical concentrations for RNA electroporation. After electroporation, cells were incubated on ice.

The cells were then transferred to a expression medium (e.g., Expi293) and counted. The cells were seeded at the typical concentration used for expression and incubated at 37°C for, for example, 48 hours. The supernatant was then harvested by centrifugation, carefully aspirated to avoid disturbing the cell sediment, and stored at 4°C.

For example, the performance of incretins can be quantified using ELISA or Western blot analysis of cell culture supernatant. Example 6: In vitro functionality of additional polynucleotides encoding incretins.

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

Methods: In this example, 6 × ^10⁴ HEK293t17 cells were seeded per well in three different 48-well plates and grown overnight at 37°C in a 5% CO₂ incubator. Cells were transfected with 0.6 μg of polynucleotide candidates using the Lipofectamine Messenger MAX kit (ThermoFisher Scientific, catalog number LMRNA003).

Cells were further incubated. Supernatants were collected at 3, 6, 24, 48, and 72 hours post-transfection. At 3 and 6 hours post-transfection, the supernatants were collected and frozen at -80°C. At 24 hours post-transfection, the supernatants were collected and the medium in the wells was replaced with fresh medium. The wells were further incubated until 48 and 72 hours post-transfection. The supernatants collected at 24, 48, and 72 hours post-transfection were stored at -80°C until further analysis.

The concentrations of GIP and GLP1 in the supernatant were then quantified using ELISA (Human GIP (Total) ELISA Kit and GLP1 (7-36) Active ELISA Kit, Merck Millipore). Statistical analysis was performed using one-way ANOVA followed by a post-hoc Tukey test.

Results : Figure 18 shows the concentration (pg/ml) of exemplary GLP1 incretin in the supernatant of HEK29t17 cells transfected with polynucleotides encoding exemplary GLP1 incretins containing either the viral signaling peptide (“viral SP”) or the husec signaling peptide (“husec”). The polynucleotides encoding the incretins: (1) husec GLP1 (7-37) with the A8G mutation and (2) husec GLP1 (7-37) with the A8G mutation and a linker showed significantly higher concentrations at 24 hours post-transfection. These results indicate that incretins including the husec signaling peptide and the A8G mutation exhibit superior translation. Furthermore, for both (1) husec GLP1 (7-37) with the A8G mutation and (2) husec GLP1 (7-37) with the A8G mutation, linker, and incretin, the codon-optimized variant 1 ("opt1") showed better translation than the codon-optimized variant 2 ("optp").

Figure 19 shows the concentration (ng/ml) of exemplary GIP incretin in the supernatant of HEK29t17 cells transfected with polynucleotides encoding incretins containing viral signaling peptides or husec signaling peptides. Polynucleotides encoding viral SP-GIP (1-42) showed the highest concentrations, particularly at 24 and 48 hours post-transfection. These results indicate superior transfection of incretins, including those induced by viral signaling peptides. Codon-optimized variants containing husec signaling peptides showed similar transfection efficiencies. Example 7: Bioactivity of incretins transfected from polynucleotides.

This example confirms the biological activity of the incretins described herein as polynucleotides delivered to and subsequently translated into cells. Various incretins encoded by the polynucleotides described in Example 5 were tested.

Methods: The purpose of this experiment was to determine the biological activities of GIP and GLP1 incretins translated into the GLP1R-CRE and GIPR-CRE luciferase reporter gene-HEK293 cell lines (see Figure 22 ). Day 0: Flat-lay HEK293 CRE reporter cells expressing GLP1R and GIPR.

HEK293 cells carrying the GLP1R-CRE and GIPR-CRE luciferase reporter genes were seeded at a density of approximately 38,000 cells/well in 100 μl of their specific culture medium in 96-well white clear plates. The cells were incubated at 37°C in a CO2 incubator for 2 days. Day 1: Cell stimulation and luciferase assay

24 hours after transfection, gently remove 100 μl of culture medium from each well and incubate the cells at 37°C with 5% CO2 for 6 hours. Add 100 μl of ONE-Step™ luciferase reagent to each well. Gently shake the cells at room temperature for 15 min and then measure the fluorescence. The results are expressed as the fold increase relative to the control sample.

The bioactivity of GIP and GLP1 candidates was tested in 100 μl of DMEM at concentrations of 1.75 nM and 50 pM, respectively. GLP1, smegglutinin, and telposide were used as controls for GLP1 assay. GIP and telposide were used as controls for GIP assay. Results:

The results of the bioactivity assay of GLP1 incretins are shown in Figure 23. The results show that GLP1 incretins with the husec signaling peptide and the A8G mutation exhibit better bioactivity than those with the viral signaling peptide. Furthermore, the results show that codon optimization of GLP1 incretins does not affect bioactivity. The specific bioactivity of the GLP1 variant encoded by the viral signaling peptide mRNA is comparable to that of the controls (GLP1, smegrubin, and telperidine). Surprisingly, the specific bioactivity of the GLP1 variant encoded by the husec signaling peptide mRNA is superior to that of the controls (GLP1, smegrubin, and telperidine).

The results of the bioactivity assays of GIP incretins are shown in Figure 24. The results show that GIP incretins with the husec signaling peptide and the A2G mutation exhibit better bioactivity than those with the viral signaling peptide. Furthermore, the results indicate that the codon optimization strategy does not affect the bioactivity of the GIP incretins. The specific bioactivity of mRNA-encoded GIP incretins (using husec or the viral signaling peptide) is lower than that of the controls (GIP and telpolide).

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

Without being bound by any theory, in the context of the polynucleotides encoding incretins described herein, the choice of signal peptides can influence the manner in which the N-terminus of incretin peptides is cleaved. Certain signal peptides can lead to substitution of processing or cleavage sites, ultimately altering the final amino acid sequence of the mature incretin peptide. In such relatively small peptides, changes in amino acid sequence can significantly affect biological activity. These results support the view that, when designing polynucleotide constructs encoding incretin peptides (or other similar peptides), signal peptides should be chosen to influence the appropriate cleavage of the N-terminus of the incretin peptide, or in other words, to produce a scarless N-terminus to maintain the biological activity of the incretin peptide. Figures 20 and 21 illustrate the theoretical cleavage sites of various signal peptides. Figure 20 shows that the A8G mutation promotes the correct N-terminal processing of a GLP1 incretin containing the husec signal peptide. Figure 21 shows that the A2G mutation promotes the correct N-terminal processing of a GIP incretin containing the husec signal peptide. The results in this example show that signal peptide selection affects translation and biological activity, possibly due to the manner in which the signal peptide is cleaved from incretin peptides. Example 8: Generation of polynucleotides encoding exemplary incretins.

This example describes the production of polynucleotides encoding various incretins. This example further describes the design of polynucleotides that enable transient in vivo production of incretins after ip/iv/im/sc administration. The exemplary incretin produced in this example utilizes the various strategies described herein to improve the activity and half-life of incretins.

The method of this example includes: (1) Selecting a DNA fragment encoding an incretin (e.g., GLP1, GIP, or a variant thereof) into a DNA plasmid suitable for RNA expression. A suitable 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 typically also includes restriction sites that allow selection of a DNA fragment encoding an incretin downstream of the region encoding the 5' UTR and Kozak sequence and upstream of the region encoding the 3' UTR and polyA tail sequence. Examples of suitable DNA plasmids can also be found in WO2021/214204, which is hereby incorporated herein by reference in its entirety. (2) Verification of the selected strains by control digestion and conditional sequencing. (3) Linearization of DNA plastids encoding incretins. (4) Synthesis of polynucleotides encoding incretins. (5) Biochemical characterization of polynucleotides encoding incretins. (6) Transfection of polynucleotides encoding incretins into HEK cells and quantification of incretin levels.

