Chimeric proteins and uses thereof

Abstract

The present disclosure relates to, inter alia, compositions and methods, including heterologous chimeric proteins that find use, inter alia, in the treatment of diabetes, obesity, or metabolic syndrome.

Description

[0001] CHIMERIC PROTEINS AND USES THEREOF

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to, inter alia, compositions and methods, including heterologous chimeric proteins that find use, inter alia, in the treatment management of hyperglycemia, diabetes, including type II diabetes, obesity, metabolic syndrome and the reduction of cardiovascular risk.

[0004] PRIORITY

[0005] This application claims the benefit of, and priority to, International Application No. PCT / US2023 / 082572, filed December 5, 2023, and U.S. Provisional Application No. 63 / 656,437, filed June 5, 2024, the contents of each of which are hereby incorporated by reference in their entirety.

[0006] SEQUENCE LISTING

[0007] The instant application contains a sequence listing, which has been submitted in XML format via Patent Center. The contents of the XML copy named “SHK-096PC_116981 -5096_Sequence_Listing”, which was created on November 26, 2024, and is 125,976 bytes in size, are incorporated herein by reference in their entirety.

[0008] BACKGROUND

[0009] The worldwide prevalence of diabetes mellitus and obesity has been on the rise for past decades possibly because of stressful and sedentary lifestyles and unhealthy eating habits. Based on an estimate from the World Health Organization (WHO), the people with diabetes rose from 108 million in 1980 to 422 million in 2014. According to the Center for Disease Control (CDC), diabetes affects about 37.3 million people in the US, which is 11.3% of the US population, and this number includes about 8.5 million people that have undiagnosed diabetes. Similarly, obesity has nearly tripled worldwide since 1975, with more than 1.9 billion overweight, and over 650 million obese adults worldwide as of 2016. likewise, US obesity prevalence increased from about 30.5% in year 2000 to about 41 .9% in 2017 according to the CDC. Currently, over 20% children and over 40% adults suffer from obesity. Obesity is estimated to cost health services US $990 billion, which is 13% healthcare expenditure, per year globally. For example, the aggregate medical cost due to obesity among adults in the United States was $260.6 billion in 2016. Cawley et al., Direct medical costs of obesity in the United States and the most populous states, J Manag Care Spec Pharm 2021 ;27(3):354-366.

[0010] Diabetes is linked to a number of health problems, including microvascular complications, such as retinopathy, neuropathy, nephropathy, blindness in working-age adults, end-stage renal disease, peripheral artery disease (PAD), cardiovascular complications, and cardiovascular disease (CVD). Similarly, obesity itself increases risk for many serious diseases, including hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, metabolic syndrome, fatty liver disease, stroke, gallstones, cholecystitis, osteoarthritis, kidney disease, sleep apnea and breathing problems, clinical depression, anxiety, and many types of cancers.

[0011] Therefore, there remains a need for effective and accessible methods of treating diabetes, obesity, and related diseases.

[0012] SUMMARY

[0013] In various aspects, the present disclosure provides compositions and methods that are useful, inter alia, in the treatment or prevention of hyperglycemia, diabetes, including type II diabetes, obesity, metabolic syndrome and the reduction of cardiovascular risk.

[0014] Accordingly, in aspects, the present disclosure provides a chimeric protein comprising a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (A) (a) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, (c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge-CH2-CH3 Fc domain; or (B) (a) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), (c) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, and (b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2-CH3 Fc domain.

[0015] In embodiments, the GLP-1 receptor agonist is selected from GLP-1 , a DPP4 degradation resistant derivative of GLP-1, exenatide, lixisenatide, albiglutide, dulaglutide, or a variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71 , or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71. In embodiments, the GLP-1 receptor agonist is capable of binding a GLP-1 receptor. In embodiments, the GLP-1 receptor agonist is capable of activate the GLP-1 receptor. In embodiments, the GLP-1 receptor agonist is capable of stimulating and / or increasing insulin secretion, and / or inhibiting and / or decreasing glucagon secretion.

[0016] In embodiments, the portion of ACVR2B comprises substantially the entire extracellular domain of ACVR2B. In embodiments, the portion of ACVR2B comprises the entire extracellular domain of ACVR2B. In embodiments, the portion of ACVR2B is capable of binding activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B is capable of reducing or inhibiting the binding of one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11) to one or more of their receptors. In embodiments, the portion of ACVR2B is capable of sequestering one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B is capable of reducing or inhibiting ACVR2B signaling induced by one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73.

[0017] In embodiments, the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence. In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG1. In embodiments, the lgG1 is human lgG1. In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG4. In embodiments, the lgG4 is human lgG4. In embodiments, the hinge-CH2-CH3 Fc domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 53. In embodiments, the linker further comprises the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 4 to 52. In embodiments, the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 4 to 52; wherein one joining linker is N terminal to the hinge-CH2-CH3-Fc domain and another joining linker is C terminal to the hinge-CH2-CH3-Fc domain.

[0018] In embodiments, the chimeric protein is a recombinant fusion protein.

[0019] In embodiments, the chimeric protein is capable of inducing insulin production by pancreatic islet beta cells. In embodiments, the chimeric protein is capable of reducing one or more of subcutaneous white adipose tissue (sWAT), peri-renal adipose tissue, mesenteric adipose tissue, epididymal white adipose tissue (EWAT), cervical adipose tissue, visceral adipose tissue, and subcutaneous adipose tissue. In embodiments, he chimeric protein is capable of reducing or inhibiting adipocyte progenitor proliferation. In embodiments, the chimeric protein is capable of promoting adipocyte progenitor differentiation. In embodiments, the chimeric protein is capable of reducing fat mass. In embodiments, the chimeric protein is capable of inducing skeletal muscle hypertrophy. In embodiments, the chimeric protein is capable of promoting muscle growth. In embodiments, the chimeric protein is capable of reducing hepatic glucose production. In embodiments, the chimeric protein is capable of increasing lean tissue. In embodiments, the chimeric protein is capable of preventing diet-induced obesity and insulin resistance.

[0020] In aspects, the present disclosure provides a polypeptide comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113. In embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 74 or 113.

[0021] In aspects, the present disclosure provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113. In embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 74 or 113.

[0022] In aspects, the present disclosure provides an isolated polynucleotide encoding the chimeric protein of any of the embodiments disclosed herein or the polypeptide of any of the embodiments disclosed herein (e.g., comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 74 or 113), or the polypeptide of any of the embodiments disclosed herein. In embodiments, the polynucleotide is DNA. In embodiments, the polynucleotide is selected from mRNA, circular RNA (circRNA) and self-amplifying RNA (saRNA), optionally wherein the polynucleotide comprises at least one modified nucleotide selected from a modified sugar, a modified base and / or a modified internucleotide linkage.

[0023] In embodiments, the polynucleotide is an mmRNA. In embodiments, the mmRNA comprises one or more nucleoside modifications. In embodiments, the nucleoside modifications are selected from pseudouridine, 1- methylpseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl- uridine, 1 -propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl- 2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1 -methylpseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1 -deazapseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5- aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio- cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2- methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio- adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6- thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and a combination thereof. In embodiments, the mmRNA further comprises one or more of a 5'-cap, a poly A tail, a 5' UTR, optionally selected from SEQ ID NOs: 132-153, and / or a 3’ UTR, optionally selected from SEQ ID NOs: 118- 131.

[0024] In embodiments, the polynucleotide is DNA. In embodiments, the polynucleotide comprises a liver, skin and / or muscle-specific control element. In embodiments, the liver-specific control element is a liver-specific promoter selected from albumin promoter, thyroxine-binding globulin (TBG) promoter, hybrid liver-specific promoter (HLP), human a 1 -antitrypsin promoter, LP1 promoter, and hemopexin promoter.

[0025] In aspects, the present disclosure provides an expression vector comprising the isolated polynucleotide of any of the embodiments disclosed herein.

[0026] In aspects, the present disclosure provides an expression vector, wherein the expression vector is a mammalian expression vector.

[0027] In aspects, the present disclosure provides a host cell comprising the isolated polynucleotide of any of the embodiments disclosed herein or the expression vector of any of the embodiments disclosed herein.

[0028] In aspects, the present disclosure provides a pharmaceutical composition comprising the chimeric protein of any of the embodiments disclosed herein or the polypeptide of any of the embodiments disclosed herein (e.g, comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 74 or 113), the polypeptide of any of the embodiments disclosed herein, the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, the host cell of any of the embodiments disclosed herein, and a pharmaceutically acceptable carrier.

[0029] In aspects, the present disclosure provides a pharmaceutical composition comprising the mmRNA of any of the embodiments disclosed herein, and a pharmaceutically acceptable carrier. In embodiments, the carrier is a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric nanoparticle, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP), a lipoplex, or a liposome. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP).

[0030] In aspects, the present disclosure provides a pharmaceutical composition comprising mRNA (mmRNA) comprising at least one modified nucleotide selected from a modified base (without limitation, e.g., 1- methylpseudouridine, N1 -methylpseudouridine), a modified sugar (without limitation, e.g., 2'fluoro-2'- deoxyribose and 2'-0-methyl-ribose), and / or a modified internucleotide linkage (without limitation, e.g., phosphorothioate), wherein the mmRNA encodes a chimeric protein comprising a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (A) (a) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, wherein the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71, or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71 , (c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), wherein the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73, and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge-CH2-CH3 Fc domain; or (B) (a) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), wherein the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73, (c) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, wherein the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71 , or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71 , and (b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2-CH3 Fc domain, wherein the mmRNA comprises one or more nucleoside modifications and wherein the pharmaceutical composition is formulated as a lipid nanoparticle (LNP).

[0031] In embodiments, the lipid nanoparticle comprises lipids selected from an ionizable lipid e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG- ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA. In embodiments, the lipid nanoparticle comprises (a) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the particle; (b) a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and (c) a conjugated lipid that inhibits aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle. In embodiments, the lipid nanoparticle comprises a lipid selected from SM-102, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12- 5, and C12-200; a cholesterol; and a PEG-lipid.

[0032] In embodiments, the pharmaceutical composition is formulated for parenteral administration. In embodiments, the pharmaceutical composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial or transdermal administration.

[0033] In aspects, the present disclosure provides a method of preventing and / or treating hyperglycemia, diabetes, obesity, metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, or for reducing blood glucose compared to a pre-treatment blood glucose, or for reducing fed and fasting blood glucose compared to a pre-treatment fed and fasting blood glucose, or for reducing cardiovascular risk compared to a pre-treatment cardiovascular risk, or for decreasing body weight compared to a pre-treatment body weight, for decreasing food intake compared to a pre-treatment food intake, for decreasing blood glucose compared to a pre-treatment blood glucose, for decreasing liver adiposity compared to a pre-treatment liver adiposity, for decreasing liver fibrosis compared to a pre-treatment liver fibrosis, for decreasing liver weight compared to a pre-treatment liver weight, for decreasing an amount of subcutaneous white adipose tissue (sWAT) compared to a pre-treatment amount of sWAT, for decreasing an amount of epididymal white adipose tissue (eWAT) compared to a pre-treatment amount of eWAT, for decreasing liver steatosis compared to a pre-treatment liver steatosis, for decreasing hepatocellular ballooning compared to a pre-treatment hepatocellular ballooning, and / or for increasing glucose tolerance compared to a pre-treatment glucose tolerance in a subject in need thereof, the method comprising administering to the subject the chimeric protein of any of the embodiments disclosed herein, the polypeptide of any of the embodiments disclosed herein (e.g., comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113), the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, the host cell of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

[0034] In embodiments, the method decreases an amount of connective tissue within muscle of the subject compared to a pre-treatment amount of connective tissue within muscle in the subject. In embodiments, the method decreases an amount of phagocytising necrotic muscle fibers of the subject compared to a pretreatment amount of phagocytising necrotic muscle fibers in the subject. In embodiments, the method decreases an amount of fragmented sarcoplasm of the subject compared to a pre-treatment amount of fragmented sarcoplasm in the subject. In embodiments, the method retains or increases muscle size in the subject compared to a pre-treatment muscle size in the subject. In embodiments, the method retains or increases muscle striation in the subject compared to a pre-treatment muscle striation in the subject. In embodiments, the method retains or increases muscle mass and / or muscle density in the subject compared to a pre-treatment muscle mass and / or muscle density in the subject. In embodiments, the method prevents and / or treats sarcopenia, muscle weakness and / or muscle loss in the subject. In embodiments, the method improves one or more of muscle strength, physical frailty, aerobic capacity, physical performance, limitations in mobility, and physical function in the subject compared to pre-treatment level of one or more of muscle strength, physical frailty, aerobic capacity, physical performance, limitations in mobility, and physical function.

[0035] In aspects, the present disclosure provides a method of preventing and / or treating sarcopenia, physical frailty, aerobic resistance, muscle weakness, muscle loss, limitations in mobility and / or physical function, of decreasing a level of connective tissue within muscle, phagocytising necrotic muscle fibers, and / or fragmented sarcoplasm compared to a pre-treatment levels of connective tissue within muscle, phagocytising necrotic muscle fibers, and / or fragmented sarcoplasm, or of retaining or increasing muscle size, muscle striation, muscle mass, muscle density, diminished muscle strength, and / or physical performance compared to pre-treatment level of muscle size, muscle striation, muscle mass, muscle density, diminished muscle strength, or physical performance in a subject in need thereof, the method comprising administering to the subject the chimeric protein of any of the embodiments disclosed herein, the polypeptide of any of the embodiments disclosed herein (e.g., comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113), the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, the host cell of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

[0036] In embodiments, the method prevents, decreases or treats one or more of hyperglycemia, diabetes, obesity, metabolic syndrome, elevated cardiovascular risk, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) in the subject. In embodiments, the method prevents, decreases or treats one or more of one or more of increased liver weight, liver adiposity, liver fibrosis, liver steatosis, hepatocellular ballooning, and / or liver fibrosis compared to pre-treatment levels of liver weight, liver adiposity, liver fibrosis, liver steatosis, hepatocellular ballooning, and / or liver fibrosis in the subject.