Design of Exemplary Incretin Agents: To best express exemplary incretins, DNA sequences were generated based on the amino acid sequences of GLP1 (7-37), GIP (1-42), and their truncated or mutant variants fused to exemplary signal peptides (SP). These signal peptides include the viral signal peptide gD1 of SEQ ID NO: 66 and the husec (δGS) signal peptide of SEQ ID NO: 65 ("husec"), as shown in Tables 10 and 11 above. The incretin agents generated in this example were also examined for different half-life extensions and combinations of multiple incretin peptides. The exemplary incretin agents generated in this example are shown in Table 20 below. Table 20: Exemplary incretin preparations, where examples x2 and x4 include linkers between the repeating units and furin cleavage sites. Architectural ID describe SEQ ID NO: 3815 husec SP, GLP1 with A8G mutation (7-37) 55 3817 husec SP, GIP with A2G mutation (1-42) 54 4071 Husec SP, GLP1 (7-37) 52 4072 Husec SP, GIP (1-42) 53 4073 husec SP, GLP1 with H7Y and A8G mutations (7-37) 56 4074 gD1 SP, GLP1 (7-37) 57 4075 gD1 SP, GLP1 with K34R mutation (7-37) 161 4076 gD1 SP, GLP1 with A8G mutation (7-37) 60 4077 gD1 SP, GLP1 with H7Y and A8G mutations (7-37) 61 4078 gD1 SP, GIP (1-42) 58 4079 gD1 SP, GIP with A2G mutation (1-42) 59 4080 husec SP, GLP1 (7-37)-linker-furin protease-GIP (1-42) with H7Y, A8G and R36G mutations 124 4081 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation. 126 4082 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x2 127 4083 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 128 4084 husec SP, GLP1 (7-37) linker with H7Y, A8G and R36G mutations- Dula_IgG4 136 4085 husec SP, GLP1(7-37)-linker-Dula_IgG4 (LS) with H7Y, A8G, R36G mutations. 162 4086 husec SP, GIP (1-42) with A2G mutation - linker - Dula_IgG4 138 4087 husec SP, GIP (1-42) with A2G mutation - linker - Dula_IgG4 (LS) 163 4088 husec SP, GLP1 (7-37) connector with H7Y, A8G, R36G mutations- FcKIH-a (LS and STR) 131 4089 husec SP, GIP (1-42) with A2G mutation - connector - FcKIH-b LS and STR) 133 4090 husec SP, GLP1 (7-37) connector with H7Y, A8G, R36G mutations- hAlbumin 143 4091 husec SP, GLP1 (7-37) connector with H7Y, A8G, R36G mutations - aHSA-VHH 144 4092 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - furin protease - GIP (1-42) with A2G mutation - linker - Dula_IgG4 142 4093 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations, furin protease, GIP (1-42) with A2G mutation, linker, Dula_IgG4 (LS) 164 4094 husec SP, GIP (1-42) with A2G mutation - furin protease - GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - Dula_IgG4 (LS) 165 4095 husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 - linker - Dula_IgG4 (LS) 166 4096 husec SP, GIP with A2G mutation (1-42) - connector - hAlbumin 145 4097 husec SP, GIP with A2G mutation (1-42) - connector - aHSA-VHH 146 4098 husec SP, GLP1 (7-37) furin protease-GIP (1-42) linker-hAlbumin with H7Y, A8G, R36G mutations 147 4099 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker-furin protease-GIP (1-42) with A2G mutation - linker-hAlbumin 149 4100 husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x2 - linker - hAlbumin 151 4101 Husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin-GIP (1-42) with A2G mutation] x4 - linker - hAlbumin 152 Password optimization

The amino acid sequence is translated into a DNA nucleotide sequence. If any of Eam1104I (GAAGAG), BamHI (GGATCC), PstI (CTGCAG), SbfI (CCTGCAGG), XhoI (CTCGAG), SpeI (ACTAGT), BspEI (TCCGGA), SacI (GAGCTC), Ear1 (CTCTTCN^NNN), and NheI (GCTAGC) (or other enzymes) are used for linearization or for selecting ploids, restriction sites targeting these enzymes are eliminated as appropriate after optimization. The presence of regions showing high homology to the T7 RNA polymerase termination signal sequence "ATCTGTT" and subsequent multiple "T" residues is also examined in the sequence.

Optimization was performed using GeneOptimizer® software provided by Life Technologies GmbH's GeneArt®, employing two strategies. Both strategies generally optimize codon usage by using the most frequently used codons and adjusting the GC content of the uploaded sequence for the chosen phenotype (in this case, Homo sapiens ). Simultaneously, codon optimization removes sequence duplications, introns, hidden 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 stabilizing RNA secondary structures and unwanted sequences such as restriction sites. The output sequence is then used for sequencing the DNA fragment string. Those skilled in this technique will recognize that alternative methods for codon optimization are available. In addition, this article provides extra information on cryptographic optimization methods. (Selection/breeding)

Each intestinal insulin sequence was selected and colonized into a DNA plasmid (e.g., pST5). This can be accomplished, for example, by in vivo assembly. Garcia-Nafria, "IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly," Scientific Reports , Vol. 6, Article No. 27459 (2016).

The nucleotide sequences of the incretins generated from the plasmids produced in this example are shown in Table 21 below. Table 21: Exemplary polynucleotides encoding incretins, where examples x2 and x4 include linkers between repeating units and furin cleavage sites. Architectural ID describe Nucleic acid sequence - payload SEQ ID NO 3815 husec SP, GLP1 (7-37) with A8G mutation (codec-optimized variant 1 or "opt1") 179 3817 husec SP, GIP with A2G mutation (1-42) (code-optimized variant 1 or "opt1") 184 4071 husec SP, GLP1 (7-37) (a codec-optimized variant 1 or "opt1") 226 4072 husec SP, GIP (1-42) (a variant 1 or "opt1" optimized with cryptography) 227 4073 husec SP, GLP1 (7-37) with H7Y and A8G mutations (codec-optimized variant 1 or "opt1") 228 4074 gD1 SP, GLP1 (7-37) (Variant 1 or "opt1" optimized by the codeword) 229 4075 gD1 SP, GLP1 (7-37) with K34R mutation (codec-optimized variant 1 or "opt1") 230 4076 gD1 SP, GLP1 (7-37) with A8G mutation (codec-optimized variant 1 or "opt1") 231 4077 gD1 SP, GLP1 (7-37) with H7Y and A8G mutations (codecoin-optimized variant 1 or "opt1") 232 4078 gD1 SP, GIP (1-42) (Variant 1 or "opt1" optimized by the code) 233 4079 gD1 SP, GIP (1-42) with A2G mutation (code-optimized variant 1 or "opt1") 234 4080 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations - linker-furin protease-GIP (1-42) (codon-optimized variant 1 or "opt1") 235 4081 husec SP, GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation (codon-optimized variant 1 or "opt1") 236 4082 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x2 237 4083 husec SP, [GLP1 (7-37) with H7Y, A8G and R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 (codon-optimized variant 1 or "opt1") 238 4084 husec SP, GLP1 (7-37)-linker-Dula_IgG4 with H7Y, A8G and R36G mutations (codon-optimized variant 1 or "opt1") 239 4085 husec SP, GLP1 (7-37)-linker-Dula_IgG4 (LS) with H7Y, A8G, R36G mutations (codon-optimized variant 1 or "opt1") 240 4086 husec SP, GIP (1-42) with A2G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") 241 4087 husec SP, GIP (1-42) with A2G mutation - linker - Dula_IgG4 (LS) (codon-optimized variant 1 or "opt1") 242 4088 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - FcKIH-a (codecoin-optimized variant 1 or "opt1") 243 4089 husec SP, GIP with A2G mutation (1-42) - FcKIH-b (code-optimized variant 1 or "opt1") 244 4090 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - connector - hAlbumin (code-optimized variant 1 or "opt1") 245 4091 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - connector - aHSA-VHH (code-optimized variant 1 or "opt1") 246 4092 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation - linker - Dula_IgG4 (codon-optimized variant 1 or "opt1") 247 4093 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation - linker - Dula_IgG4 (LS) (codon-optimized variant 1 or "opt1") 248 4094 husec SP, GIP (1-42) with A2G mutation - linker - furin protease - GLP1 (7-37) with H7Y, A8G, R36G mutation - linker - Dula_IgG4 (LS) (codon-optimized variant 1 or "opt1") 249 4095 husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 - linker - Dula_IgG4 (LS) (codon-optimized variant 1 or "opt1") 250 4096 husec SP, GIP with A2G mutation (1-42) - connector - hAlbumin (code-optimized variant 1 or "opt1") 251 4097 husec SP, GIP (1-42) with A2G mutation - connector - aHSA-VHH (code-optimized variant 1 or "opt1") 252 4098 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker-furin protease-GIP (1-42) - linker-hAlbumin (codon-optimized variant 1 or "opt1") 253 4099 husec SP, GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation - linker - hAlbumin (codon-optimized variant 1 or "opt1") 254 4100 husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x2 - linker - hAlbumin (codon-optimized variant 1 or "opt1") 255 4101 husec SP, [GLP1 (7-37) with H7Y, A8G, R36G mutations - linker - furin protease - GIP (1-42) with A2G mutation] x4 - linker - hAlbumin (codon-optimized variant 1 or "opt1") 256 Plastid DNA Preparation

Plasmid DNA is prepared by selecting a strain to be inoculated into the culture medium. The selected strain is verified, if necessary, by control digestion and sequencing. After cell harvesting, the culture is purified according to the manufacturer's instructions, for example, using the QIAGEN Plasmid Plus Maxi kit. DNA concentration is determined by UV spectroscopy. The DNA is stored in certified RNase-free and DNase-free reaction tubes. Linearization and DNA purification are then performed.

Linearization of plastid DNA was 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 was measured by UV spectroscopy, control digestion, and sequencing as needed. In vivo extracellular transcription.

RNA was then generated, for example, following the process described in Kreiter et al., Cancer Immunol. Immunother . 2007, 56, 1577–87 and WO2021/214204, with capped RNA as appropriate. All of these references are incorporated herein by reference in their entirety. Methylpseudouridine was used in the in vivo transcription reaction and incorporated into the generated RNA. The resulting RNA was purified by cellulose to separate single-stranded RNA, followed by concentration determination by UV spectroscopy. RNA integrity was determined by microfluidic-based electrophoresis. Further biochemical characterization of the resulting RNA was performed as needed. Transfection and Expression

RNA encoding an incretin was transfected into HEK cells, for example, via electroporation, and the resulting incretin levels were quantified. HEK cells, such as HEK293T cells, were washed with chilled medium. Electroporation was performed in pre-chilled cuvettes. Cells and RNA in each sample were at typical concentrations for RNA electroporation. After electroporation, cells were incubated on ice.

The cells were then transferred to a expression medium (e.g., Expi293) and counted. The cells were seeded at the typical concentration used for expression and incubated at 37°C for, for example, 48 hours. The supernatant was then harvested by centrifugation, carefully aspirated to avoid disturbing the cell sediment, and stored at 4°C.

For example, the performance of incretins can be quantified using ELISA or Western blot analysis of cell culture supernatant. Example 9: In vitro functionality and bioactivity of additional polynucleotides encoding incretins.

This example examines the in vitro functionality and biological activity of the polynucleotides encoding incretins produced as described in Example 8. This example compares peptide secretion levels and demonstrates that the polynucleotides produced in Example 8 can induce different levels of incretin production. Furthermore, this example confirms the biological activity of the incretins described herein as polynucleotides delivered to cells and subsequently translated. The biological activity of various incretins encoded by the polynucleotides described in Example 8 was tested. This example also examines various design strategies to improve the properties of the expressed incretins, such as half-life, N-terminal cleavage, stability, translation efficiency, and biological activity. Methods:

In vivo appearance: In this example, 6 × ^10⁴ HEK293t17 cells were seeded per well in three different 48-well plates and grown overnight at 37°C in a 5% CO₂ incubator. Cells were transfected with 0.6 μg of polynucleotide candidates using the Lipofectamine Messenger MAX kit (ThermoFisher Scientific, catalog number LMRNA003).

Cells were further incubated. Supernatants were collected at 3, 6, 24, 48, and 72 hours post-transfection. At 3 and 6 hours post-transfection, the supernatants were collected and frozen at -80°C. At 24 hours post-transfection, the supernatants were collected and the medium in the wells was replaced with fresh medium. The wells were further incubated until 48 and 72 hours post-transfection. The supernatants collected at 24, 48, and 72 hours post-transfection were stored at -80°C until further analysis.

The concentrations of GIP and GLP1 in the supernatant were then quantified using ELISA (Human GIP (Total) ELISA Kit and GLP1 (7-36) Active ELISA Kit, Merck Millipore). Statistical analysis was performed using one-way ANOVA followed by a post-hoc Tukey test.

Bioactivity: The purpose of this experiment was to determine the bioactivity of GIP and GLP1 incretins translated into the GLP1R-CRE and GIPR-CRE luciferase reporter gene-HEK293 cell lines (see Figure 22 ). Day 0: Flatbed display of HEK293 CRE reporter cells expressing GLP1R and GIPR.

HEK293 cells carrying the GLP1R-CRE and GIPR-CRE luciferase reporter genes were seeded at a density of approximately 38,000 cells/well in 100 μl of their specific culture medium in 96-well white clear plates. The cells were incubated at 37°C in a CO2 incubator for 2 days. Day 1: Cell stimulation and luciferase assay.

24 hours after transfection, gently remove 100 μl of culture medium from each well and incubate the cells at 5% CO2 and 37°C for 6 hours. Add 100 μl of ONE-Step™ luciferase reagent to each well. Gently shake the cells at room temperature for 15 min and then measure the fluorescence. The results are expressed as the fold increase relative to the control sample.

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

Figure 25 shows the GIP performance of all the incretins tested. The constructs comprising two or more incretin peptides (i.e., 4081 and 4082) and those fused to Dula_IgG4 (i.e., 4092 and 4093) showed the highest performance, including at a later time point of 72 hours.

Figure 26 shows the GIP bioactivity of all the GIP-containing incretins tested (in each of the three replicates). Each construct used the same volume of supernatant (with different concentrations of incretins as shown in Figure 25), and therefore different amounts of proteins exhibited bioactivity in this experiment. For example, constructs 4081 and 4082 showed high GIP activity in Figure 25, which may have led to the high GIP bioactivity shown in Figure 26.

Figure 27 shows the GLP1 performance of all the incretins tested.

Figure 28 shows the GLP1 bioactivity of all the GLP1-containing incretins tested. Each construct used the same volume of supernatant (as shown in Figure 27, incretins of different concentrations), and therefore different amounts of proteins were observed when assessing bioactivity in this experiment.

Figure 29 shows a comparison of GIP performance ( A ) and GIP bioactivity ( B ) among candidates with different signaling peptides (husec versus gD1). Incretins with the gD1 signaling peptide showed increased GIP performance (approximately 2X) and increased GIP bioactivity (approximately 5X) compared to the same incretin with the husec signaling peptide. As described herein, this disclosure recognizes that the selection of the signaling peptide used in the polynucleotide construct encoding incretins can be important for promoting proper expression and cleavage of the signaling peptide, resulting in an N-terminus of the incretin that is "scarless" and retains its functionality to bind to its homologous receptor (e.g., GIPR).

Figure 30 shows a comparison of GLP1 performance ( A ) and GLP1 bioactivity ( B ) among candidates with different signaling peptides (husec vs. gD1). Incretins containing the gD1 signaling peptide showed increased GLP1 performance compared to incretins containing the husec signaling peptide (see Figure 30A). However, incretins utilizing the gD1 signaling peptide do not necessarily exhibit better GLP1 bioactivity. Incretin formulations utilizing the husec signaling peptide (4071, 3815, and 4073) exhibited lower GLP1 expression, and therefore used less GLP1 peptide in bioactivity assays. However, even with the lower GLP1 peptide content, 3815 showed comparable bioactivity to gD1-containing incretin formulations 4074, 4075, and 4076 (which contained higher levels of GLP1 peptide in bioactivity assays). Therefore, although gD1-containing incretin formulations appear to have increased GLP1 expression compared to those containing the husec signaling peptide, increased GLP1 expression does not necessarily lead to increased GLP1 bioactivity.

Figure 31 shows a comparison of GIP performance (A) and GIP bioactivity (B) with and without various half-life extension (HLE) moieties. The results show that the incretins containing Dula_IgG4 (4086 and 4087) performed well ( A ). Without being bound by any theory, the high performance could be due to the overall size of the polynucleotides. However, the results show that higher performance does not necessarily lead to improved GIP bioactivity, which was relatively low ( B ). The albumin-containing incretin (4096) showed consistent GIP performance and bioactivity compared to other incretins. In addition, the incretin agent (4097) containing VHH bound to albumin (a-HSA VHH) exhibited a relatively low level of GIP performance ( A ), but showed higher GIP bioactivity even with fewer proteins detected ( B ). The incretin agent (4089) containing the Fc fusion with the KIH mutation (FcKIH-b) also showed consistent GIP performance and GIP bioactivity.

Figure 32 shows a comparison of GLP1 performance (A) and GLP1 bioactivity (B) with and without various half-life extension (HLE) moieties. The results show that the incretin agent (4088) with the KIH-mutated Fc domain fusion (FcKIH-a) exhibits consistent GLP1 performance ( A ) and GLP1 bioactivity ( B ), similar to the GLP1 performance/bioactivity (4089) of FcKIH-b in Figure 31 (1:1 translation/bioactivity), indicating that the HLE moiety (FcKIH) does not interfere with bioactivity. Incretins containing Dula_IgG4 (4084 and 4085) performed well ( A ), possibly due to the overall size of the polynucleotide, but exhibited relatively low GLP1 bioactivity ( B ), similar to the GIP performance/bioactivity results in Figure 31. Additionally, the results showed that the incretin containing albumin-bound VHH (a-HSA VHH) (4091) had a relatively low performance level ( 32A ), but showed higher bioactivity even with fewer detected GLP1 peptides ( 32B ), consistent with the results in Figure 31.

In summary, the results in Figures 31 and 32 surprisingly show that the HLE moiety described herein does not reduce the bioactivity of incretins, even though the HLE moiety is larger in size compared to incretin peptides.

Figures 33 and 34 show a comparison of the performance ( Figures 33A and 34A ) and biological activity ( Figures 33B and 34B ) of GIP and GLP1 in exemplary incretin formulations containing GIP and GLP1, respectively, where the order of the GIP and GLP1 peptides encoded by a single polynucleotide changes. For example, incretin 4093 contains, from the N-terminus to the C-terminus: a GLP1 peptide (with H7Y, A8G, R36G mutations), a furin cleavage site, a GIP peptide (with A2G mutation), fused to Dula_IgG4 (with LS mutation), while incretin 4094 contains, from the N-terminus to the C-terminus: a GIP peptide (with A2G mutation), a furin cleavage site, a GLP1 peptide (with H7Y, A8G, R36G mutations), fused to Dula_IgG4 (with LS mutation).