[0037] In embodiments, the method reduces or treats one or more of the cardiovascular risk, body weight, food intake, subcutaneous white adipose tissue (sWAT), and epididymal white adipose tissue (eWAT) compared to pre-treatment elevated cardiovascular risk, body weight, food intake, sWAT and eWAT in the subject.

[0038] In aspects, the present disclosure provides a method of preventing and / or treating hyperglycemia, diabetes, obesity, metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, muscle weakness and / or muscle loss, or for reducing blood glucose compared to a pretreatment blood glucose, or for reducing fed and fasting blood glucose compared to a pre-treatment fed and fasting blood glucose, or for reducing cardiovascular risk compared to a pre-treatment cardiovascular risk, or for decreasing body weight compared to a pre-treatment body weight, for decreasing food intake compared to a pre-treatment food intake, for decreasing blood glucose compared to a pre-treatment blood glucose, for decreasing liver adiposity compared to a pre-treatment liver adiposity, for decreasing liver fibrosis compared to a pre-treatment liver fibrosis, for decreasing liver weight compared to a pre-treatment liver weight, for decreasing an amount of subcutaneous white adipose tissue (sWAT) compared to a pre-treatment amount of sWAT, for decreasing an amount of epididymal white adipose tissue (eWAT) compared to a pre-treatment amount of eWAT, for decreasing liver steatosis compared to a pre-treatment liver steatosis, for decreasing hepatocellular ballooning compared to a pre-treatment hepatocellular ballooning, decreasing connective tissue within muscle, phagocytising necrotic muscle fibers and / or fragmented sarcoplasm compared to pre- treatment connective tissue within muscle, phagocytising necrotic muscle fibers and / or fragmented sarcoplasm, and / or for increasing glucose tolerance compared to a pre-treatment glucose tolerance, retaining or increasing muscle size, muscle striation, and / or muscle mass compared to pre-treatment muscle size, muscle striation, and / or muscle mass in a subject in need thereof, the method comprising administering to the subject the chimeric protein of any of the embodiments disclosed herein, the polypeptide of any of the embodiments disclosed herein (e.g., comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113), the isolated polynucleotide of any of the embodiments disclosed herein, or the expression vector of any of the embodiments disclosed herein, the host cell of any of the embodiments disclosed herein, or the pharmaceutical composition of any of the embodiments disclosed herein.

[0039] In embodiments, the method reduces fat mass in the subject compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method induces skeletal muscle hypertrophy compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method reduces body weight compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method reduces glucose tolerance compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method increases insulin secretion compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method promotes muscle growth compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method reduces hepatic glucose production compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method increases lean tissue compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method prevents diet-induced obesity and insulin resistance compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist. In embodiments, the method increases muscle weight compared to another subject that has been treated with a activin receptor type-2B (ACVR2B) antagonist. In embodiments, the method reduces phagocytising necrotic fibers and / or fragmented sarcoplasm compared to another subject that has been treated with a activin receptor type-2B (ACVR2B) antagonist. In embodiments, the method reduces body weight compared to another subject that has been treated with a glucagon-like peptide-1 (GLP-1) receptor agonist, an activin receptor type-2B (ACVR2B) antagonist or a combination thereof.

[0040] Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1A to FIG. 1B show the non-limiting schematic illustrations of the chimeric proteins of the extracellular domain of activin receptor type-2B (ACVR2B)-Fc-glucagon-like peptide-1 (GLP-1) (FIG. 1A) and GLP-1-Fc- ACVR2B (FIG. 1B).

[0043] FIG. 2 demonstrates the construction of the GLP-1 -Fc-ACVR2B chimeric protein. Western blots showing the GLP-1 -Fc-ACVR2B chimeric protein are shown. A molecular weight ladder is loaded in first lane of each gel. The samples of the GLP-1 -Fc-ACVR2B chimeric protein that were not reduced or deglycosylated but only boiled with SDS were loaded into the lane marked as NR of each of the blots. Samples in the lane marked as R were treated with a reducing agent, |3-mercaptoethanol and boiled. Samples in the lane marked as DG were treated with a deglycosylation agent, the reducing agent, and boiled. Each individual domain of the chimeric protein was probed using an anti-human GLP antibody (left blot), an anti-Fc antibody (center blot), or an anti-human ACVR2B antibody (right blot).

[0044] FIG. 3A to FIG. 3C demonstrate binding of the binding of the GLP-1 -Fc-ACVR2B chimeric protein to ACVR2B ligands and / or GLP-1 receptor. FIG. 3A is a line graph showing simultaneous binding of the binding of the GLP-1 -Fc-ACVR2B chimeric protein to an anti-Fc antibody and one of the ACVR2B ligands: activin A, GDF- 5 (BMP-14), GDF-8 (myostatin) or GDF-11, as detected using an ELISA-based assay. FIG. 3B is a line graph showing simultaneous binding of the binding of the GLP-1 -Fc-ACVR2B chimeric protein to an anti-Fc antibody and GLP-1 receptor (GLP-1 R), as detected using an ELISA-based assay. FIG. 3C is a line graph showing simultaneous binding of the binding of the GLP-1 -Fc-ACVR2B chimeric protein to myostatin and GLP-1 R, as detected using an ELISA-based assay.

[0045] FIG. 4A to FIG. 4C demonstrate the inhibition by the GLP-1 -Fc-ACVR2B chimeric protein of the ACVRB2 signaling induced by ACVR2B ligands. FIG. 4A and FIG. 4B are line graphs showing the inhibition by the GLP-1 -Fc-ACVR2B chimeric protein of activin A-mediated stimulation of ACVRB2 signaling. FIG. 4C is a line graph showing the inhibition by the GLP-1 -Fc-ACVR2B chimeric protein of myostatin (GDF-8)-mediated stimulation of ACVRB2 signaling. FIG. 5A and FIG. 5B demonstrate the inhibition by the GLP-1-Fc-ACVR2B chimeric protein of apoptosis induced by ACVR2B ligands in murine plasmacytoma MPC-11 cells. FIG. 5A is line graphs showing dosedependent induction of apoptosis by GDF-11 and Activin A in MPC-11 cells. FIG. 5B is a line graph showing the inhibition by the GLP-1-Fc-ACVR2B chimeric protein of apoptosis in MPC-11 cells induced by GDF-11 and Activin A.

[0046] FIG. 6 shows a dose-dependent and saturable activation of GLP-1 R signaling by the GLP-1-Fc-ACVR2B chimeric protein.

[0047] FIG. 7A to FIG. 7C are the bar graphs that demonstrate binding by the GLP-1-Fc-ACVR2B chimeric protein to GLP-1 R (FIG. 7A), Activin A (FIG. 7B), and Myostatin (FIG. 7C) as determined by Biolayer Interferometry (BLI).

[0048] FIG. 8 shows the contemporaneous binding to an anti-ACVR2B antibody and an anti-GLP-1 antibody by the GLP-1-Fc-ACVR2B chimeric protein present in culture supernatant of CHO-K1 cells transfected with mRNA encoding the GLP-1-Fc-ACVR2B chimeric protein.

[0049] FIG. 9 shows the contemporaneous GLP-1-Fc-ACVR2B chimeric protein present in culture supernatant of CHO-K1 cells transfected with mRNA encoding the GLP-1-Fc-ACVR2B chimeric protein blocks the activin A-mediated stimulation of ACVRB2 signaling.

[0050] FIG. 10A to FIG. 10P demonstrate the efficacy of the GLP-1 -Fc-ACVR2B chimeric protein or mRNA encoding the chimeric protein in mouse models of diet-induced obesity, hepatic steatosis and early-stage liver fibrosis. FIG. 10A shows the schematic representation of generation of mouse models for diet-induced obesity (DIO) and glucose tolerance test (GTT) and experiments using the models. FIG. 10B is a line graph showing the change in body weight of the DIO model mice in response to the indicated treatments. FIG. 10C and FIG. 10D are line graphs showing the changes blood glucose levels (FIG. 10C) and insulin secretion (FIG. 10D) in the GTT model mice after a bolus injection of glucose and the indicated treatments. FIG. 10E is a line graph showing the change in body weight of the DIO model mice in response to the indicated treatments. FIG. 10F is a bar graph showing the change in muscle weight in the DIO model mice in response to the indicated treatments. FIG. 10G and FIG. 10H show muscle histology in the DIO model mice in response to the indicated treatments, as detected by hematoxylin and eosin (H&E) staining. FIG. 101 to FIG. 10L are bar graphs showing changes in food intake (FIG. 10I), liver weight (FIG. 10 J), subcutaneous white adipose tissue (sWAT) weight (FIG. 10K) and epididymal white adipose tissue (eWAT) weight (FIG. 10L) in the DIO model mice in response to the indicated treatments. FIG. 10M shows liver histology in the DIO model mice (showing changes in steatosis and ballooning of liver) in response to the indicated treatments, as detected by H&E staining. FIG. 10N and FIG. 100 are bar graphs showing microvascular steatosis (FIG. 10N), and hepatocellular ballooning (FIG. 100) in the DIO model mice in response to the indicated treatments. FIG. 10P shows liver histology in the DIO model mice (showing changes in liver fibrosis) in response to the indicated treatments, as detected by picrosirius red (PSR) staining.

[0051] FIG. 11 A to FIG. 11 E show the characterization of mRNA constructs encoding the GLP-1 -Fc-Activin receptor type-2B (ACVR2B) chimeric protein. FIG. 11A is a bar graph comparing the production of the GLP-1 -Fc- ACVR2B chimeric protein by cells transfected with mRNA constructs encoding the GLP-1 -Fc-ACVR2B chimeric protein and harboring different 5’ UTR sequences and 3’UTR sequences. FIG. 11 B is a line graph showing the binding by increasing amounts of purified recombinant human ACVR2B-Fc fusion protein to recombinant human GDF-8 as measured using a Meso Scale Discovery (MSD) platform-based assay. FIG. 11C is a line graph showing the binding to recombinant human Activin A by the GLP-1 -Fc-ACVR2B chimeric protein increasing dilutions of the culture supernatants of HEK293T cells transfected with mRNA constructs encoding the GLP-1 -Fc-ACVR2B chimeric protein and harboring different 5’ UTR sequences and 3’UTR sequences. FIG. 11 D is a line graph showing the binding by increasing amounts of purified recombinant human ACVR2B-Fc fusion protein to recombinant human GDF-8 as measured using a Meso Scale Discovery (MSD) platform-based assay. FIG. 11 E is a line graph showing the binding to recombinant human GDF-8 by the GLP-1 -Fc-ACVR2B chimeric protein increasing dilutions of the culture supernatants of HEK293T cells transfected with mRNA constructs encoding the GLP-1 -Fc-ACVR2B chimeric protein and harboring different 5’ UTR sequences and 3’UTR sequences.

[0052] DETAILED DESCRIPTION

[0053] The present disclosure is based, in part, on the creation of a fusion protein comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist or a variant thereof, which is connected via an optional protease- cleavable linker to a polypeptide comprising a hinge-CH2-CH3 Fc domain to a portion of activin receptor type-2B (ACVR2B).

[0054] Fusion Proteins of the Present Disclosure

[0055] In aspects, the present disclosure provides a chimeric protein having a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein (a) is the second domain comprising a portion of activin receptor type-2B (ACVR2B), (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises one or more protease-cleavable polypeptide linkers, and / or a hinge-CH2-CH3 Fc domain, and (c) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist or a variant thereof, or an analog thereof. In embodiments, the portion of ACVR2B is capable of binding activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B is the extracellular domain of ACVR2B. In embodiments, the first domain comprises the GLP-1 receptor agonist of any of the embodiments disclosed herein. In embodiments, the chimeric protein is administered to a patient. In embodiments, a nucleic acid encoding the chimeric protein (e.g., modified mRNA or DNA) is administered to a patient. In embodiments, the nucleic acid encoding harbors control elements that enable the expression of the chimeric protein (e.g., modified mRNA or DNA) is the expressed in liver, skin and / or muscle.

[0056] In aspects, the present disclosure provides a polypeptide comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113. In embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 74 or 113. In embodiments, the chimeric protein is administered to a patient. In embodiments, a nucleic acid encoding the chimeric protein (e.g., modified mRNA or DNA) is administered to a patient. In embodiments, the nucleic acid encoding harbors control elements that enable the expression of the chimeric protein (e.g., modified mRNA or DNA) is the expressed in liver, skin and / or muscle.

[0057] Glucagon-Like Peptide 1 (GLP-1) Receptor Agonists

[0058] Glucagon-like peptide 1 (GLP-1) is a 30-amino acid peptide hormone produced in the intestine. GLP-1 is normally produced after meals and stimulates insulin secretion and inhibits glucagon secretion. It is also involved in the regulation of p-cell growth and survival, gastric emptying, and appetite. In the body, GLP-1 is degraded by dipeptidyl peptidase IV and has a short half-life of around 2 minutes. Reduced GLP-1 secretion is associated with type 2 diabetes and the development of obesity.

[0059] In embodiments, the chimeric proteins disclosed herein have a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein (a) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist. In these embodiments, (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises one or more protease-cleavable polypeptide linkers, and / or a hinge-CH2-CH3 Fc domain. In these embodiments, (c) is the second domain comprising a portion of activin receptor type-2B (ACVR2B).

[0060] In alternative embodiments, the chimeric proteins disclosed herein have a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein (c) is a first domain comprising a portion of activin receptor type-2B (ACVR2B). In these embodiments, (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises one or more protease-cleavable polypeptide linkers, and / or a hinge-CH2-CH3 Fc domain. In these embodiments, (c) is a second domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist or a variant thereof, or an analog thereof.

[0061] In embodiments, the GLP-1 receptor agonist signals through its receptor, GLP-1 receptor (GLP-1 R), a G- protein coupled receptor. In embodiments, the GLP-1 receptor agonist activates the GLP-1 R on the surface of pancreatic p-cells. In embodiments, the GLP-1 receptor agonist induces increased insulin production by the pancreatic p-cells in a glucose dependent manner in response to the GLP-1 R activation. In embodiments, the GLP-1 receptor agonist activates the GLP-1 R on the surface of pancreatic a-cells. In embodiments, the GLP-1 receptor agonist activates GLP-1 R suppresses glucose-dependent glucagon secretion by the pancreatic a-cells in response to the GLP-1 R activation.

[0062] In embodiments, any of a number of drugs that mimic the action of GLP-1 by binding and activating the GLP- 1 receptor is suitable. In embodiments, the GLP-1 receptor agonist is a short acting form (without limitation, e.g., exenatide). In embodiments, the GLP-1 receptor agonist is a long-acting forms (without limitation, e.g., dulaglutide and liraglutide). In embodiments, the GLP-1 receptor agonist is GLP-1.

[0063] GLP-1 is produced by the alpha cells of the pancreas and in the intestinal L cells in the distal ileum and colon in form of a precursor called preglucagon that is cleaved in different organs into glicentin, glicentin-related pancreatic polypeptide (GRPP), oxyntomodulin, glucagon, glucagon-like peptide 1 (GLP-1 , indicated in a boldface-underlined font), and glucagon-like peptide 2 (GLP-2). Preglucagon has the following sequence:

[0064] MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSR RAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAI VEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK (SEQ ID NO: 60)

[0065] In embodiments, the GLP-1 receptor agonist is wild-type human GLP-1. In embodiments, the GLP-1 receptor agonist is GLP-1 receptor agonists. In embodiments, the GLP-1 receptor agonist is variant of these peptides that can activate the GLP-1 receptor. Suitable GLP-1 receptor agonists are disclosed in US Patent Nos. 5,188,666, 5,120,712, 5,523,549, 5,512,549, 5,977,071 , 6,191 ,102; 6,956,026; 6,506,724; 6,703,359; 6,858,576; 6,872,700; 6,902,744; 7,157,555; 7,223,725; 7,220,721; 9,161 ,953; PCT International Patent Application Publication Nos: WO 1998 / 008871 ; WO 1998 / 05351 ; WO 1999 / 07404; WO 1999 / 25727; WO 1999 / 25728; WO 1999 / 40788; WO 2000 / 034331; WO 2000 / 41546; WO 2000 / 41548; WO 2000 / 069911; WO 2000 / 73331; WO 2001 / 004156; WO 2001 / 51078; WO 2003 / 018516; WO 2003 / 099314; U.S. Patent Application Publication Nos. 2003 / 0036504; and 2006 / 0094652, the entire contents of which are hereby incorporated by reference in their entirety.

[0066] In embodiments, the GLP-1 receptor agonist is GLP-1 having the following sequence, and is also referred to herein as GLP-1 (1-37):

[0067] HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 61)

[0068] In embodiments, the GLP-1 receptor agonist is a GLP-1 variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 61.

[0069] In embodiments, the GLP-1 receptor agonist is GLP-1 having the following sequence, and is also referred to herein as GLP-1 (1-36):

[0070] HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 62)

[0071] In embodiments, the GLP-1 receptor agonist is a GLP-1 variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 62.

[0072] In embodiments, the GLP-1 receptor agonist is GLP-1 having the following sequence, and is also referred to herein as GLP-1 (7-36) or GLP-1 :

[0073] HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 63)

[0074] In embodiments, the GLP-1 receptor agonist is a GLP-1 variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 63.

[0075] In embodiments, the GLP-1 receptor agonist is exenatide having the following sequence:

[0076] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 64) In embodiments, the GLP-1 receptor agonist is an exenatide variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 64.

[0077] In embodiments, the GLP-1 receptor agonist lixisenatide is having the following sequence:

[0078] HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK (SEQ ID NO: 65)

[0079] In embodiments, the GLP-1 receptor agonist is a lixisenatide variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 65.

[0080] In embodiments, the GLP-1 receptor agonist is the GLP-1 receptor agonist portion of albiglutide having the following sequence:

[0081] HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 66)

[0082] In embodiments, the GLP-1 receptor agonist is an albiglutide variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 66.

[0083] In embodiments, the GLP-1 receptor agonist is liraglutide having the following sequence:

[0084] HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 67)

[0085] In embodiments, the GLP-1 receptor agonist is an liraglutide variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 67.

[0086] In embodiments, the GLP-1 receptor agonist is exendin-4 having the following sequence: HGEGTFTSDLSKQMEEEAVRLFEWLKNGGPSSGAPPPS (SEQ ID NO: 68)

[0087] In embodiments, the GLP-1 receptor agonist is an exendin-4 variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 68.

[0088] Dulaglutide (GLP-1 moiety-Fc fusion protein; GLP-1 moiety underlined, (GGGGS)3 shown in a boldface font) has the following sequence:

[0089] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 69)

[0090] In embodiments, the GLP-1 receptor agonist is an GLP-1 moiety of dulaglutide variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 69.

[0091] In embodiments, the GLP-1 receptor agonist is the GLP-1 moiety of dulaglutide having the following sequence:

[0092] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG (SEQ ID NO: 70)

[0093] In embodiments, the GLP-1 receptor agonist is an GLP-1 moiety of dulaglutide variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 70.

[0094] In embodiments, the GLP-1 receptor agonist is GLP-1 derivative that is resistant to DPP4 degradation. In embodiments, the GLP-1 receptor agonist comprises a mutation that confers resistance to DPP4. In embodiments, the GLP-1 receptor agonist is DPP4 degradation resistant GLP-1 (7-37, A8G) having the following sequence:

[0095] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRG (SEQ ID NO: 71)

[0096] In embodiments, the GLP-1 receptor agonist is a GLP-1 , or a variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 71.

[0097] In embodiments, the GLP-1 receptor agonist is an GLP-1 moiety of liraglutide, or an amino acid sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence of the GLP-1 moiety of liraglutide. In embodiments, the GLP-1 receptor agonist is an GLP-1 moiety of semaglutide, or an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence of the GLP-1 moiety of semaglutide. In embodiments, the GLP-1 receptor agonist is an GLP-1 moiety of taspoglutide, or an amino acid sequence having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence of the GLP-1 moiety of taspoglutide.

[0098] In embodiments, the chimeric protein of the disclosure binds to human GLP-1 receptor with a KD of less than about 1 JJM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 550 nM, about 530 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 55 nM, about 50 nM, about 45 nM, about 40 nM, about 35 nM, about 30 nM, about 25 nM, about 20 nM, about 15 nM, about 10 nM, or about 5 nM, or about 1 nM (as measured, for example, by surface plasmon resonance or biolayer interferometry). In embodiments, the chimeric protein binds to human GLP-1 receptor with a KD of less than about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM about 55 pM about 50 pM about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, or about 10 pM, or about 1 pM (as measured, for example, by surface plasmon resonance or biolayer interferometry). In embodiments, the chimeric protein binds to human GLP-1 receptor with a KD of from about 300 pM to about 700 pM.

[0099] GLP-1 derivatives can be constructed from available structural data, including that described by Chang et al., Structure and Folding of Glucagon-like Peptide-1 -(7-36)-amide in Trifluoroethanol Studied by NMR, Magn Reson Chem 39: 477-483 (2001); Underwood et al., Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor, J Biol Chem 285: 723-730 (2010); Lau et al., Discovery of the Once-Weekly Glucagon-Like Peptide-1 (GLP-1) Analogue Semaglutide, J Med Chem 58: 7370-7380 (2015); Oddo et al., alpha-Helix or beta-Turn? An Investigation into N-Terminally Constrained Analogues of Glucagon-like Peptide 1 (GLP-1) and Exendin-4, Biochemistry 57: 4148-4154 (2018); Zhang et al., Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein, Nature 546: 248-253 (2017); Bueno et al., Structural insights into probe-dependent positive allosterism of the GLP-1 receptor, Nat Chem Biol 16: 1105-1110 (2020); Zhang et al., Differential GLP-1 R Binding and Activation by Peptide and Non-peptide Agonists, Mol Cell 80: 485 (2020).

[0100] In embodiments, the GLP-1 receptor agonist is selected from GLP-1 (1-37), GLP-1 (1-36) GLP-1 (7-36), exenatide, lixisenatide, exendin-4, albiglutide, liraglutide, dulaglutide, dulaglutide, or a variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71 , or a variant having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71. In embodiments, the mutations are independently selected from substitutions, insertions, deletions, and truncations. In embodiments, first domain is capable of binding a GLP-1 receptor. In embodiments, the GLP- 1 receptor agonist is capable of stimulating and / or increasing insulin secretion, and / or inhibiting and / or decreasing glucagon secretion. In embodiments, the GLP-1 receptor agonist is capable of stimulating and / or increasing insulin secretion, and / or inhibiting and / or decreasing glucagon secretion, compared to a chimeric protein lacking the first domain (e.g., having a structure: (a) a second domain comprising a portion of activin receptor type-2B (ACVR2B) of any of the embodiments disclosed herein and optionally (b) a linker comprising one or more protease-cleavable polypeptide linkers and / or a hinge-CH2-CH3 Fc domain.

[0101] Activin Receptor Type-2B (ACVR2B)

[0102] Activin receptor type-2B is a transmembrane serine / threonine kinase activin type-2 receptor, which transduces regulates muscle physiology, neuron physiology, hair follicle development and cycling, FSH production, wound healing, extracellular matrix production, etc. ACVR2B ligands include activin A and GDF- 8 / myostatin, both of which negatively regulate the muscle size. McPherron et al., Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member, Nature 387, 83-90 (1997); Chen et al. Elevated expression of activins promotes muscle wasting and cachexia. FASEB J. 28, 1711-1723 (2014). Concomitant inhibition of activin A and GDF8 has been shown to synergistically increase muscle mass in mice and non-human primates. Nissinen ef al., Systemic blockade of ACVR2B ligands prevents chemotherapy-induced muscle wasting by restoring muscle protein synthesis without affecting oxidative capacity or atrogenes, Sci Rep 6: 32695 (2016); Latres et al., Activin A more prominently regulates muscle mass in primates than does GDF8, Nat Commun. 8: 15153 (2017).

[0103] As shown in FIG. 3A, ligands like activin A, GDF-5 (BMP-14), GDF-8 (myostatin) or GDF-11 bind the ACVR2B receptor. When bound to its ligands, ACVR2B, which forms an activin receptor complex along with a activin type-1 receptor such as ACVR1, ALK4 (ActRIB) and ALK7 (ActRIC), recruits and phosphorylates a serine residues of the C-terminal tail of SMAD2 and / or SMAD3. SMAD2 and SMAD3 are released into the cytoplasm where they interact with the common partner SMAD4. This SMAD complex translocates into the nucleus where it mediates activin-induced transcription. Deacu et al., Activin Type II Receptor Restoration in ACVR2- Deficient Colon Cancer Cells Induces Transforming Growth Factor-p Response Pathway Genes, Cancer Res 64 (21): 7690-7696 (2004); Olsen et a / ., Activin A Inhibits BMP-Signaling by Binding ACVR2A and ACVR2B, Cell Commun Signal. 13: 27 (2015); Morianos ef al., Activin-A in the Regulation of Immunity in Health and Disease, J. Autoimmun. 104: 102314 (2019).

[0104] In aspects, the present disclosure provides a chimeric protein comprising a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (A) (a) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, (c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge- CH2-CH3 Fc domain; or (B) (a) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), (c) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, and (b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2-CH3 Fc domain.

[0105] In embodiments, the GLP-1 receptor agonist is selected from GLP-1 , a DPP4 degradation resistant derivative of GLP-1, exenatide, lixisenatide, albiglutide, dulaglutide, or a variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71 or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71. In embodiments, the GLP-1 receptor agonist is capable of binding a GLP-1 receptor. In embodiments, the GLP-1 receptor agonist is capable of stimulating and / or increasing insulin secretion, and / or inhibiting and / or decreasing glucagon secretion.

[0106] In embodiments, the portion of ACVR2B is capable of binding activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B comprises substantially the entire extracellular domain of ACVR2B. In embodiments, the portion of ACVR2B comprises the entire extracellular domain of ACVR2B. In embodiments, the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73.

[0107] In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG1. In embodiments, the lgG1 is human lgG1. In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG4. In embodiments, the lgG4 is human lgG4. In embodiments, the hinge-CH2-CH3 Fc domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 53. In embodiments, the linker further comprises the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 4 to 52. In embodiments, the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 4 to 52; wherein one joining linker is N terminal to the hinge-CH2-CH3-Fc domain and another joining linker is C terminal to the hinge-CH2-CH3-Fc domain.

[0108] In aspects, the present disclosure provides an isolated polynucleotide encoding the chimeric protein of any of the embodiments disclosed herein, or the polypeptide of any of the embodiments disclosed herein (e.g., comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113). In embodiments, the polynucleotide is selected from mRNA, circular RNA (circRNA) and self-amplifying RNA (saRNA), optionally wherein the polynucleotide comprises at least one modified nucleotide selected from a modified sugar, a modified base and / or a modified internucleotide linkage. In embodiments, the polynucleotide is an mmRNA. In embodiments, the mmRNA comprises one or more nucleoside modifications, optionally wherein the mmRNA comprises one or more of 1 -methylpseudouridine nucleotides.

[0109] In embodiments, the chimeric protein comprises a portion of the full length human activin receptor type- 26 (ACVR2B), which comprises the following illustrative amino acid sequence: MTAPWVALALLWGSLCAGSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSG TIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLTVL AYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMN DFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITW NELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGD THGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEIGQHPSL EELQEVWHKKMRPTIKDHWLKHPGLAQLCVTIEECWDHDAEARLSAGCVEERVSLIRRSVNGTTSDCLVS LVTSVTNVDLPPKESSI (SEQ ID NO: 72).

[0110] In embodiments, the chimeric protein comprises a portion of the full length ACVR2B, which comprises a variant or functional fragment SEQ ID NO: 72. For instance, the chimeric protein may comprise a portion of the full length ACVR2B having a sequence as provided above, or a variant or functional fragment thereof having at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) sequence identity with the amino acid sequence of SEQ ID NO: 72.