Figure 33 shows the GIP performance in both 4093 and 4094 ( A ), however, considering the amount of GIP peptide tested, 4094 (where GIP is at the N-terminus) exhibits slightly better GIP bioactivity ( B ). In Figure 34, GLP1 performs better in the 4094 construct (where GLP1 is after GIP and adjacent to the furin cleavage site) than in the 4093 construct ( A ), however, GLP1 shows higher bioactivity in the 4093 construct, where GL1 is adjacent to the husec signaling peptide at the N-terminus ( B ). These results indicate a position-dependent effect at the N-terminus, including GLP1 versus GIP. Specifically, GIP appears to exhibit better bioactivity upon furin cleavage, and GLP1 appears to exhibit better bioactivity upon processing with the husec signaling peptide. As described herein, for incretin peptides to function and interact with their homologous receptors GIPR and GLP1R, they are preferably properly cleaved (i.e., cleaved with signal peptides or furin) and have a scarless N-terminus (i.e., cleavage/processing at the N-terminus should not alter the peptide structure). Equivalents

Those skilled in the art will recognize or be able to identify many equivalents of the specific embodiments of the invention described herein using only conventional experiments. The scope of the invention is not intended to be limited to the above description, but rather as set forth in the following patent claims:

Figure 1 illustrates an exemplary therapeutic strategy for delivering and expressing in vivo incretin agents using polynucleotides as described herein. Figure 2 shows a schematic diagram of an exemplary polynucleotide encoding an incretin agent. Figure 3 illustrates an exemplary design of the incretin agent described herein. Specifically, Figure 3 shows a schematic diagram (top) of the polynucleotide encoding the signal peptide (“SP”) and a single incretin peptide (referred to herein as “I:1x”), and a schematic diagram (bottom) of the translated incretin protein. Figure 4 illustrates an exemplary design of the incretin agent described herein. Specifically, Figure 4 shows a schematic diagram (top) of the polynucleotides encoding the signal peptide (“SP”) and two incretin peptides (this configuration is referred to herein as “I:2x”) separated by a linker (“L1”) and a furin cleavage site (“F”), and a schematic diagram of the translated protein (bottom). Figure 5 shows an exemplary design of the incretin agent described herein. Specifically, Figure 5 shows a schematic diagram (top) of the polynucleotides encoding the signal peptide (“SP”) and four incretin peptides (this configuration is referred to herein as “I:4x”) separated by furin cleavage sites (“F”) of each linker (“L1”), and a schematic diagram of the translated protein (bottom). Figures 6A-6B illustrate exemplary incretin formulations, including an incretin formulation having a signal peptide (“SP”), a GLP1 incretin peptide, and a (GGGGS) ₂ linker ( Figure 6A ), and an incretin formulation having a signal peptide (“SP”), a GLP1 incretin peptide, a (GGGGS) ₂ linker, a furin cleavage site, and a GIP incretin peptide ( Figure 6B ). The signal peptide cleavage site is indicated in Figures 6A and 6B . Additionally, the furin cleavage site is indicated in Figure 6B , such that, upon expression, the GIP incretin peptide cleaves from the GLP1 incretin peptide. Figure 7 illustrates an exemplary design of the incretin formulations described herein. Specifically, Figure 7 shows a schematic diagram (top) of the polynucleotide encoding a signal peptide (“SP”) and an incretin (e.g., I:1x, I:2x, or I:4x incretins as described herein) and a schematic diagram (bottom) of the translated protein, which is fused via a linker (“L2”) to a half-life extension (“HLE”) domain, such as human serum albumin (“HSA”) or an albumin-binding domain (“ABD”). Figures 8A-8B show exemplary incretins that can be encoded by the polynucleotides described herein, comprising more than one incretin peptide and an HLE domain. Figure 8A shows an incretin formulation containing a signal peptide ("SP"), a first GLP1 incretin peptide, a linker, a second GLP1 incretin peptide, a second linker (GGGGS) ₃ , and a half-life extension (HLE) domain of human serum albumin (HSA). Figure 8B shows an incretin formulation containing a signal peptide ("SP"), a first GLP1 incretin peptide, a linker, a second GLP1 incretin peptide, a second linker (GGGGS) ₃ , and a half-life extension (HLE) domain bound to the VHH domain of HSA. The furin and SP cleavage sites within the incretin agent are indicated by arrows, such that after expression, the signal peptide cleaves and the first GLP1 incretin peptide cleaves from the second GLP1 incretin peptide, while the second GLP1 incretin peptide remains fused to the HLE domain (HSA or anti-HSA VHH). This design produces two incretin peptides with different half-lives and activities. Figure 9 illustrates an exemplary incretin agent that can be encoded by one or more of the polynucleotides described herein, comprising more than one incretin peptide and a half-life extension (HLE) domain. Specifically, the incretin agent in Figure 9 comprises a signal peptide ("SP"), a first GLP1 incretin peptide, a linker (GGGGS) ₂ , a first GIP incretin peptide, a second linker (GGGGS) ₂ , a second GLP1 incretin peptide, a third linker (GGGGS) ₂ , a second GIP incretin peptide, a fourth linker (GGGGS) ₃ , and a human serum albumin (HSA) half-life extension (HLE) domain. The furin and SP cleavage sites within the incretin agent are indicated by arrows. This design produces four separate incretin peptides, with the second GIP incretin peptide remaining fused to the HLE domain. Figure 10 illustrates an exemplary design of a polynucleotide encoding an incretin agent comprising an incretin peptide fused to the Fc domain, wherein the incretin peptide can be one (I:1x), two (I:2x), or four (I:4x) incretin peptides (top). When encoding two of two separate polynucleotide chains, the two polypeptide chains attach and form a dimer (e.g., homodimer) structure (bottom). Each polypeptide chain also includes a signal peptide (SP) and a linker (L2). In some embodiments, the Fc domain includes mutations that eliminate effector function (e.g., mutations in STR, LALA, LALAPG, etc.) and/or mutations that prolong half-life (e.g., mutations in YTE, LS, etc.). Figure 11 illustrates exemplary incretin agents encoded by one or more polynucleotides described herein, comprising more than one incretin peptide on more than one polypeptide chain. Specifically, each polypeptide chain of the incretin agent in Figure 11 has a signal peptide (“SP”), a GLP1 incretin peptide, a linker (GGGGS) ₃ , and an Fc domain. One or both Fc domains contain an “LS” mutation (M428L/N434S according to the EU designation scheme) to prolong the half-life of the incretin agent. When two polypeptide chains are represented, they condense to form a homodimeric structure as shown in Figure 11. SP cleavage sites within the incretin agent are indicated by arrows. Figure 12 illustrates an exemplary incretin agent encoded by one or more of the polynucleotides described herein, comprising more than one incretin peptide on more than one polypeptide chain. Specifically, each polypeptide chain of the incretin agent has a signal peptide (SP), a GLP1 incretin peptide, a linker, a GIP peptide, a second linker (GGGGS) ₃ , and an Fc domain. One or two Fc domains contain an LS mutation. When two polypeptide chains are expressed, they condense to form a homodimeric structure as shown in Figure 12. Frin and SP cleavage sites within the incretin agent are indicated by arrows. Figure 13 illustrates exemplary designs of two polynucleotides, each encoding a polypeptide chain (top) comprising an incretin peptide fused to an Fc domain. Within each polypeptide chain (incretin-Fc fusion), a signal peptide (SP) and one, two, or four incretin peptides (I:1x, I:2x, or I:4x) fused to the Fc domain via a linker (L2) are present, and each Fc domain is modified to induce heterodimerization (e.g., a knock-in-hole mutation). When both polypeptide chains are represented, they condense together to form a heterodimeric incretin. Figure 14 illustrates exemplary incretin agents encoded by one or more polynucleotides described herein, comprising more than one incretin peptide on more than one polypeptide chain. Specifically, each polypeptide chain of the incretin agents in Figure 14 has a signal peptide (SP), a GLP1 or GIP incretin peptide, a linker (GGGGS) ₃ , and an Fc domain. One or two Fc domains contain "LS" mutations (M428L/N434S), "STR" mutations (L234S, L235T, and G236R mutations according to the EU numbering scheme) to silence Fc effector function, and "mortar and pestle" mutations to induce heterodimerization. When two polypeptide chains are expressed, they condense to form a heterodimeric incretin agent containing two polypeptide chains with different incretin peptides. SP cleavage sites within the incretin agent are indicated by arrows. Figure 15 shows the exemplary concentrations (pg/ml) of GLP1 (7-37) in the supernatant of HEK293t17 cells transfected with polynucleotides encoding GLP1 (7-37) at 3, 6, 24, 48, and 72 hours post-transfection (GLP1 n=4 +/- SD; ns = not significant; * p < 0.05, ** p < 0.01). Figure 16 shows the exemplary concentration (pg/ml) of K34R-mutated GLP1 (7-37) in the supernatant of HEK293t17 cells transfected with polynucleotides encoding GLP1 (7-37)-(K34R) at 3, 6, 24, 48 and 72 hours post-transfection (GLP1 n=4 +/- SD; ns = not significant; * p < 0.05, ** p < 0.01). Figure 17 shows the exemplary concentrations (pg/ml) of GIP (1-42) in the supernatant of HEK293t17 cells transfected with polynucleotides encoding GIP (1-42) at 3, 6, 24, 48, and 72 hours post-transfection (GIP n=6 +/- SD; ns = not significant; * p < 0.05, ** p < 0.01). Figure 18 shows the exemplary concentrations (pg/ml) of GLP1 in the supernatant of HEK29t17 cells transfected with polynucleotides encoding either a viral signaling peptide (“viral SP”) or a husec signaling peptide (“husec”) and codon-optimized using different strategies (“opt1” vs. “optp”). Specific intestinal prokinetic agents include: viral SP-GLP1 (7-37), viral SP-GLP1 (7-37)-(K34R), hube-GLP-1 (7-37)-A8G (opt1), hube-GLP-1 (7-37)-A8G-linker (opt1), hube-GLP-1 (7-37)-A8G (optp), and hube-GLP-1 (7-37)-A8G-linker (optp) intestinal prokinetic agents. Figure 19 shows the concentration (ng/ml) of exemplary GIP in the supernatant of HEK29t17 cells transfected with polynucleotides encoding either a viral signaling peptide (“viral SP”) or a husec signaling peptide (“husec”) and codon-optimized using different strategies (“opt1” vs. “optp”). Specific intestinal agonists include: viral SP-GIP (1-42), husec-GIP (1-42)-A2G (opt1), and GIP (1-42)-A2G (optp). Figure 20 shows a schematic diagram of the theoretical cleavage sites of various signaling peptides within the amino acid sequences of the intestinal agonists. Figure 20 also indicates that the A8G mutation promotes the correct N-terminal processing of the GLP1 incretin with the husec signaling peptide. Figure 21 shows a schematic diagram of the theoretical cleavage sites of various signaling peptides located within the amino acid sequence of the incretin. Figure 21 also indicates that the A2G mutation promotes the correct N-terminal processing of the GIP incretin with the husec signaling peptide. Figures 22A-22B show schematic diagrams of the HEK293 reporter cell lines of overexpressing GLP1R (A) and GIPR (B) intended for use in assays to determine the bioactivity of the exemplary GLP1 and GIP incretins in Example 7. Figure 23 shows the results of the bioactivity assay of the exemplary GLP1 incretin. Specifically, the results are expressed as induction multiples relative to the control sample. Figure 24 shows the results of the bioactivity assays for exemplary GIP intestinal insulin agents. Specifically, the results are expressed as induction multiples relative to the control sample. Figure 25 shows the in vitro activity (GIP performance) of some of the tested exemplary intestinal insulin agents. Figure 26 shows the GIP bioactivity of some tested exemplary GIP-containing intestinal insulin agents. Figure 27 shows the in vitro activity (GLP1 performance) of some tested exemplary intestinal insulin agents. Figure 28 shows the GLP1 bioactivity of some tested exemplary GLP1-containing intestinal insulin agents. Figure 29 shows a comparison of GIP performance ( A ) and GIP bioactivity ( B ) in exemplary candidates with different signaling peptides (husec vs. gD1). Figure 30 shows a comparison of GLP1 performance ( A ) and GLP1 bioactivity ( B ) in exemplary candidates with different signaling peptides (husec vs. gD1). Figure 31 shows a comparison of GIP performance (A) and GIP bioactivity (B) with and without various half-life extension (HLE) moieties. Figure 32 shows a comparison of GLP1 performance (A) and GLP1 bioactivity (B) with and without various half-life extension (HLE) moieties. Figure 33 shows a comparison of GIP performance (A) and GIP bioactivity (B) in a demonstrative incretin formulation containing both GIP and GLP1, with variations in the order of the GIP and GLP1 peptides encoded by a single polynucleotide. Figure 34 shows a comparison of GLP1 performance (A) and GLP1 bioactivity (B) in a demonstrative incretin formulation containing both GIP and GLP1, with variations in the order of the GIP and GLP1 peptides encoded by a single polynucleotide.