[0111] In embodiments, the chimeric protein comprises an extracellular domain of human activin receptor type- 26 (ACVR2B), which comprises the following illustrative amino acid sequence:

[0112] SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDR QECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLT (SEQ ID NO: 73).

[0113] In embodiments, the chimeric protein comprises an extracellular domain of ACVR2B, which comprises a variant or functional fragment SEQ ID NO: 73. For instance, the chimeric protein may comprise an extracellular domain of ACVR2B having a sequence as provided above, or a variant or functional fragment thereof having at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) sequence identity with the amino acid sequence of SEQ ID NO: 73.

[0114] ACVR2B derivatives can be constructed from available structural data, including a homology model described by Goebel et al., Structures of activin ligand traps using natural sets of type I and type II TGF receptors, IScience 25(1 ): 103590 (2022); Morvan et al., Blockade of activin type II receptors with a dual anti- ActRIIA / IIB antibody is critical to promote maximal skeletal muscle hypertrophy, Proc Natl Acad Sci U S A 114(47): 12448-12453 (2017); Townson et al., Specificity and structure of a high affinity activin receptor-like kinase 1 (ALK1) signaling complex, J Biol Chem 2 287(33): 27313-25 (2012); Han et al., Crystal structure of activin receptor type IIB kinase domain from human at 2.0 Angstrom resolution, Protein Sci 16(10): 2272- 2277 (2007); and Weber et al., A silent H-bond can be mutationally activated for high-affinity interaction of BMP-2 and activin type IIB receptor, BMC Struct Biol 7'. 6 (2007).

[0115] In aspects, the present disclosure provides a chimeric protein having a general structure of: N terminus - (a)

[0116] - (b) - (c) - C terminus, wherein (a) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, a variant thereof, or an analog thereof, (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises one or more protease-cleavable polypeptide linkers, and / or a hinge- CH2-CH3 Fc domain, and (c) is the second domain comprising a portion of activin receptor type-2B (ACVR2B). In embodiments, the portion of ACVR2B is capable of binding activin A, GDF-5 (BMP-14), GDF- 8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B is the extracellular domain of ACVR2B. In embodiments, the first domain comprises the GLP-1 receptor agonist of any of the embodiments disclosed herein.

[0117] In aspects, the present disclosure provides a chimeric protein having a general structure of: N terminus - (a)

[0118] - (b) - (c) - C terminus, wherein (a) is the second domain comprising a portion of activin receptor type-2B (ACVR2B), (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises one or more protease-cleavable polypeptide linkers, and / or a hinge-CH2-CH3 Fc domain, and (c) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist or a variant thereof, or an analog thereof. In embodiments, the portion of ACVR2B is capable of binding activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B is the extracellular domain of ACVR2B. In embodiments, the first domain comprises the GLP-1 receptor agonist of any of the embodiments disclosed herein.

[0119] An illustrative GLP-1 -Fc-ACVR2B chimeric protein has the following sequence (a secretion signal sequence is shown in double underline. DPP4 degradation resistant GLP-1 is shown by an boldface-italicized font, a linker comprising a mutant Fc domain of human I gG1 is shown in an unmarked font, joining linkers are shown in a boldface-underlined font, and an extracellular domain of ACVR2B is shown in an italic font):

[0120] MEFGLSWVFLVAIIKGVQCHGEG7FTSDVSSYLEEQA KEF / A .VKGRGEPKSVDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K\EG^DSGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLD DFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLT (SEQ ID NO: 74).

[0121] In embodiments, the chimeric protein comprises a variant of the GLP-1 -Fc-ACVR2B chimeric protein. As examples, the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 74.

[0122] An illustrative mature GLP-1 -Fc-ACVR2B chimeric protein, which is formed after the cleavage of secretion signal sequence, has the following sequence (DPP4 degradation resistant GLP-1 is shown by an boldface- italicized font, a linker comprising a mutant Fc domain of human lgG1 is shown in an unmarked font, joining linkers are shown in a boldface-underlined font, and an extracellular domain of ACVR2B is shown in an italic font):

[0123] HGEGTFTSDYSSYLEEQAAKEF / AIVLV GRGEPKSyDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIEGRMDSGRGEAETREC / YYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQV YFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTLLT (SEQ ID NO: 113).

[0124] In embodiments, the chimeric protein comprises a variant of the GLP-1-Fc-ACVR2B chimeric protein. As examples, the variant may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 113.

[0125] Linker

[0126] In embodiments, the chimeric protein comprises a linker.

[0127] In embodiments, the chimeric proteins disclosed herein have a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein (a) is a first domain comprising a glucagon-like peptide-1 (GLP-1 ) receptor agonist and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises one or more protease-cleavable polypeptide linkers, and / or a hinge-CH2-CH3 Fc domain. In these embodiments, (c) is the second domain comprising a portion of activin receptor type-2B (ACVR2B).

[0128] In alternative embodiments, the chimeric proteins disclosed herein have a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein (c) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises one or more protease-cleavable polypeptide linkers, and / or a hinge-CH2-CH3 Fc domain. In these embodiments, (a) is the second domain comprising a portion of activin receptor type-2B (ACVR2B).

[0129] In embodiments, the present chimeric proteins may comprise variants of the protease-cleavable polypeptide linkers disclosed in Table 1, below. For instance, a linker may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 4 to 52.

[0130] Table 1 : Illustrative protease-cleavable polypeptide linkers

[0131] In embodiments, the linker comprises a protease-cleavable polypeptide linker. In embodiments, the protease- cleavable polypeptide linker cleavable by a protease that is endogenous to a mammalian expressed in liver, skin and / or muscle. In embodiments, the protease-cleavable linker is cleavable by a protease selected from caspases, kallikreins, cathepsins, legumain, matrix metalloproteinases (MMPs), cathepsin, elastase, plasmin, thrombin, trypsin, urokinase-type plasminogen activator (uPA), matriptase, meprins and hepsin. In embodiments, the protease-cleavable linker comprises a consensus recognition and / or cleavage site of a protease selected from, caspases, kallikreins, cathepsins, legumain, matrix metalloproteinases (MMPs), cathepsin, elastase, plasmin, thrombin, trypsin, urokinase-type plasminogen activator (uPA), matriptase, meprins and hepsin. In embodiments, the chimeric protein comprises one protease-cleavable polypeptide linker selected from RFRS (SEQ ID NO: 54), HSSKLQ (SEQ ID NO: 55), GPLGVRG (SEQ ID NO: 56), IPVSLRSG (SEQ ID NO: 57), VPLSLYSG (SEQ ID NO: 58), and SGESPAYYTA (SEQ ID NO: 59), or a variant thereof having about 1, 2, 3, 4, or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 54 to 59. In embodiments, the protease-cleavable polypeptide linker is C terminal to the first domain or N terminal to the second domain.

[0132] In embodiments, the chimeric protein comprises two protease-cleavable polypeptide linkers. In embodiments, wherein the first protease-cleavable polypeptide linker is C terminal to the first domain and the second protease-cleavable polypeptide linker is N terminal to the second domain. In embodiments, the two protease- cleavable polypeptide linkers are cleavable by a protease that is endogenous to a mammalian expressed in liver, skin and / or muscle. In embodiments, the two protease-cleavable polypeptide linkers are cleavable by a protease independently selected from caspases, kallikreins, cathepsins, legumain, matrix metalloproteinases (MMPs), cathepsin, elastase, plasmin, thrombin, trypsin, urokinase-type plasminogen activator (uPA), matriptase, meprins and hepsin. In embodiments, the two protease-cleavable polypeptide linkers comprise consensus recognition and / or cleavage sites of a proteases independently selected from, caspases, kallikreins, cathepsins, legumain, matrix metalloproteinases (MMPs), cathepsin, elastase, plasmin, thrombin, trypsin, urokinase-type plasminogen activator (uPA), matriptase, meprins and hepsin. In embodiments, the two protease-cleavable polypeptide linkers are cleavable by a protease independently comprises an amino acid sequence selected from RFRS (SEQ ID NO: 54), HSSKLQ (SEQ ID NO: 55), GPLGVRG (SEQ ID NO: 56), IPVSLRSG (SEQ ID NO: 57), VPLSLYSG (SEQ ID NO: 58), and SGESPAYYTA (SEQ ID NO: 59), or a variant thereof having about 1, 2, 3, 4, or more amino acid mutations with respect to an amino acid sequence selected from SEQ ID NOs: 54 to 59.

[0133] In embodiments, the protease-cleavable polypeptide linkers are cleavable by a protease that is endogenous to mammalian expressed in liver, skin and / or muscle. In embodiments, the protease is selected from, caspases, kallikreins, cathepsins, legumain, matrix metalloproteinases (MMPs), cathepsin, elastase, plasmin, thrombin, trypsin, urokinase-type plasminogen activator (uPA), matriptase, meprins and hepsin. Accordingly, in embodiments, the protease that cleaves the protease-cleavable polypeptide linkers is already present in the subject and an exogenous protease need not be administered. In embodiments, levels of the protease are elevated by liver injury, diabetes, and / or fibrosis. In embodiments, the chimeric protein comprises one protease-cleavable polypeptide linker selected from RFRS (SEQ ID NO: 54), HSSKLQ (SEQ ID NO: 55), GPLGVRG (SEQ ID NO: 56), IPVSLRSG (SEQ ID NO: 57), VPLSLYSG (SEQ ID NO: 58), and SGESPAYYTA (SEQ ID NO: 59) or a variant thereof having about 1, 2, 3, 4, or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 54 to 59. In embodiments, the protease-cleavable polypeptide linker is C terminal to the first domain or N terminal to the second domain. Additional suitable protease-cleavable polypeptide linkers are disclosed in US Publication Nos. 2009 / 0042787 and 2021 / 0130430, the contents of which are hereby incorporated by reference in their entirety.

[0134] In embodiments, the first domain comprises a glucagon-like peptide-1 (GLP-1) receptor agonist and the protease-cleavable polypeptide linker is C terminal to the first domain; or the second domain comprises a glucagon-like peptide-1 (GLP-1) receptor agonist and the protease-cleavable polypeptide linker is N terminal to the second domain. In embodiments, the chimeric protein comprises two protease-cleavable polypeptide linkers, such protease-cleavable polypeptide linker independently selected from RFRS (SEQ ID NO: 54), HSSKLQ (SEQ ID NO: 55), GPLGVRG (SEQ ID NO: 56), IPVSLRSG (SEQ ID NO: 57), VPLSLYSG (SEQ ID NO: 58), and SGESPAYYTA (SEQ ID NO: 59) or a variant thereof having about 1 , 2, 3, 4, or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 54 to 59. In embodiments, the first protease-cleavable polypeptide linker is C terminal to the first domain and the second domain is protease-cleavable polypeptide linker is N terminal to the second domain.

[0135] In embodiments, the linker comprises at least one cysteine residue capable of forming a disulfide bond. The at least one cysteine residue is capable of forming a disulfide bond between a pair (or more) of chimeric proteins. Without wishing to be bound by theory, such disulfide bond forming is responsible for maintaining a useful multimeric state of chimeric proteins. This allows for efficient production of the chimeric proteins; it allows for desired activity in vitro and in vivo.

[0136] In a chimeric protein of the present disclosure, the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence. In embodiments, the linker comprises hinge-CH2- CH3 Fc domain derived from lgG4, optionally human lgG4. In embodiments, the linker comprises hinge-CH2- CH3 Fc domain derived from lgG1 , optionally human lgG1.

[0137] In embodiments, the linker may be derived from naturally occurring multi-domain proteins or is an empirical linker as described, for example, in Chichili et al., (2013), Protein Sci. 22(2): 153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contents of which are hereby incorporated by reference. In embodiments, the linker may be designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et. al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.

[0138] In embodiments, the linker is a synthetic linker such as PEG.

[0139] In embodiments, the linker comprises a polypeptide. In embodiments, the polypeptide is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long. For example, the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11 , about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.

[0140] In embodiments, the linker is flexible.

[0141] In embodiments, the linker is rigid.

[0142] In embodiments, the linker is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines).

[0143] In embodiments, the linker comprises a hinge region of an antibody (e.g., of IgG, IgA, IgD, and I g E, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1 , and lgA2)). The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of lgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. lgG2 has a shorter hinge than lgG1 , with 12 amino acid residues and four disulfide bridges. The hinge region of lgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the I gG2 molecule. I gG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the I gG 1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In lgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in I gG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of I gG4 is shorter than that of lgG1 and its flexibility is intermediate between that of lgG1 and lgG2. The flexibility of the hinge regions reportedly decreases in the order I gG3> I gG 1 > I gG4>l gG2. In embodiments, the linker may be derived from human lgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.

[0144] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region includes amino acids from the carboxyl end of CHI to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id. The core hinge region of wild-type human lgG1 contains the sequence CPPC (SEQ ID NO: 24) which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. In embodiments, the present linker comprises, one, or two, or three of the upper hinge regions, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, I g D, and IgE, inclusive of subclasses (e.g., lgG1 , lgG2, lgG3, and lgG4, and lgA1 and lgA2)). The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, lgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin. In embodiments, the linker of the present disclosure comprises one or more glycosylation sites.

[0145] In embodiments, the linker comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1, lgG2, lgG3, and lgG4, and lgA1 and lgA2)).

[0146] In a chimeric protein of the present disclosure, the linker comprises a hinge-CH2-CH3 Fc domain derived from lgG4. In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human I gG4. In embodiments, the linker has at least about 95%, or at least about 97%, or at least about 97%, or at least about 98% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 3, e.g., at least 95% identical to the amino acid sequence of SEQ ID NO: 2. In embodiments, the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 4-50 (or a variant thereof). In embodiments, the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 4-50 (or a variant thereof); wherein one joining linker is N terminal to the hinge-CH2-CH3 Fc domain and another joining linker is C terminal to the hinge-CH2-CH3 Fc domain.

[0147] In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derived from a human lgG1 antibody. In embodiments, the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn). In embodiments, the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present chimeric proteins.

[0148] In embodiments, the Fc domain in a linker contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311 , 416, 428, 433 or 434 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference), or equivalents thereof. In embodiments, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In embodiments, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine. In embodiments, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In embodiments, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In embodiments, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In embodiments, the amino acid substitution at amino acid residue 309 is a substitution with proline. In embodiments, the amino acid substitution at amino acid residue 311 is a substitution with serine. In embodiments, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine. In embodiments, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In embodiments, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In embodiments, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine. In embodiments, the amino acid substitution at amino acid residue 416 is a substitution with serine. In embodiments, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In embodiments, the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine. In embodiments, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine.

[0149] In embodiments, the Fc domain linker (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference). In embodiments, the IgG constant region includes a triple M252Y / S254T / T256E mutation or YTE mutation. In embodiments, the IgG constant region includes a triple H433K / N434F / Y436H mutation or KFH mutation. In embodiments, the IgG constant region includes an YTE and KFH mutation in combination.

[0150] In embodiments, the linker comprises an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435 (in accordance with Kabat numbering, as in as in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) expressly incorporated herein by reference). Illustrative mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In embodiments, the IgG constant region comprises a M428L / N434S mutation or LS mutation. In embodiments, the IgG constant region comprises a T250Q / M428L mutation or QL mutation. In embodiments, the IgG constant region comprises an N434A mutation. In embodiments, the IgG constant region comprises a T307A / E380A / N434A mutation or AAA mutation. In embodiments, the IgG constant region comprises an I253A / H310A / H435A mutation or IHH mutation. In embodiments, the IgG constant region comprises a H433K / N434F mutation. In embodiments, the IgG constant region comprises a M252Y / S254T / T256E and a H433K / N434F mutation in combination.

[0151] Additional exemplary mutations in the IgG constant region are described, for example, in Robbie, et al., Antimicrobial Agents and Chemotherapy (2013), 57(12):6147-6153, Dall’Acqua et al., JBC (2006), 281 (33):23514-24, Dall’Acqua et al., Journal of Immunology (2002), 169:5171-80, Ko et al. Nature (2014) 514:642-645, Grevys etal. Journal of Immunology. (2015), 194(11):5497-508, and U.S. Patent No. 7,083,784, the entire contents of which are hereby incorporated by reference.

[0152] In embodiments, the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 1 (see the below table), or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, mutations are made to SEQ ID NO: 1 to increase stability and / or half-life. For instance, in embodiments, the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 2 (see the below table), or at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. An illustrative Fc stabilizing mutant is S228P. Illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311 S and the present linkers may comprise 1, or 2, or 3, or 4, or 5 of these mutants.

[0153] In embodiments, the chimeric protein binds to FcRn with high affinity. In embodiments, the chimeric protein may bind to FcRn with a KD of about 1 nM to about 80 nM. For example, the chimeric protein may bind to FcRn with a KD of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, or about 80 nM. In embodiments, the chimeric protein may bind to FcRn with a KD of about 9 nM. In embodiments, the chimeric protein does not substantially bind to other Fc receptors ( / .e., other than FcRn) with effector function.

[0154] In embodiments, the Fc domain in a linker has the amino acid sequence of SEQ ID NO: 1 (see Table 2, below), or at least at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. In embodiments, mutations are made to SEQ ID NO: 1 to increase stability and / or half-life. For instance, in embodiments, the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 2 (see Table 2, below), or at least at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. For instance, in embodiments, the Fc domain in a linker comprises the amino acid sequence of SEQ ID NO: 3 (see Table 2, below), or at least at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0155] In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG1. In embodiments, the lgG1 is human lgG1. In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG4. In embodiments, the lgG4 is human lgG4. In embodiments, the hinge-CH2-CH3 Fc domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 53. In embodiments, the linker further comprises the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 4 to 52. In embodiments, the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 4 to 52; wherein one joining linker is N terminal to the hinge-CH2-CH3-Fc domain and another joining linker is C terminal to the hinge-CH2-CH3-Fc domain.

[0156] Further, one or more joining linkers may be employed to connect an Fc domain in a linker (e.g., one of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or at least at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto) and the extracellular domains. For example, any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or variants thereof may connect an extracellular domain as disclosed herein and an Fc domain in a linker as disclosed herein. Optionally, any one of SEQ ID NOs: 4 to 52, or variants thereof are located between an extracellular domain as disclosed herein and an Fc domain as disclosed herein.

[0157] In embodiments, the presentchimeric proteins may comprise variants of the joining linkers disclosed in Table 2, below. For instance, a linker may have at least about 60%, or at least about 61 %, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71 %, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81 %, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 4 to 52.

[0158] In embodiments, the first and second joining linkers may be different, or they may be the same.

[0159] Without wishing to be bound by theory, including a linker comprising at least a part of an Fc domain in a chimeric protein, helps avoid formation of insoluble and, likely, non-functional protein concatamers and / or aggregates. This is in part due to the presence of cysteines in the Fc domain which are capable of forming disulfide bonds between chimeric proteins.

[0160] In embodiments, a chimeric protein may comprise one or more joining linkers, as disclosed herein, and lack an Fc domain linker, as disclosed herein. In embodiments, the first and / or second joining linkers are independently selected from the amino acid sequences of SEQ ID NOs: 4 to 52 and are provided in Table 2 below:

[0161] Table 2: Illustrative linkers (Fc domain linkers and joining linkers)

[0162] In embodiments, the joining linker substantially comprises glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99%, or about 100% glycines and serines). For example, in embodiments, the joining linker is (Gly4Ser)n, where n is from about 1 to about 8, e.g., 1 , 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO: 25 to

[0163] SEQ ID NO: 32, respectively). In embodiments, the joining linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 33). Additional illustrative joining linkers include, but are not limited to, linkers having the sequence LE, (EAAAK)n(n=1-3) (SEQ ID NO: 36 to SEQ ID NO: 38), A(EAAAK)nA (n = 2-5) (SEQ ID NO: 39 to SEQ ID NO: 42), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 43), PAPAP (SEQ ID NO: 44), KESGSVSSEQLAQFRSLD (SEQ ID NO: 45), GSAGSAAGSGEF (SEQ ID NO: 46), and (XP)n, with X designating any amino acid, e.g., Ala, Lys, or Glu. In embodiments, a joining linker has the sequence (Gly)n where n is any number from 1 to 100, for example: (Gly)s (SEQ ID NO: 34) and (Gly)e (SEQ ID NO: 35). In embodiments, the joining linker has the amino acid sequence GGS (SEQ ID NO: 3), or GS or LE. In embodiments, the joining linker has the amino acid sequence EPKSCDKTHTCP (SEQ ID NO: 51). In embodiments, the joining linker has the amino acid sequence EPKSCDKTHTCP EPKSVDKTHTCP (SEQ ID

[0164] NO: 52).

[0165] In embodiments, the joining linker is one or more of GGGSE (SEQ ID NO: 47), GSESG (SEQ ID NO: 48), GSEGS (SEQ ID NO: 49), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 50), and a joining linker of randomly placed G, S, and E every 4 amino acid intervals. In embodiments, where a chimeric protein comprises a glucagon-like peptide-1 (GLP-1), a protease- cleavable linker preceding the Fc domain, an Fc domain, a joining linker following the Fc domain, and ACVR2B, the chimeric protein may comprise the following structure:

[0166] Glucagon-like peptide-1 (GLP-1) - a protease-cleavable linker - Fc Domain - Joining Linker - extracellular domain of activin Receptor Type-2B (ACVR2B)

[0167] In embodiments, where a chimeric protein comprises ACVR2B, a joining linker preceding the Fc domain, an Fc domain, a protease-cleavable linker following the Fc domain, a glucagon-like peptide-1 (GLP-1), and the chimeric protein may comprise the following structure: extracellular domain of activin Receptor Type-2B (ACVR2B) - Joining Linker- Fc Domain - protease- cleavable linker- glucagon-like peptide-1 (GLP-1)

[0168] In embodiments, where a chimeric protein comprises a glucagon-like peptide-1 (GLP-1), a protease- cleavable linker preceding the Fc domain, an Fc domain, a joining linker following the Fc domain, and ACVR2B, the chimeric protein may comprise the following structure:

[0169] Glucagon-like peptide-1 (GLP-1 ) - Joining Linker - Fc Domain - Joining Linker - extracellular domain of activin Receptor Type-2B (ACVR2B)

[0170] In embodiments, where a chimeric protein comprises ACVR2B, a joining linker preceding the Fc domain, an Fc domain, a protease-cleavable linker following the Fc domain, a glucagon-like peptide-1 (GLP-1), and the chimeric protein may comprise the following structure: extracellular domain of activin Receptor Type-2B (ACVR2B) - Joining Linker- Fc Domain - Joining Linker - glucagon-like peptide-1 (GLP-1)

[0171] Accordingly, in aspects, the present disclosure provides a chimeric protein comprising a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (A) (a) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, (c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge-CH2-CH3 Fc domain; or (B) (a) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), (c) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, and (b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2-CH3 Fc domain.

[0172] In embodiments, the GLP-1 receptor agonist is selected from GLP-1 , a DPP4 degradation resistant derivative of GLP-1, exenatide, lixisenatide, albiglutide, dulaglutide, or a variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71 , or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71. In embodiments, the GLP-1 receptor agonist is capable of binding a GLP-1 receptor. In embodiments, the GLP-1 receptor agonist is capable of activate the GLP-1 receptor. In embodiments, the GLP-1 receptor agonist is capable of stimulating and / or increasing insulin secretion, and / or inhibiting and / or decreasing glucagon secretion.

[0173] In embodiments, the portion of ACVR2B comprises substantially the entire extracellular domain of ACVR2B. In embodiments, the portion of ACVR2B comprises the entire extracellular domain of ACVR2B. In embodiments, the portion of ACVR2B is capable of binding activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B is capable of reducing or inhibiting the binding of one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11) to one or more of their receptors. In embodiments, the portion of ACVR2B is capable of sequestering one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B is capable of reducing or inhibiting ACVR2B signaling induced by one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11). In embodiments, the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73.

[0174] Isolated Polynucleotide Encoding the Chimeric Protein

[0175] In aspects, the present disclosure provides an isolated polynucleotide encoding the chimeric protein of any one of the embodiments disclosed herein.

[0176] Accordingly, in aspects, the present disclosure provides an isolated polynucleotide encoding a chimeric protein comprising a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (A) (a) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, (c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge-CH2-CH3 Fc domain; or (B) (a) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), (c) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, and (b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2-CH3 Fc domain. Without wishing to be bound by theory, glucagon-like peptide-1 (GLP-1) has a very short half-life, limiting its potential direct use as therapeutics. For example, GLP-1 secreted in the blood has a very short half-life of less than 2 minutes, which is caused by a loss of activity due to the cleavage of amino acids at the N-terminus by the enzyme dipeptidyl peptidase-4 (DPP-4). Therefore, novel approaches to deliver GLP-1 and / or chimeric proteins comprising GLP-1 are required. The present disclosure addresses this need by delivering pharmaceutical compositions which can contain nucleic acids such as modified mRNA (mmRNA) or DNA.

[0177] In aspects, the present disclosure provides an isolated polynucleotide encoding a chimeric protein comprising a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (A) (a) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, (c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge-CH2-CH3 Fc domain; or (B) (a) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), (c) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, and (b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2- CH3 Fc domain.

[0178] In embodiments, the isolated polynucleotide encodes a GLP-1 receptor agonist is selected from GLP-1 , a DPP4 degradation resistant derivative of GLP-1 , exenatide, lixisenatide, albiglutide, dulaglutide, or a variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71 , or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71. In embodiments, the GLP-1 receptor agonist is capable of binding a GLP-1 receptor. In embodiments, the GLP-1 receptor agonist is capable of stimulating and / or increasing insulin secretion, and / or inhibiting and / or decreasing glucagon secretion.

[0179] In embodiments, the isolated polynucleotide encodes a portion of ACVR2B is capable of binding activin A and / or GDF-8. In embodiments, the portion of ACVR2B comprises substantially the entire extracellular domain of ACVR2B. In embodiments, the portion of ACVR2B comprises the entire extracellular domain of ACVR2B. In embodiments, the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73.

[0180] In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG1. In embodiments, the lgG1 is human lgG1. In embodiments, the hinge-CH2-CH3 Fc domain is derived from lgG4. In embodiments, the lgG4 is human lgG4. In embodiments, the hinge-CH2-CH3 Fc domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 53. In embodiments, the linker further comprises the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 4 to 52. In embodiments, the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 4 to 52; wherein one joining linker is N terminal to the hinge-CH2-CH3-Fc domain and another joining linker is C terminal to the hinge-CH2-CH3-Fc domain.

[0181] In embodiments, the polynucleotide is RNA, optionally, an mRNA. In embodiments, the polynucleotide is codon optimized.

[0182] In embodiments, the polynucleotide is selected from mRNA, circular RNA (circRNA) and self-amplifying RNA (saRNA), optionally wherein the polynucleotide comprises at least one modified nucleotide selected from a modified sugar, a modified base and / or a modified internucleotide linkage. In embodiments, the polynucleotide may include a polynucleotide modification including, but not limited to, a nucleoside modification. In embodiments, the polynucleotide is an mmRNA. In embodiments, the mmRNA comprises one or more nucleoside modifications. In embodiments, the nucleoside modifications are selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, pseudouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-tau ri nomethyl-2-th io-uri di ne, 1 -taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1 - deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4- acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 2-aminoadenine, 7-deaza-adenine, 7- deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6- thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7- methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.

[0183] In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues of the mmRNA are replaced by modified cytosine residues. In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the uracil residues of the mmRNA are replaced by modified uracil residues. In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the adenine residues of the mmRNA are replaced by modified adenine residues. In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the guanine residues of the mmRNA are replaced by modified guanine residues.