TW202540154A_113134412_SEQL.xml

Claims (223)

A composition comprising a polynucleotide encoding an incretin. The combination of claim 1, wherein the incretin is a GLP-1 receptor agonist. The combination of claim 1, wherein the incretin is a GIP receptor agonist. The combination of claim 1, wherein the incretin is a GLP1/GIP dual receptor agonist. The combination of claim 1, wherein the incretin is a GLP1/GCG dual receptor agonist. The combination of claim 1, wherein the incretin is a GLP1/GIP/GCG triple receptor agonist. 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 NO: 5-7, 63-64, 69-70 and 74-75. 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 NO: 8-9, 62 and 72. The composition of claim 2, wherein the incretin agent comprises an incretin peptide having an amino acid sequence according to SEQ ID NO: 11. The composition of claim 4, wherein the incretin agent comprises an incretin peptide having an amino acid sequence according to any one of SEQ ID NO: 12-14. The composition of claim 6, wherein the incretin agent comprises an incretin peptide having an amino acid sequence according to SEQ ID NO: 15. Combinations of any of claims 7 to 11, wherein the incretin peptide is fused to a signal peptide via the N-terminus of the incretin peptide, or via a linker, as appropriate. The combination of claim 12, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 16-39 and 65-67. The combination of claim 12, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 16-21 and 65-67. The combination of claim 12, wherein the signal peptide has an amino acid sequence according to SEQ ID NO: 17. The combination of claim 12, wherein the signal peptide has an amino acid sequence according to SEQ ID NO: 65. The combination of claim 12, wherein the signal peptide has an amino acid sequence according to SEQ ID NO: 66. The composition of claim 1, wherein the incretin agent comprises an amino acid sequence according to any one of SEQ ID NO: 41-45, 52-61 and 108-152. Combinations of any of claims 1 to 18, wherein the incretin agent comprises, where appropriate, an incretin peptide fused to one or more additional incretin peptides via one or more linkers. The combination of claim 19, wherein the one or more connectors comprises an amino acid sequence according to any one of SEQ ID NO: 1-5, 68 or 156. Combinations of claims 19 or 20, wherein the incretin agent comprises an incretin peptide fused to two or more incretin peptides. The combination of any one of claims 19 to 21, wherein the incretin agent comprises at least one GLP1 receptor agonist and at least one GIP receptor agonist. Combinations of any of claims 19 to 22, wherein the incretin agent comprises at least two GLP1 receptor agonists. Combinations of any of claims 19 to 23, wherein the incretin agent comprises at least two GIP receptor agonists. Combinations of any of claims 19 to 24, wherein the incretin agent contains one or more furin cleavage sites. As in claim 25, the combination wherein the one or more furin cleavage sites are located between adjacent incretin peptides. Combinations of claims 27 or 28, wherein the one or more furin cleavage sites comprise an amino acid sequence according to SEQ ID NO: 153. Combinations of any of claims 19 to 27, wherein the incretin agent comprises one or more units, each of which comprises, from the N-terminus to the C-terminus: a GLP1 receptor agonist-linker-furin cleavage site-GIP receptor agonist, for example, wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 76, 77, 78, 79, 80, 81), two units (e.g., SEQ ID NO: 82); or four units (e.g., SEQ ID NO: 83). Combinations of any of claims 19 to 30, wherein the incretin agent comprises an amino acid sequence according to any of SEQ ID NO: 76-83, 94-97, 102-107. Combinations of any of claims 1 to 29, wherein the incretin agent includes a half-life extension portion. As in claim 30, the composition wherein the extended half-life portion comprises albumin (e.g., human serum albumin). The combination of claim 31, wherein the human serum albumin comprises an amino acid sequence having at least 90%, 95% or 99% similarity to SEQ ID NO: 159. Combinations of claims 31 or 32, wherein the human serum albumin comprises an amino acid sequence according to SEQ ID NO: 159. Combinations of any of claims 31 to 33, wherein the incretin agent comprises albumin (e.g., human serum albumin) fused to one or more units, each of the one or more units comprising, from the N-terminus to the C-terminus: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 98); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 100); (iii) a GLP1 receptor agonist-linker-furin cleavage site (e.g., SEQ ID NO: 102); or (iv) a GLP1 receptor agonist-linker-furin cleavage site-GIP receptor agonist, for example, wherein the incretin agent comprises one unit (e.g., SEQ ID NO: 104) and two units (e.g., SEQ ID NO: 104). 106) or four units (e.g., SEQ ID NO: 107). The combination of any of claims 31 to 34, wherein the incretin agent comprises an amino acid sequence according to any of SEQ ID NO: 98, 100, 102, 104, 106, 107, or any combination thereof. As in claim 30, the composition wherein the half-life extension portion comprises an albumin-binding domain (ABD). As in the composition of claim 36, wherein the ABD is derived from protein G of Streptococcus strain GI48 and/or protein PAB of Finegoldia magna , such as ABD035 and SA21. The composition of claim 36, wherein the half-life extension portion comprises ABD, which binds to domain II of human serum albumin without overlapping or interfering with the binding of FcRn binding sites on albumin. As in claim 36, the combination wherein the half-life extension portion comprises ABDCon. As in the composition of claim 36, the extended half-life portion comprises albumin-binding domains (ABDs) of bacterial proteins Sso7d derived from the hyperthermophilic archaeon Sulfolobus solfataricus , such as M11.12 and M18.2.5. As in claim 36, the composition wherein the extended half-life portion comprises DARPin, which binds to albumin. The combination of claim 36, wherein the ABD comprises an immunoglobulin domain or a fragment thereof that binds to albumin. Combinations such as those in claims 36 or 42, wherein the ABD comprises a fully human structural domain antibody (dAb) that binds to albumin, such as AlbudAb. Combinations such as those in claims 36 or 42 to 43, wherein the ABD contains Fab that binds albumin, such as dsFv CA645. Combinations such as claims 36 or 42 to 44, wherein the ABD comprises a heavy-chain only (VHH) antibody that binds to albumin, such as a nano-antibody. The composition of claim 45, wherein the VHH antibody comprises a VHH domain having complementary determinant (CDR) sequences HCDR1, HCDR2 and/or HCDR3 according to SEQ ID NO: 191 (GFTLDYYA), SEQ ID NO: 192 (IASSGGST) and/or SEQ ID NO: 193 (AAAVLECRTVVRGYDY). The combination of claim 46, wherein the VHH antibody comprises an amino acid sequence having at least 90%, 95% or 99% similarity to SEQ ID NO: 154. Combinations of claims 46 or 47, wherein the VHH antibody comprises an amino acid sequence according to SEQ ID NO: 154. Combinations of any of claims 45 to 47, wherein the incretin agent comprises a VHH antibody bound to an albumin fused to a unit, the unit comprising, from the N-terminus to the C-terminus: (i) a GLP1-linker (e.g., SEQ ID NO: 99); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 101); or (iii) a GLP1 receptor agonist-linker-furin protease-GIP receptor agonist-linker (e.g., SEQ ID NO: 103 or 105). Combinations of any of claims 45 to 49, wherein the incretin agent comprises an amino acid sequence according to any of SEQ ID NO: 99, 101, 103, 105. As in the composition of claim 30, wherein the half-life extension portion does not contain an Fc domain, such as that derived from human IgG, or, where appropriate, from human IgG1, IgG2, IgG3, or IgG4. As in the composition of claim 30, wherein the half-life extension portion comprises an Fc domain, such as that derived from human IgG, or, where appropriate, from human IgG1, IgG2, IgG3, or IgG4. The combination of claim 52, wherein the human IgG is human IgG4. Combinations of claims 52 or 53, wherein the incretin agent comprises an IgG4 Fc domain fused to a unit, the unit comprising, from the N-terminus to the C-terminus: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 10, 89, 90, 91); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 92, 93); or (iii) a GLP1 receptor agonist-linker-furin protease-GIP receptor agonist-linker (e.g., SEQ ID NO: 94, 95, 96, 97). Combinations of claims 53 or 54, wherein the IgG4 Fc domain comprises an amino acid sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 155. The combination of claim 55, wherein the IgG4 Fc domain comprises the amino acid sequence according to SEQ ID NO: 155. Combinations of any of claims 53 to 56, wherein the incretin agent comprises an amino acid sequence according to any of SEQ ID NO: 10 and 89-97. Combinations