[0184] In embodiments, the polypeptide the at least one chemically modified nucleoside is selected from pseudouridine ('+’), N1 -methylpseudouridine (m1l), 2-thiouridine (s2U), 4’ -thiouridine, 5-methylcytosine, 2- th io- 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl-pseudouridine, 2-th io-5-aza-uridi ne, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4- methoxy-pseudouridine, 4-thio-1 -methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2’-O-methyl uridine, 1 -methyl-pseudouridine (ml ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C). alpha-thio-guanosine, alpha.-thio-adenosine, 5- cyano uridine, 4’-thio uridine 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6- methyl-adenosine (m6A), and 2,6-Diaminopurine, (I), 1-methylinosine (ml I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, and two or more combinations thereof.

[0185] In embodiments, the mmRNA does not cause a substantial induction of the innate immune response of a cell into which the mmRNA is introduced. In embodiments, the modification in the mmRNA enhance one or more of the efficiency of production of the chimeric protein, intracellular retention of the mmRNA, and viability of contacted cells, and possess reduced immunogenicity.

[0186] In embodiments, the mmRNA has a length sufficient to include an open reading frame encoding the chimeric protein of the present disclosure.

[0187] In embodiments, the mmRNA is not uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification may also be a 5' or 3' terminal modification. The nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least about 50% modified nucleotides, at least about 80% modified nucleotides, or at least about 90% modified nucleotides.

[0188] In embodiments, the mmRNA may contain a modified pyrimidine such as uracil or cytosine. In embodiments, at least about 5%, at least about 10%, at least about 25%, at least about 50%, In embodiments, the modified uracil may be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures disclosed above (e.g., same mmRNA may contain 2, 3, 4 or more types of uniquely modified uracil). In embodiments, at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 80%, at least about 90% or 100% of the cytosine in the nucleic acid may be replaced with a modified cytosine. The modified cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures disclosed above (e.g., same mmRNA may contain 2, 3, 4 or more types of uniquely modified cytosine).

[0189] In embodiments, the mmRNA comprises at least one chemically modified nucleoside. In embodiments, wherein the at least one chemically modified nucleoside is selected from pseudouridine (1), N1- methylpseudouridine (mI ), 2-thiouridine (s2U), 4’-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza- pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1- methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methoxyuridine, 2’-O-methyl uridine, 1-methyl-pseudouridine (ml ), 5-methoxy-uridine (mo5U), 5-methyl- cytidine (m5C). alpha-thio-guanosine, alpha.-thio-adenosine, 5-cyano uridine, 4’-thio uridine 7-deaza- adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), and 2,6- Diaminopurine, (I), 1-methylinosine (ml I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7- cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),

[0190] 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, and two or more combinations thereof. In embodiments, the mmRNA comprises at least one chemically modified nucleoside, wherein the at least one chemically modified nucleoside is selected from pseudouridine, N1 -methylpseudouridine, 5- methylcytosine, 5-methoxyuridine, and a combination thereof. In embodiments, the mmRNA comprises at least one chemically modified nucleoside is N1 -methylpseudouridine. In embodiments, the mmRNA is fully modified with chemically-modified uridines. In embodiments, the mmRNA is a fully modified N1- methylpseudouridine mRNA. Additional chemical modifications are disclosed in US Patent Application Publication No. 2019 / 0111003, the entire contents of which are hereby incorporated by reference.

[0191] In embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza- uridine, 2-thiouridine, pseudouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2- thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine,

[0192] 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1- methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1 -methyl- pseudoisocytidine. In embodiments, modified nucleosides include 2-aminopurine, 2, 6-diaminopurine, 2-aminoadenine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza- 2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine.

[0193] In embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0194] In embodiments, the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.

[0195] In embodiments, a modified nucleoside is 5'-O-(1 -Thiophosphatej-Adenosine, 5'-O-(1 -Thiophosphate)- Cytidine, 5'-O-(1-Thiophosphate)-Guanosine, 5'-O-(1-Thiophosphate)-Uridine or 5'-O-(1-Thiophosphate)- Pseudouridine.

[0196] Further examples of modified nucleotides and modified nucleotide combinations are disclosed in US Patent Nos. 8,710,200; 8,822,663; 8,999,380; 9,181,319; 9,254,311; 9,334,328; 9,464,124; 9,950,068; 10,626,400; 10,808,242; 11,020,477, and US Patent Application Publication Nos. 2022 / 0001026, 2021 / 0318817, 2021 / 0283262, 2020 / 0360481, 2020 / 0113844, 2020 / 0085758, 2017 / 0204152, 2019 / 0114089, 2019 / 0114090, 2018 / 0369374, 2018 / 0318385, 2019 / 0111003, and PCT International Application Publication Nos. WO / 2017112943, WO 2014 / 028429, WO 2017 / 201325 the entire contents of which are hereby incorporated by reference. The methods for synthesizing the modified mRNAare disclosed, e.g., in US Patent Application Publication Nos. 2017 / 0204152, the entire contents of which are hereby incorporated by reference.

[0197] In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues of the mmRNA are replaced by modified cytosine residues. In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the uracil residues of the mmRNA are replaced by modified uracil residues. In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the adenine residues of the mmRNA are replaced by modified adenine residues. In embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the guanine residues of the mmRNA are replaced by modified guanine residues.

[0198] In embodiments, the mmRNA further comprises a 5' untranslated region (UTR) and / or a 3'UTR, wherein either or both may independently contain one or more different nucleoside modifications. In such embodiments, nucleoside modifications may also be present in the translatable region. In embodiments, the mmRNA further comprises a Kozak sequence. In embodiments, the mmRNA further comprises a internal ribosome entry site (IRES).

[0199] In embodiments, the mmRNA further comprises a 5'-cap and / or a poly A tail. In embodiments, the mmRNA further comprises a 5' UTR, optionally comprising a nucleotide sequence that is at least about 90%, or about 95% identical to the nucleotide sequence selected from SEQ ID NOs: 128-149, and / or a 3’ UTR, optionally comprising a nucleotide sequence that is at least about 90%, or about 95% identical to the nucleotide sequence selected from SEQ ID NOs: 114-127. In embodiments, the mmRNA further comprises 5’ UTR_1 (SEQ ID NO: 89) and 3’ UTR_1 (SEQ ID NO: 75); 5’ UTR_1 (SEQ ID NO: 89) and 3’ UTR_2 (SEQ ID NO: 76); 5’ UTR_2 (SEQ ID NO: 90) and 3’ UTR_1 (SEQ ID NO: 75); 5’ UTR_3 (SEQ ID NO: 91) and 3’ UTR_1 (SEQ ID NO: 75); 5’ UTR_7 (SEQ ID NO: 95) and 3’ UTR_3 (SEQ ID NO: 77); 5’ UTR_8 (SEQ ID NO: 96) and 3’ UTR_4 (SEQ ID NO: 78); 5’ UTR_9 (SEQ ID NO: 97) and 3’ UTR_1 (SEQ ID NO: 75); 5’ UTR 0 (SEQ ID NO: 98) and 3’ UTR_1 (SEQ ID NO: 75); 5’ UTR_11 (SEQ ID NO: 99) and 3’ UTR_5 (SEQ ID NO: 79); 5’ UTR 2 (SEQ ID NO: 100) or 3’ UTR_6 (SEQ ID NO: 80); 5’ UTRJ4 (SEQ ID NO: 102) and 3’ UTRJ0 (SEQ ID NO: 84). In embodiments, the 5'-cap contains a 5'-5'-triphosphate linkage between the 5'-most nucleotide and guanine nucleotide. In embodiments, the 5'-cap comprises a methylation of the ultimate and penultimate most 5'- nucleotides on the 2'-hydroxyl group. In embodiments, the 5'-cap facilitates binding the mRNA Cap Binding Protein (CBP), confers mRNA stability in the cell and / or confers translation competency.

[0200] In embodiments, the poly-A tail is greater than about 30 nucleotides, or greater than about 40 nucleotides in length. In embodiments, the poly-A tail at least about 40 nucleotides, or at least about 45 nucleotides, or at least about 55 nucleotides, or at least about 60 nucleotides, or at least about 80 nucleotides, or at least about

[0201] 90 nucleotides, or at least about 100 nucleotides, or at least about 120 nucleotides, or at least about 140 nucleotides, or at least about 160 nucleotides, or at least about 180 nucleotides, or at least about 200 nucleotides, or at least about 250 nucleotides, or at least about 300 nucleotides, or at least about 350 nucleotides, or at least about 400 nucleotides, or at least about 450 nucleotides, or at least about 500 nucleotides, or at least about 600 nucleotides, or at least about 700 nucleotides, or at least about 800 nucleotides, or at least about 900 nucleotides, or at least about 1000 nucleotides in length.

[0202] In embodiments, the mmRNA comprises a 3’ untranslated region (UTR). In embodiments, the 3’ UTR comprises a nucleic acid sequence at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to a sequence listed in Table 4A or Table 4B of US Patent Application Publication No. 2019 / 0114089, which is incorporated herein in its entirety. In embodiments, the 3’ UTR comprises at least one microRNA-122 (miR-122) binding site, wherein the miR-122 binding site is a miR-122-3p binding site or a miR-122-5-binding site. In embodiments, the mmRNA comprises a nucleic acid sequence comprising a miRNA binding site. In some embodiments, the miRNA binding site binds to miR-122. In a particular embodiment, the miRNA binding site binds to miR- 122-3p or miR-122-5p. In embodiments, the mmRNA comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten miRNA binding sites.

[0203] In embodiments, the 3’ UTR sequence is 3’ UTR_1 having the following nucleotide sequence:

[0204] GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGG GATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCAATTGCCATG TGTATGTGGGTTCGCCCACATACTCTGATGATCCCCAATCGTGGCGTGTCGGCCTGCTTCGGCAGGCA CTGGCGCCGGGATCATTCATGGCAA (SEQ ID NO: 75).

[0205] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 75. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 75 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 75 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 75 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0206] In embodiments, the 3’ UTR sequence is 3’ UTR_2 having the following nucleotide sequence:

[0207] AAAGCAAAACTAACATGAAACAAGGCTAGAAGTCAGGTCGGATTAAGCCATAGTACGGAAAAAACTATG CTACCTGTGAGCCCCGTCCAAGGACGTTAAAAGAAGTCAGGCCATCATAAATGCCATAGCTTGAGTAAA CTATGCAGCCTGTAGCTCCACCTGAGAAGGTGTAAAAAATCCGGGAGGCCACAAACCATGGAAGCTGT ACGCATGGCGTAGTGGACTAGCGGTTAGAGGAGACCCCTCCCTTACAAATCGCAGCAACAATGGGGGC CCAAGGCGAGATGAAGCTGTAGTCTCGCTGGAAGGACTAGAGGTTAGAGGAGACCCCCCCGAAACAA AAAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG AACGCCAGAAAATGGAATGGTGCTGTTGAATCAACAGGTTCT (SEQ ID NO: 76).

[0208] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 76. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 76 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 76 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 76 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0209] In embodiments, the 3’ UTR sequence is 3’ UTR_3 having the following nucleotide sequence:

[0210] GCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGC ACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA (SEQ ID NO: 77).

[0211] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 77. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 77 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 77 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 77 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0212] In embodiments, the 3’ UTR sequence is 3’ UTR_4 having the following nucleotide sequence:

[0213] UUCUAGAGCGGCCGCUUCGAGCCGGUUGAAUCGCUGAUCUCACGCCGUGGUGAGCUCGCUUUCUU GCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAU GAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAGUUCCGCGU ACGUACGGCGUC (SEQ ID NO: 78).

[0214] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 78. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 78, with at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or about 100% U residues replaced with T residues, modified U residues or a combination thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 78 with one or more U and / or T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 78 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% U or T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 78 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0215] In embodiments, the 3’ UTR sequence is 3’ UTR_5 having the following nucleotide sequence:

[0216] CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCC CCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGAC ACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCA GTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGC CAGCCACACCCTGGAGCTAGC (SEQ ID NO: 79).

[0217] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 79. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 79 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 79 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 79 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0218] In embodiments, the 3’ UTR sequence is 3’ UTR_6 having the following nucleotide sequence:

[0219] GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUU GGUCUUUGAAUAAAGCCUGAGUAGGAAGU (SEQ ID NO: 80).

[0220] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 80. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 80, with at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or about 100% U residues replaced with T residues, modified U residues or a combination thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 80 with one or more U or T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 80 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% U or T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 80 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0221] In embodiments, the 3’ UTR sequence is 3’ UTR_7 having the following nucleotide sequence:

[0222] GGGCCTTTCCAAGATTGCTGTTTTTGTTTTGGAGCTTCAAGACTTTGCATTTCCTAGTATTTCTGTTTGTC AGTTCTCAATTTCCTGTGTTTGCAATGTTGAAATTTTTTGGTGAAGTACTGAACTTGCTTTTTTTCCGGTT TCTACATGCAGAGATGAATTTATACTGCCATCTTACGACTATTTCTTCTTTTTAATACACTTAACTCAGGCC ATTTTTTAAGTTGGTTACTTCAAAGTAAATAAACTTTAAAATTCAA (SEQ ID NO: 81).

[0223] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 81. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 81 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 81 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 81 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0224] In embodiments, the 3’ UTR sequence is 3’ UTR_8 having the following nucleotide sequence:

[0225] CCGCTACGCCCCAATGACCCGACCAGCAAAACTCGACGTACTACCGAGGAACCGATGTGCATAACGCA TCGGGCTGGTACATTAGATCCCCGTCATCAGACGGGCTCATAGCGACGCTAAAACTCGACGTATTCCCG AGGAAGTGCAGTGCATAATGCTGAGCAGCGTCGTCATATATTCACTTATTATTCAATATAGAGTAGACACC AAAACTCAATGTATTTCTGAGGAAGCGTGGTGCATAATGCCACGCAGTGTCTACATAATCAATTTATTATT TTCTTTTATTTTATTCACATAATTTTGTTTTTAATATTTC (SEQ ID NO: 82).

[0226] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 82. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 82 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 82 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 82 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0227] In embodiments, the 3’ UTR sequence is 3’ UTR_9 having the following nucleotide sequence:

[0228] CATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGCTTATTCA TCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGC CTCTTTTCTCT (SEQ ID NO: 83).