of any of claims 52 to 57, wherein the Fc domain contains one or more mutations in one or both constant Fc domains that increase the half-life of the incretin and/or induce dimerization. As in claim 58, the combination wherein the one or more mutations comprise one or more mutations in the CH3 domain. Combinations such as those in claims 58 or 59, wherein the one or more mutations inducing dimerization comprise: (i) according to EU designations, Y349C, T366S, L368A and/or Y407V; or (ii) according to EU designations, S354C and/or T366W. Combinations of any of claims 58 to 60, wherein the one or more mutations comprise, according to EU designations, Y349C, T366S, L368A and Y407V (“FcKIH-b”); or according to EU designations, S354C and T366W (“FcKIH-a”). The composition of any one of claims 58 to 61, wherein the incretin agent comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an incretin peptide fused to a first Fc domain, wherein the first Fc domain comprises mutants Y349C, T366S, L368A and Y407V (“FcKIH-b”) according to EU designations, and wherein the second polypeptide chain comprises an incretin peptide fused to a second Fc domain, wherein the second Fc domain comprises mutants S354C and T366W (“FcKIH-a”) according to EU designations. Combinations of any of claims 58 to 62, wherein the one or more mutations that increase the half-life of the incretin agent include, according to EU designations, M428L and N434S (“LS”). Compositions of any of claims 58 to 63, wherein the incretin agent comprises an Fc domain having an FcKIH-a mutation on a first polypeptide chain and an Fc domain having an FcKIH-b mutation on a second polypeptide chain, wherein the Fc domain on each polypeptide chain is independently fused to one or more units, the one or more units comprising, from the N-terminus to the C-terminus: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 84, 85, 86, 87); or (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 88). Combinations of any of claims 58 to 64, wherein the incretin agent comprises an amino acid sequence according to any of SEQ ID NO: 84-88. Combinations of any of claims 58 to 64, wherein the Fc domain comprises one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to an Fcγ receptor or C1q). As in claim 66, the one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to the Fcγ receptor or C1q) include the following mutations: L234S, L235T and G236R (“STR”) according to EU designations. As in the composition of claim 66, the one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to the Fcγ receptor or C1q) include the following mutations: according to EU designations, L234A and L235A (“LALA”). As in claim 66, the one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to the Fcγ receptor or C1q) include the following mutations: according to EU designation, L234A/L235A/P329G (“LALAPG”). As in claim 30, the composition wherein the extended half-life portion comprises albumin-binding VNAR. Combinations such as claim 30, wherein the half-life extension portion comprises an XTEN sequence. Combinations of any of claims 1 to 71, wherein the polynucleotide has a ribonucleic acid sequence that is at least 90% identical to any of SEQ ID NO: 177-185 and 224-256. The composition of claim 72, wherein the polynucleotide has a ribonucleic acid sequence according to any one of SEQ ID No: 177-185 and 224-256. Combinations of any of claims 1 to 73, wherein the polynucleotide comprises at least one non-coding sequence element that enhances RNA stability and/or translation efficiency. Combinations such as claim 74, wherein the at least one non-coded sequence element comprises a 5' cap structure, a 5' UTR, a 3' UTR, and/or a polyA tail. The composition of claim 75, wherein the polynucleotide comprises, in the 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. The composition of claim 75, wherein the polynucleotide comprises, in the 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 extension 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 extension coding sequence; d. a linker coding sequence; e. an incretin peptide coding sequence; f. a 3' UTR; and g. a polyA tail. Combinations of any of claims 1 to 77, wherein the incretin peptide is encoded by a coding sequence optimized by codons and/or by an increase in its G/C content compared to the wild-type coding sequence, wherein the codon optimization and/or the increase in the G/C content does not alter the sequence of the encoded amino acid sequence. Combinations of any of claims 1 to 78, wherein the polynucleotide comprises at least one modified ribonucleotide. As in claim 79, the polynucleotide comprises a modified nucleoside that replaces uridine. As in claim 80, the polynucleotide comprises a modified nucleoside replacing each uridine. As in the composition of claim 81, wherein the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U). Combinations of any of claims 80 to 83, wherein the modified nucleoside is N1-methyl-pseudouridine (m1ψ). Combinations of any of claims 1 to 83, wherein the polynucleotide comprises a 5' cap structure. Combinations of any of claims 1 to 84, wherein the polynucleotide contains 5' UTR. Combinations of any of claims 1 to 85, wherein the polynucleotide contains 3' UTR. A combination of any of claims 1 to 86, wherein the polynucleotide comprises a polyA tail. Combinations such as those in claim 87, wherein the polyA tail contains at least 100 nucleotides. A combination of any of the claims 1 to 88, wherein the polynucleotide is mRNA. Combinations of any of claims 1 to 89, wherein the polynucleotide is formulated as a liquid, formulated as a solid, or a combination thereof. Combinations of any of claims 1 to 90, wherein the polynucleotide is formulated for injection. Combinations of any of claims 1 to 91, wherein the polynucleotide is formulated for intraperitoneal or intravenous administration. Combinations of any of claims 1 to 92, wherein the polynucleotide is configured or intended to be configured as lipid particles. As in claim 93, the polynucleotide is formulated or intended to be formulated as lipid nanoparticles. As in claim 94, the polynucleotide is encapsulated within the lipid nanoparticles. Combinations of claims 94 or 95, wherein the lipid nanoparticles are lipid nanoparticles targeting the pancreas and/or the intestine. Combinations of any of claims 94 to 96, wherein the lipid nanoparticles are cationic lipid nanoparticles. As in claim 97, the lipids forming the lipid nanoparticles comprise: a. polymer-coupled lipids; b. cationic lipids; and c. neutral lipids. The composition of claim 98, wherein the polymer-coupled lipid is a PEG-coupled lipid. Combinations such as those in claims 98 or 99, wherein the cationic lipid is an ionizable lipid-like material (lipid). The composition of claim 100, wherein the cationic lipid has one of the following structures: X-1 X-2 X-3 X-4 Combinations of any of claims 99 to 101, wherein the neutral lipid comprises co-lipids such as 1,2-distearyl-sn-glycerol-3-phosphate choline (DPSC) and/or cholesterol. Combinations of any of claims 99 to 102, wherein the cationic lipid is selected from cationic lipids X-2, X-3 or X-4, and the neutral lipid includes auxiliary lipids such as DOTAP, DOPE or PS and cholesterol. The composition of claim 103, wherein the polymer-coupled lipid is C14-PEG2000. Compositions of any of claims 99 to 104, wherein the lipid nanoparticles comprise: i) about 30 mol% to about 50 mol% cationic lipids; ii) about 1 mol% to 5 mol% PEG-coupled lipids; iii) about 30 mol% to about 50 mol% co-lipids; and iv) about 20 mol% to about 40 mol% cholesterol. Compositions of any of claims 99 to 104, wherein the lipid nanoparticles comprise about 35 mol% cationic lipids; about 40 mol% co-lipids; about 22.5 mol% cholesterol; and about 2.5 mol% PEG-coupled lipids. The composition of claim 106, wherein the lipid nanoparticles comprise about 35 mol% of cationic lipids 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. The composition of claim 107, 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. The composition of claim 107, 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. The composition of claim 107, 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. The composition of claim 107, 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. The composition of claim 107, 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. The composition of claim 107, 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. The composition of claim 107, 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. The composition of claim 107, 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. The composition of claim 107, 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. Combinations of any of claims 94 to 116, wherein the lipid nanoparticles are formulated for intraperitoneal (i.p.) delivery. Combinations of any of claims 94 to 117, wherein the lipid nanoparticles have an average size