[0229] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 83. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 83 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 83 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 83 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0230] In embodiments, the 3’ UTR sequence is 3’ UTR_10 having the following nucleotide sequence:

[0231] ATATTATCCCTAATACCTGCCACCCCACTCTTAATCAGTGGTGGAAGAACGGTCTCAGAACTGTTTGTTT CAATTGGCCATTTAAGTTTAGTAGTAAAAGACTGGTTAATGATAACAATGCATCGTAAAACCTTCAGAAGG AAAGGAGAATGTTTTGTGGACCACTTTGGTTTTCTTTTTTGCGTGTGGCAGTTTTAAGTTATTAGTTTTTA AAATCAGTACTTTTTAATGGAAACAACTTGACCAAAAATTTGTCACAGAATTTTGAGACCCATTAAAAAAG TTAAATGAGAAA (SEQ ID NO: 84).

[0232] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 84. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 84 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 84 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 84 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0233] In embodiments, the 3’ UTR sequence is 3’ UTR_11 having the following nucleotide sequence:

[0234] GCGCCTGCCCACCTGCCACCGACTGCTGGAACCCAGCCAGTGGGAGGGCCTGGCCCACCAGAGTCC TGCTCCCTCACTCCTCGCCCCGCCCCCTGTCCCAGAGTCCCACCTGGGGGCTCTCTCCACCCTTCTC AGAGTTCCAGTTTCAACCAGAGTTCCAACCAATGGGCTCCATCCTCTGGATTCTGGCCAATGAAATATC TCCCTGGCAGGGTCCTCTTCTTTTCCCAGAGCTCCACCCCAACCAGGAGCTCTAGTTAATGGAGAGCT CCCAGCACACTCGGAGCTTGTGCTTTGTCTCCACGCAAAGCGATAAATAAAAGCATTGGTGGCCTTA (SEQ ID NO: 85).

[0235] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 85. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 85 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 85 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 85 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0236] In embodiments, the 3’ UTR sequence is 3’ UTR_12 having the following nucleotide sequence:

[0237] GTGTGTGGAGGACACCCTGAACCCCCCGCTTTCAAACAAGTTTTCAAATTGTTTGAGGTCAGGATTTCT CAAACTGATTCCTTTCTTTGCATATGAGTATTTGAAAATAAATATTTTCCCAGAATATAAATAAATCATCACA TGATTATTTTAACTATA (SEQ ID NO: 86).

[0238] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 86. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 86 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 86 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 86 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0239] In embodiments, the 3’ UTR sequence is 3’ UTR_13 having the following nucleotide sequence:

[0240] GCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGC ACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA (SEQ ID NO: 87).

[0241] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 87. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 87 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 87 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 87 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0242] In embodiments, the 3’ UTR sequence is 3’ UTR_14 having the following nucleotide sequence:

[0243] UUCUAGAGCGGCCGCUUCGAGCCGGUUGAAUCGCUGAUCUCACGCCGUGGUGAGCUCGCUUUCUU GCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAACUACUAAACUGGGGGAUAUUAU GAAGGGCCUUGAGCAUCUGGAUUCUGCCUAAUAAAAAACAUUUAUUUUCAUUGCAAAGUUCCGCGU ACGUACGGCGUC (SEQ ID NO: 88).

[0244] In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 88. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 88, with at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or about 100% U residues replaced with T residues, modified U residues or a combination thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 88 with one or more U or T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 88 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% U or T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 3' UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 88 at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0245] In embodiments, the miRNA binding site is inserted within the 3’ UTR. In embodiments, the polynucleotide is DNA. In embodiments, the further comprises a spacer sequence between the open reading frame and the miRNA binding site. In aspects, the spacer sequence comprises at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, or at least about 100 nucleotides.

[0246] In embodiments, the mmRNA further comprises a 5’ UTR. In embodiments, the 5’ UTR comprises a nucleic acid sequence at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to a sequence listed in Table 3 of US Patent Application Publication No. 2019 / 0114089, or a sequence disclosed in PCT International Application Publication Nos. WO 2017 / 201325 and WO 2014 / 164253, each of which is incorporated herein in its entirety. In embodiments, the 5’ UTR bears features, which play roles in translation initiation. In embodiments, the 5’ UTR harbors signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. In embodiments, the 5’ UTR forms secondary structures which are involved in elongation factor binding. In embodiments, the 5’ UTR of mRNA known to be upregulated in cancers, such as c-myc, may be used to enhance expression of a nucleic acid molecule, such as a polynucleotides, in cancer cells. In embodiments, the 5’ UTR of mRNA known to be upregulated in liver and / or spleen may be used to enhance expression of a nucleic acid molecule, such as a polynucleotides, in liver and / or spleen. In embodiments, the mRNA comprises a GC-rich element comprising, or derivatives or analogs thereof, located upstream of a Kozak consensus sequence or a Kozak-like sequence that is located in the 5' UTR. In embodiments, GC-rich element is located at least about 30, or at least about 25, or at least about 20, or at least about 15, or at least about 10, or at least about 5, or at least about 4, or at least about 3, or at least about 2, or at least about 1 nucleotide(s) upstream of a Kozak consensus sequence or a Kozak-like sequence that is located in the 5' UTR. In embodiments, the GC-rich element is located about 15- 30, or about 15-20, or about 15-25, or about 10-15, or about 5-10, or about 1-8 nucleotides upstream of a Kozak consensus sequence or a Kozak-like sequence that is located in the 5' UTR. In embodiments, the GC- rich element is located upstream of and immediately abutting to Kozak consensus sequence or a Kozak-like sequence that is located in the 5' UTR. In embodiments, the GC-rich element is about 20-30, about 10-20, about 10-15, about 5-15, or about 3-15 nucleotides long. In embodiments, the GC-rich element comprises a sequence of about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11 , or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 nucleotides long. In embodiments, the GC-rich element is located about 60, or about 55, or about 50, or about 45, or about 40, or about 35, or about 30, or about 25, or about 20, or about 15, or about 10, or about 5, or about 4, or about 3, or about 2, or about 1 nucleotide upstream of the initiation codon. In embodiments, the GC-rich element comprises about 30% to about 50%, or about 40% to about 60%, or about 50% to about 70%, or about 60% to about 80%, or about 70% to about 90% cytosine. In embodiments, the GC-rich element comprises about 30% to about 50%, or about 40% to about 60%, or about 50% to about 70%, or about 60% to about 80%, or about 70% to about 90% guanine. Additional properties of the GC-rich element are described in US Patent Application No. 2020 / 0208145, which is incorporated herein in its entirety.

[0247] In embodiments, the GC-rich element comprises the nucleotide sequence CCCCGGCGCC (SEQ ID NO:

[0248] 111). In embodiments, the GC-rich element is a variant of CCCCGGCGCC (SEQ ID NO: 111) having one or more nucleotide mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GC-rich element has a nucleotide sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotide mutations with respect to the nucleotide sequence CCCCGGCGCC (SEQ ID NO: 111).

[0249] In embodiments, the GC-rich element comprises the nucleotide sequence GGGGCGCCCG (SEQ ID NO:

[0250] 112). In embodiments, the GC-rich element is a variant of GGGGCGCCCG (SEQ ID NO: 112) having one or more nucleotide mutations, independently selected from substitutions, insertions, deletions, and truncations. In embodiments, the GC-rich element has a nucleotide sequence having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotide mutations with respect to the nucleotide sequence GGGGCGCCCG (SEQ ID NO: 112). Additional GC-rich element sequences are described in US Patent Application No. 2020 / 0208145, which is incorporated herein in its entirety.

[0251] In embodiments, the 5’ UTR sequence is 5’ UTR_1 having the following nucleotide sequence:

[0252] ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC (SEQ ID NO: 89).

[0253] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 89. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 89 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 89 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 89 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0254] In embodiments, the 5’ UTR sequence is 5’ UTR_2 having the following nucleotide sequence:

[0255] ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCCCCCGGCGCCGCCACCAT G (SEQ ID NO: 90). In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 90 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, but lacking a GC-rich element. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element comprising the nucleotide sequence CCCCGGCGCC (SEQ ID NO: 111) . In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element that is a variant of CCCCGGCGCC (SEQ ID NO: 111) having one or more nucleotide mutations, independently selected from substitutions, insertions, deletions, and truncations.

[0256] In embodiments, the 5’ UTR sequence is 5’ UTR_3 having the following nucleotide sequence:

[0257] ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACCGGGGCGCCCGGCCACCAT G (SEQ ID NO: 91).

[0258] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 91. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 91 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 91 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 91 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element comprising the nucleotide sequence GGGGCGCCCG (SEQ ID NO: 112). In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element that is a variant of GGGGCGCCCG (SEQ ID NO: 112) having one or more nucleotide mutations, independently selected from substitutions, insertions, deletions, and truncations.

[0259] In embodiments, the 5’ UTR sequence is 5’ UTR_4 having the following nucleotide sequence:

[0260] TTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCT AGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGT CTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATTAACCCGCTCAA TGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTT GTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCATCATGAGCAC AAATCCTAAACCTCAAAGAAAAACCAAACGTAACAAGGGCGAATTCGTTGGTAAAGCCACC (SEQ ID NO: 92).

[0261] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 92. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 92 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 92 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 92 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0262] In embodiments, the 5’ UTR sequence is 5’ UTR_5 having the following nucleotide sequence:

[0263] TTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCT AGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGT CTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATTAACCCGCTCAA TGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTT GTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCATCATGAGCAC AAATCCTAAACCTCAAAGAAAAACCAAACGTAACAAGGGCGAATTCGTTGGTAAACCCCGGCGCCGCC ACC (SEQ ID NO: 93).

[0264] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 93. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 93 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 93 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 93 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element comprising the nucleotide sequence CCCCGGCGCC (SEQ ID NO: 111). In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element that is a variant of CCCCGGCGCC (SEQ ID NO: 111) having one or more nucleotide mutations, independently selected from substitutions, insertions, deletions, and truncations.

[0265] In embodiments, the 5’ UTR sequence is 5’ UTR_6 having the following nucleotide sequence:

[0266] TTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCT AGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGT CTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATTAACCCGCTCAA TGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTT GTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCATCATGAGCAC AAATCCTAAACCTCAAAGAAAAACCAAACGTAACAAGGGCGAATTCGTTGGTAAAGGGGCGCCCGGCC ACC (SEQ ID NO: 94).

[0267] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 94. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 94 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 94 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 94 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element comprising the nucleotide sequence GGGGCGCCCG (SEQ ID NO: 112). In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element that is a variant of GGGGCGCCCG (SEQ ID NO: 112)having one or more nucleotide mutations, independently selected from substitutions, insertions, deletions, and truncations.

[0268] In embodiments, the 5’ UTR sequence is 5’ UTR_7 having the following nucleotide sequence:

[0269] GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACC (SEQ ID NO: 95).

[0270] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 95. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 95 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 95 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 95 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element comprising the nucleotide sequence CCCCGGCGCC (SEQ ID NO: 111). In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 90, and comprising a GC-rich element that is a variant of CCCCGGCGCC (SEQ ID NO: 111) having one or more nucleotide mutations, independently selected from substitutions, insertions, deletions, and truncations.

[0271] In embodiments, the 5’ UTR sequence is 5’ UTR_8 having the following nucleotide sequence:

[0272] GGGACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACC (SEQ ID NO: 96).

[0273] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 96. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 96 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 96 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 96 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0274] In embodiments, the 5’ UTR sequence is 5’ UTR_9 having the following nucleotide sequence:

[0275] TTTAAAATCTGTGTGGCTGTCACTCGGCTGCTTGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTA CTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCA GCCGATCATCAGCACATCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGTTGGAGAGCCTTGTCCCT GGTTTCAACGAGAAAAC (SEQ ID NO: 97).

[0276] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 97. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 97 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 97 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 97 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0277] In embodiments, the 5’ UTR sequence is 5’ UTR_10 having the following nucleotide sequence:

[0278] GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC (SEQ ID NO: 98).

[0279] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 98. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 98 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 98 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 98 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0280] In embodiments, the 5’ UTR sequence is 5’ UTR_11 having the following nucleotide sequence:

[0281] GAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC (SEQ ID NO: 99).

[0282] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 99. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 99 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 99 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 99 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0283] In embodiments, the 5’ UTR sequence is 5’ UTR_12 having the following nucleotide sequence:

[0284] GGGAAAAAGAGAGAAAAGAAGAGAAGAAGAAAAAAGAGCCACC (SEQ ID NO: 100). In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 100. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 100 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 100 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 100 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0285] In embodiments, the 5’ UTR sequence is 5’ UTR_13 having the following nucleotide sequence:

[0286] AACGGCTAGCCTGAGGAGCTGCTGCGACAGTCCACTACCTTTTTCGAGAGTGACTCCCGTTGTCCCAA GGCTTCCCAGAGCGAACCTGTGCGGCTGCAGGCACCGGCGCGTCGAGTTTCCGGCGTCCGGAAGGA CCGAGCTCTTCTCGCGGATCCAGTGTTCCGTTTCCAGCCCCCAATCTCAGAGCGGAGCCGACAGAGA GCAGGGAACCGGCCCGCCACC (SEQ ID NO: 101).

[0287] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 101. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 101 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 101 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 101 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0288] In embodiments, the 5’ UTR sequence is 5’ UTR_14 having the following nucleotide sequence:

[0289] TAGTCAGTGTAATATACAGTAACTGACCAAACCACATCCACCGTAAACCCGCCACC (SEQ ID NO: 102).

[0290] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 102. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 102 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 102 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 102 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0291] In embodiments, the 5’ UTR sequence is 5’ UTR_15 having the following nucleotide sequence:

[0292] GAGACCCAAGCTGGCTAGCGGGAGAAAGCTTACCGGCTAGCGCCGCCACC (SEQ ID NO: 103).

[0293] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 103. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 103 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 103 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 103 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0294] In embodiments, the 5’ UTR sequence is 5’ UTR_16 having the following nucleotide sequence:

[0295] TTTAAAATCTGTGTGGCTGTCACTCGGCTGCTTGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTA CTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCA GCCGATCATCAGCACATCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGTTGGAGAGCCTTGTCCCT GGTTTCAACGAGAAAACGCCACC (SEQ ID NO: 104).