of about 50-150 nm. Combinations of any of claims 1 to 118 may further comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients. The composition of claim 119 further includes a freeze protectant. The composition of claim 120, wherein the freeze protectant is sucrose. Combinations of any of claims 119 to 121 further include an aqueous buffer solution. The composition of claim 122, wherein the aqueous buffer solution comprises sodium ions. A method for treating a disease state in an individual in need, comprising administering to the individual a therapeutically effective amount of any combination of any of the items 1 to 123. The method of claim 124 further comprises administering one or more DPP-4 inhibitors. The method of claim 125, wherein the one or more DPP-4 inhibitors and the combination are administered simultaneously. The method of claim 125, wherein the one or more DPP-4 inhibitors and the combination are administered sequentially. The method of claim 127, wherein the one or more DPP-4 inhibitors are administered prior to the composition. The method of claim 127, wherein the one or more DPP-4 inhibitors are administered after the composition. The method of any of claims 125 to 129, wherein the one or more DPP-4 inhibitors comprises sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin, teneligliptin, alogliptin, and trelagliptin. Omagliptin, evogliptin, gosogliptin, dutogliptin, neogliptin, retagliptin, denagliptin, cofroglipin, fotagliptin, prusogliptin, berberine, or any combination thereof. The method of any of claims 125 to 130, wherein the one or more DPP-4 inhibitors are administered orally. The method of request item 124, wherein the disease condition is obesity or obesity-related conditions. The method of claim 132, wherein the obesity-related condition is prediabetes, type 2 diabetes (T2D), early type 1 diabetes (T1D), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular (CV) disease, kidney disease, or increased risk of premature death. The method of claim 133, wherein the cardiovascular (CV) disease includes major cardiovascular events (MACE), including CV death, nonfatal myocardial infarction, nonfatal stroke and/or heart failure with preserved ejection fraction (HFpEF). The method described in Request 132, wherein the method improves the individual’s weight management. The method of claim 132, wherein the method reduces the individual’s weight gain or induces weight loss. The method of request item 124, wherein the disease condition is diabetes. The method described in claim 137 improves the individual's blood glucose control. The method of claim 137, wherein the method reduces the individual's HbA1c. The method of claim 137, wherein the diabetes is prediabetes, type 2 diabetes (T2D), or early type 1 diabetes (T1D). The method of request item 124, wherein the disease condition is cardiovascular (CV) disease. The method of claim 141, wherein the cardiovascular disease includes major cardiovascular events (MACE), including CV death, nonfatal myocardial infarction, nonfatal stroke and/or heart failure with preserved ejection fraction (HfpEF). The method of claim 141, wherein the method improves the individual’s blood pressure and/or blood lipids. The method of request item 124, wherein the disease condition is kidney disease. The method of request item 124, wherein the disease condition is non-alcoholic fatty liver disease (NAFLD). The method of claim 124, wherein the disease condition is non-alcoholic steatohepatitis (NASH) and, if applicable, its sequelae, liver fibrosis and cirrhosis. The method of any of claims 124 to 146, wherein administering the composition to the individual comprises administering one or more doses of the composition to the individual. The method of claim 147, wherein one or more doses of the composition are administered to the individual daily, every other day or weekly. The method of claim 147, wherein the one or more doses of the composition are administered to the individual at a frequency of less than once per week. The method of claim 147, wherein one or more doses of the composition are administered to the individual every 2, 3 or 4 weeks. The method of any of claims 124 to 150, wherein the composition is administered by injection. The method of claim 151, wherein the composition is administered subcutaneously, intravenously, intramuscularly, or intraperitoneally. The method of claim 152, wherein the composition is administered intraperitoneally. The method of any of claims 124 to 150, wherein the composition is administered non-invasively (e.g., orally or nasally). The method of any of claims 124 to 154, wherein administration of the composition results in the expression of the incretin in the individual. The method of any of claims 124 to 155, wherein the composition is administered in a volume of less than 0.5 mL. Use of a combination of any of claims 1 to 123 for the treatment of a disease condition in an individual in need. A method for producing an incretin, comprising administering to cells a combination of any one of claims 1 to 123, such that the cells express and secrete the incretin. An incretin agent comprising an incretin peptide fused to a signaling peptide. Such as the incretin preparation of claim 159, wherein the incretin peptide is fused to the signal peptide via a linker at the N-terminus of the incretin peptide, if applicable. The incretin agent of claim 159 or 160, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 16-39 and 65-67. Such as the incretin agent of claim 161, wherein the signal peptide has an amino acid sequence according to any one of SEQ ID NO: 16-21 and 65-67. Such as the incretin agent of claim 161, wherein the signal peptide has an amino acid sequence according to SEQ ID NO: 17. Such as the incretin agent of claim 161, wherein the signal peptide has an amino acid sequence according to SEQ ID NO: 65. Such as the incretin agent of claim 161, wherein the signal peptide has an amino acid sequence according to SEQ ID NO: 66. An incretin preparation of any one of claims 159 to 165, wherein the incretin preparation comprises an incretin peptide fused to a signal peptide, which comprises an amino acid sequence according to any one of SEQ ID NO: 41-45, 52-61 and 108-152. The incretin preparation of any of claims 159 to 166, wherein the incretin preparation comprises, where appropriate, an incretin peptide fused to one or more additional incretin peptides via one or more linkers. The incretin agent of claim 167, wherein the one or more linkers comprise an amino acid sequence according to any one of SEQ ID NO: 1-5, 68 or 156. The incretin preparation of claim 167 or 168, wherein the incretin preparation comprises an incretin peptide fused to two or more incretin peptides. The intestinal insulin agent of any of claims 167 to 169, wherein the intestinal insulin agent comprises at least one GLP1 receptor agonist and at least one GIP receptor agonist. An intestinal insulin agent as described in any of claims 167 to 170, wherein the intestinal insulin agent contains at least two GLP1 receptor agonists. The intestinal insulin agent of any of claims 167 to 171, wherein the intestinal insulin agent contains at least two GIP receptor agonists. Combinations of any of claims 167 to 172, wherein the incretin agent comprises one or more furin cleavage sites. Such as the incretin preparation of claim 173, wherein the one or more furin cleavage sites are located between adjacent incretin peptides. The incretin agent of claim 173 or 174, wherein the one or more furin cleavage sites contain an amino acid sequence according to SEQ ID NO: 153. The incretin formulation of any of claims 167 to 175, wherein the incretin formulation comprises one or more units, each of which comprises, from the N-terminus to the C-terminus: a GLP1 receptor agonist-linker-furin cleavage site-GIP receptor agonist, for example, wherein the incretin formulation comprises one unit (e.g., SEQ ID NO: 76, 77, 78, 79, 80, 81), two units (e.g., SEQ ID NO: 82); or four units (e.g., SEQ ID NO: 83). An incretin preparation of any one of claims 167 to 176, wherein the incretin preparation comprises an amino acid sequence according to any one of SEQ ID NO: 76-83, 94-97, 102-107. An incretin preparation such as any of claims 159 to 177, wherein the incretin preparation includes a portion with an extended half-life. For example, incretins such as claim 178, wherein the extended half-life portion comprises albumin (e.g., human serum albumin). The incretin preparation of claim 180, wherein the human serum albumin contains an amino acid sequence having at least 90%, 95%, or 99% similarity to SEQ ID NO: 159. The incretin agent of claim 179 or 180, wherein the human serum albumin contains the amino acid sequence according to SEQ ID NO: 159. An incretin formulation as described in any of claims 172 to 174, wherein the incretin formulation comprises albumin (e.g., human serum albumin) fused to one or more units, each of the one or more units comprising, from the N-terminus to the C-terminus: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 98); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 100); (iii) a GLP1 receptor agonist-linker-furin cleavage site (e.g., SEQ ID NO: 102); or (iv) a GLP1 receptor agonist-linker-furin cleavage site-GIP receptor agonist, for example, wherein the incretin formulation comprises one unit (e.g., SEQ ID NO: 98). 104), two units (e.g., SEQ ID NO: 106), or four units (e.g., SEQ ID NO: 107). An incretin preparation of any one of claims 179 to 182, wherein the incretin preparation comprises an amino acid sequence according to any one of SEQ ID NO: 98, 100, 102, 104, 106, 107, or any combination thereof. For example, in the incretin formulation of claim 178, the extended half-life portion includes an albumin-binding domain (ABD). For example, in the incretin preparations of claim 184, wherein the ABD is derived from protein G of Streptococcus strain GI48 and/or protein PAB of Fungoldii macrophage , such as ABD035 and SA21. For example, in the incretin formulation of claim 184, wherein the extended half-life portion comprises ABD, which binds to domain II of human serum albumin without overlapping or interfering with binding to FcRn binding sites on albumin. For example, in the case of the incretin agent in claim 184, wherein the extended half-life portion includes ABDCon. For example, in the incretin agent of claim 184, the extended half-life portion comprises the ABD of bacterial protein Sso7d derived from the hyperthermophilic archaea *Sulphozoa sulfadiazine* , such as M11.12 and M18.2.5. For example, in the incretin formulation of claim 178, wherein the extended half-life portion comprises DARPin bound to albumin. Such as the incretin preparations in claim 184, wherein the ABD comprises an immunoglobulin domain or a fragment thereof that binds to albumin. For example, incretin preparations such as those in claims 184 or 190, wherein the ABD contains a fully human domain antibody (dAb) that binds to albumin, such as AlbudAb. Such as the incretin agent of any of claims 184 or 190 to 191, wherein the ABD contains Fab that binds to albumin, such as dsFv CA645. Such as incretins in claims 184 or 190 to 192, wherein the ABD contains a heavy-chain-only (VHH) antibody that binds to albumin, such as a nano-antibody. The incretin agent of claim 193, wherein the VHH antibody comprises a VHH domain having complementary determinant (CDR) sequences HCDR1, HCDR2 and/or HCDR3 according to SEQ ID NO: 191 (GFTLDYYA), SEQ ID NO: 192 (IASSGGST) and/or SEQ ID NO: 193 (AAAVLECRTVVRGYDY). The incretin agent of claim 193 or 194, wherein the VHH antibody comprises an amino acid sequence having at least 90%, 95% or 99% similarity to SEQ ID NO: 154. Such as the incretin agent of claim 195, wherein the VHH antibody comprises an amino acid sequence according to SEQ ID NO: 154. An incretin agent as described in any of claims 193 to 196, wherein the incretin agent comprises a VHH antibody bound to an albumin fused to a unit, the unit comprising, from the N-terminus to the C-terminus: (i) a GLP1-linker (e.g., SEQ ID NO: 99); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 101); or (iii) a GLP1 receptor agonist-linker-furin protease-GIP receptor agonist-linker (e.g., SEQ ID NO: 103 or 105). An incretin preparation of any one of claims 193 to 197, wherein the incretin preparation comprises an amino acid sequence according to any one of SEQ ID NO: 99, 101, 103, 105. For example, in the request for an incretin agent, wherein the extended half-life portion does not contain an Fc domain, such as that derived from human IgG, or, where applicable, from human IgG1, IgG2, IgG3, or IgG4. For example, in the incretin formulation of claim 178, wherein the extended half-life portion includes an Fc domain, such as that derived from human IgG, or, in some cases, from human IgG1, IgG2, IgG3, or IgG4. For example, in the case of the incretin agent in claim 200, the human IgG is human IgG4. The incretin agent of claim 200 or 201, wherein the incretin agent comprises an IgG4 Fc domain fused to a unit, the unit comprising, from the N-terminus to the C-terminus: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 10, 89, 90, 91); (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 92, 93); or (iii) a GLP1 receptor agonist-linker-furin protease-GIP receptor agonist-linker (e.g., SEQ ID NO: 94, 95, 96, 97). An incretin agent of any of claims 200 to 202, wherein the IgG4 Fc domain comprises an amino acid sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 155. The incretin agent of claim 203, wherein the IgG4 Fc domain comprises an amino acid sequence according to SEQ ID NO: 155. An incretin preparation of any one of claims 200 to 204, wherein the incretin preparation comprises an amino acid sequence according to any one of SEQ ID NO: 10, 89-97. An incretin agent as described in any of claims 200 to 205, wherein the Fc domain contains one or more mutations in one or both constant Fc domains that increase the half-life of the incretin agent and/or induce dimerization. As in the incretin agent of claim 206, wherein the one or more mutations include one or more mutations in the CH3 domain. For example, in the case of incretin agents according to claims 206 or 207, the one or more mutations inducing dimerization include: (i) according to EU designations, Y349C, T366S, L368A and/or Y407V; or (ii) according to EU designations, S354C and/or T366W. The incretin agent of any of claims 206 to 208, wherein the one or more mutations include, according to EU designations, Y349C, T366S, L368A and Y407V (“FcKIH-b”); or according to EU designations, S354C and T366W (“FcKIH-a”). The incretin preparation of any one of claims 206 to 209, wherein the incretin preparation comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an incretin peptide fused to a first Fc domain, wherein the first Fc domain comprises mutants Y349C, T366S, L368A and Y407V (“FcKIH-b”) according to EU designations, and wherein the second polypeptide chain comprises an incretin peptide fused to a second Fc domain, wherein the second Fc domain comprises mutants S354C and T366W (“FcKIH-a”) according to EU designations. For example, in any of claims 206 to 210, the one or more mutations that increase the half-life of the intestinal insulin include those according to EU designations M428L and N434S (“LS”). An incretin agent as described in any of claims 206 to 211, wherein the incretin agent comprises an Fc domain having an FcKIH-a mutation on a first polypeptide chain and an Fc domain having an FcKIH-b mutation on a second polypeptide chain, wherein the Fc domain on each polypeptide chain is independently fused to one or more units, the one or more units comprising, from the N-terminus to the C-terminus: (i) a GLP1 receptor agonist-linker (e.g., SEQ ID NO: 84, 85, 86, 87); or (ii) a GIP receptor agonist-linker (e.g., SEQ ID NO: 88). An incretin preparation of any one of claims 206 to 212, wherein the incretin preparation comprises an amino acid sequence according to any one of SEQ ID NO: 84-88. An incretin agent such as any of claims 206 to 213, wherein the Fc domain comprises one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to an Fcγ receptor or C1q). As in the composition of claim 214, the one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to the Fcγ receptor or C1q) include the following mutations: L234S, L235T and G236R (“STR”) according to EU designations. As in the composition of claim 215, the one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to the Fcγ receptor or C1q) include the following mutations: according to EU designations, L234A and L235A (“LALA”). As in the composition of claim 216, the one or more mutations that eliminate the effector activity of the Fc domain (e.g., binding to the Fcγ receptor or C1q) include the following mutations: according to EU designation, L234A/L235A/P329G (“LALAPG”). For example, in the incretin formulation of claim 178, wherein the extended half-life portion comprises albumin-bound VNAR. For example, in the incretin formulation of claim 178, wherein the extended half-life portion contains an XTEN sequence. An incretin agent comprising: a husec signaling peptide; an incretin peptide comprising a GLP1 incretin peptide or a fragment or variant thereof; wherein the GLP1 incretin peptide comprises an amino acid sequence having an A8G substitution mutation compared to the wild-type GLP1 amino acid sequence. A polynucleotide encoded as an incretin agent as requested in claim 220. An incretin agent comprising: a husec signaling peptide; an incretin peptide comprising a GIP incretin peptide or a fragment or variant thereof; wherein the GIP incretin peptide comprises an amino acid sequence having an A2G substitution mutation compared to the wild-type GIP amino acid sequence. A polynucleotide encoded as an incretin agent as specified in claim 222.