[0296] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 104. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 104 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 104 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 104 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0297] In embodiments, the 5’ UTR sequence is 5’ UTR_17 having the following nucleotide sequence: GGGTCAGTGTCACCTCCAGGATACAGACAGCCCCCCTTCAGCCCAGCCCAGCCAGGTCTCCTACACC GCCACC (SEQ ID NO: 105).

[0298] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 105. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 105 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 105 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 105 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0299] In embodiments, the 5’ UTR sequence is 5’ UTR_18 having the following nucleotide sequence:

[0300] ACTCCTCCCCATCCTCTCCCTCTGTCCCTCTGTCCCTCTGACCCTGCACTGTCCCAGCACC (SEQ ID NO: 106).

[0301] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 106. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 106 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 106 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 106 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0302] In embodiments, the 5’ UTR sequence is 5’ UTR_19 having the following nucleotide sequence:

[0303] GGGACTCCCGGGCTGGCAGCAGGGCCCCAGCGGCACC (SEQ ID NO: 107).

[0304] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 107. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 107 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 107 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 107 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0305] In embodiments, the 5’ UTR sequence is 5’ UTR_20 having the following nucleotide sequence:

[0306] AGCAATCCTTTCTTTCAGCTGGAGTGCTCCTCAGGAGCCAGCCCCACCCTTAGAAAAG (SEQ ID NO: 108).

[0307] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 108. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 108 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 108 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 108 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0308] In embodiments, the 5’ UTR sequence is 5’ UTR_21 having the following nucleotide sequence:

[0309] GGGTCAGTGTCACCTCCAGGATACAGACAGCCCCCCTTCAGCCCAGCCCAGCCAGGTCTCCTACACC GCCACC (SEQ ID NO: 109).

[0310] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 109. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 109 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 109 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 109 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0311] In embodiments, the 5’ UTR sequence is 5’ UTR_22 having the following nucleotide sequence: TTTAAAATCTGTGTGGCTGTCACTCGGCTGCTTGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTA CTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCA GCCGATCATCAGCACATCTAGGTTTCGTCCGGGTGTGACCGAAAGGTAAGTTGGAGAGCCTTGTCCCT GGTTTCAACGAGAAAACGCCACC (SEQ ID NO: 110).

[0312] In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 110. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or 100% identical to the nucleotide acid sequence of SEQ ID NO: 110 with one or more T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 110 with at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% T nucleotide replaced with a U nucleotide, a modified U nucleotide or a homolog thereof. In embodiments, the 5’ UTR comprises a nucleotide acid sequence that is at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, or about 100% identical to the nucleotide acid sequence of SEQ ID NO: 110 having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the cytosine residues, thymidine residues, adenosine residues and / or guanosine residues are replaced by modified cytidine residues, modified uridine residues, modified adenosine residues and / or modified guanosine residues, respectively.

[0313] In embodiments, at least one of the regions of linked nucleosides of A comprises a sequence of linked nucleosides which functions as a 5’ UTR and at least one of the regions of linked nucleosides of C comprises a sequence of linked nucleosides which functions as a 3’ UTR. In embodiments, the 5’ UTR and the 3’ UTR are from the same or different species. In embodiments, the 5’ UTR and the 3’ UTR may be the native untranslated regions from different proteins from the same or different species. In embodiments, the 5’ UTR and the 3’ UTR may have synthetic sequences. In embodiments, the mmRNA further comprises a 3’ polyadenylation (polyA tail).

[0314] In embodiments, the mmRNA further comprises a 5’ terminal cap. In embodiments, the 5’ terminal cap is a CapO, Cap1 , ARCA, inosine, N1-methyl-guanosine, 2’fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5’ methylG cap, or an analog thereof.

[0315] In embodiments, the polynucleotide is in vitro transcribed (IVT). In embodiments, the polynucleotide is chimeric. In embodiments, the polynucleotide is circular.

[0316] In embodiments, the polynucleotide is or comprises DNA. In embodiments, the polynucleotide is or comprises a minicircle or a plasmid DNA. In embodiments, the plasmid DNA is devoid of any prokaryotic components. In embodiments, the polynucleotide comprises a tissue-specific control element. In embodiments, the tissuespecific control element is a promoter or an enhancer. In embodiments, the plasmid DNA is an expression vector. In embodiments, the DNA is or comprises a minicircle. In embodiments, the minicircle is a circular molecule, which is optionally small. In embodiments, the minicircle utilizes a cellular transcription and translation machinery to produce an encoded gene product. In embodiments, the minicircle is devoid of any prokaryotic components. In embodiments, the minicircle only comprises substantially only sequences of mammalian origin (or those that have been optimized for mammalian cells). In embodiments, the minicircle lacks or has reduced amount of DNA sequence elements that are recognized by the innate immune system and / or toll-like receptors. In embodiments, the minicircle is produced by excising any bacterial components of from a parental plasmid, thereby making it smaller than a parental DNA sequence. In embodiments, the minicircle is of non-viral origin. In embodiments, the minicircle remains episomal. In embodiments, the minicircle does not replicate with a host cell. In embodiments, expression of the chimeric protein in nondividing cells harboring a minicircle lasts for at least 2 days, or at least 4 days, or at least 6 days, or at least 8 days, or at least 10 days, or at least 12 days, or at least 14 days, or at least 16 days, or at least 18 days, or at least 20 days, or at least 22 days, or at least 24 days, or longer in dividing cells. In embodiments, expression of the chimeric protein in non-dividing cells harboring a minicircle lasts for at least 4 days, or at least 6 days, or at least 8 days, or at least 10 days, or at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 1 month, or at least 2 months, or at least 3 months, or at least 4 months, or at least 5 months, or at least 6 months, or at least 8 months, or longer in dividing cells. In embodiments, the mmRNAs of the present disclosure are produced by means available in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. Enzymatic IVT, solid-phase, liquid-phase, combined synthetic methods, small region synthesis, and ligation methods may be utilized. In embodiments, mmRNAs are made using IVT enzymatic synthesis methods. Methods of making polynucleotides by IVT are known in the art and are described in International Application PCT International Patent Publication No. WO 2013 / 151666, the contents of which are incorporated herein by reference in their entirety. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs and vectors that may be used to in vitro transcribe an mRNA described herein.

[0317] In embodiments, the polynucleotide is DNA. In embodiments, the polynucleotide comprises a expressed in liver, skin and / or muscle-specific control element. In embodiments, the liver-specific control element is a liverspecific promoter selected from albumin promoter, thyroxine-binding globulin (TBG) promoter, hybrid liverspecific promoter (HLP), human a 1 -antitrypsin promoter, LP1 promoter, and hemopexin promoter. The gene therapy in accordance with the present disclosure can be performed using vector systems. In embodiments, the liver-specific promoter is an LP1 promoter. The LP1 promoter can be a human LP1 promoter, which can be constructed as described, e.g., in Nathwani et al. Blood vol. 107(7) (2006):2653-61, which is incorporated herein by reference in its entirety.

[0318] In aspects, the present disclosure provides a vector comprising the polynucleotide of any one of the embodiments disclosed herein. In embodiments, the chimeric protein can be provided as an expression vector. In embodiments, the expression vector is a DNA expression vector or an RNA expression vector. In embodiments, the expression vector is a viral expression vector. In embodiments, the expression vector is a non-viral expression vector (without limitation, e.g., a plasmid).

[0319] In embodiments, the present non-viral vectors are linear or circular DNA molecules that comprise a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the polynucleotide encoding the polypeptide. In embodiments, the non- viral vector comprises a promoter sequence, and transcriptional and translational stop signal sequences. In embodiments, the expression vector may include, among others, chromosomal and episomal vectors, e.g., vectors derived from bacterial plasmids, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, and vectors derived from combinations thereof. The present constructs may contain control regions that regulate as well as engender expression. A vector generally comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In embodiments, the expression vector is an autonomously replicating plasmid or a virus (e.g., AAV vectors). In embodiments, the expression vector is non-plasmid and non-viral compounds that facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0320] In embodiments, the polynucleotide or cell therapy may employ expression vectors, which comprise the nucleic acid encoding the chimeric protein operably linked to an expression control region that is functional in the host cell. The expression control region is capable of driving expression of the operably linked encoding nucleic acid such that the chimeric protein is produced in a human cell transformed with the expression vector. Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, which influence expression of an operably linked nucleic acid. An expression control region of an expression vector is capable of expressing operably linked encoding nucleic acid in a human cell. In an embodiment, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. In various embodiments, the chimeric protein expression is inducible or repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

[0321] Expression systems functional in human cells are well known in the art and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA. A promoter will have a transcription-initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

[0322] Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacterio!., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667- 678, 2004), sleeping beauty, transposases of the mariner family, and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences including CRISPR / CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.

[0323] In aspects, the present disclosure provides a host cell comprising the vector of any of the embodiments disclosed herein. A host cell comprising the mmRNA of any of the embodiments disclosed herein.

[0324] Pharmaceutical Compositions

[0325] In aspects, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier, and the chimeric protein of any of the embodiments disclosed herein, the polypeptide of any of the embodiments disclosed herein (e.g., comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113), the isolated polynucleotide of any of the embodiments disclosed herein, the mmRNA of any of the embodiments disclosed herein, or the vector of any of the embodiments disclosed herein, or the host cell of any of the embodiments disclosed herein. In embodiments, the pharmaceutical composition comprises the mmRNA of any of the embodiments disclosed herein. Suitable pharmaceutical compositions are disclosed in US Patent Nos. 8,710,200; 8,822,663; 8,999,380; 9,181 ,319; 9,254,311 ; 9,334,328; 9,464,124; 9,950,068; 10,626,400; 10,808,242; 11,020,477, US Patent Application Publication Nos. 2022 / 0001026, 2021 / 0318817, 2021 / 0283262, 2020 / 0360481, 2020 / 0113844, 2020 / 0085758, 2017 / 0204152, 2019 / 0114089, 2019 / 0114090, 2018 / 0369374, 2018 / 0318385, 2019 / 0111003, and PCT International Application Publication Nos. WO / 2017112943, WO 2014 / 028429, WO 2017 / 201325 the entire contents of which are hereby incorporated by reference.

[0326] In aspects, the present disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the isolated polynucleotide of any of the embodiments disclosed herein, the mmRNA of any of the embodiments disclosed herein, or the vector of any of the embodiments disclosed herein. In embodiments, the pharmaceutically acceptable carrier is mmRNA comprises a modification (e.g., an RNA element), wherein the modification provides a desired translational regulatory activity. Such modifications are described in PCT Application No. PCT International Application Publication No. WO 2018 / 213789, the entire contents of which are herein incorporated by reference.

[0327] In embodiments, the mmRNA further comprises a 3’ untranslated region (UTR). In embodiments, the 3’ UTR comprises at least one microRNA-122 (miR-122) binding site. In embodiments, the miR-122 binding site is a miR-122-3p binding site or a miR-122-5-binding site. In embodiments, the mmRNA further comprises a spacer sequence between the open reading frame and the miRNA binding site. In embodiments, the spacer sequence comprises at least about 10 nucleotides, at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, or at least about 100 nucleotides.

[0328] In embodiments, the mmRNA further comprises a 5’ UTR. In embodiments, the 5’ UTR harbors a Kozak sequence and / or forms a secondary structure that stimulate elongation factor binding.

[0329] In embodiments, the mmRNA further comprises a 5’ terminal cap. In embodiments, the 5’ terminal cap is a CapO, Cap1 , ARCA, inosine, N1-methyl-guanosine, 2’fluoro-guanosine, 7-deaza-guanosine, 8-oxo- guanosine, 2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5’ methylG cap, or an analog thereof.

[0330] In any of the embodiments disclosed herein, the mmRNA may comprise one or more modifications. In any of the embodiments disclosed herein, the mmRNA may comprise at least one modification. In embodiments, the modification is nucleoside modification. In embodiments, the modification is a base modification. In embodiments, the modification is a sugar-phosphate backbone modification.

[0331] In embodiments, the modifications are selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, pseudouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2- thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio- cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6- diaminopurine, 2-aminoadenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7- deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7- methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6- thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6- methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6- thio-guanosine, and a combination of any two or more thereof. In embodiments, the modifications are selected from pseudouridine (1), N1 -methylpseudouridine (mW), 2-thiouridine (s2U), 4’ -thiouridine, 5- methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2’-O-methyl uridine, 1-methyl-pseudouridine (m14J), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C). alpha-thio-guanosine, alpha.-thio-adenosine, 5- cyano uridine, 4’-thio uridine 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6- methyl-adenosine (m6A), and 2,6-Diaminopurine, (I), 1-methylinosine (ml I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, and a combination of any two or more thereof. In embodiments, modification is selected from pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and a combination thereof.

[0332] In embodiments, the mmRNA comprises at least one N1 -methylpseudouridine. In embodiments, the mmRNA is fully modified with chemically-modified uridines. In embodiments, the mmRNA is a fully modified with N1- methylpseudouridine.

[0333] In embodiments, the modifications are selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5- aza-uridine, 2-thiouridine, pseudouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2- thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine or a combination of any two or more thereof.

[0334] In embodiments, the modifications are selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4- thio-pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio-1 -methyl- 1 -deaza-pseudoisocytidine, 1 - methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy- 5-methyl-cytidine, 4-methoxy- pseudoisocytidine, and 4-methoxy-1 -methyl-pseudoisocytidine.

[0335] In embodiments, the modifications are selected from 2-aminopurine, 2, 6-diaminopurine, 2-aminoadenine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza- 2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine.

[0336] In embodiments, the modifications are selected from inosine, 1 -methyl-inosine, wyosine, wybutosine, 7- deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8- aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0337] In embodiments, the modifications are present on the major groove face. In embodiments, a hydrogen on C- 5 of uracil is replaced with a methyl group or a halo group.

[0338] In embodiments, the mmRNA further comprises one or more modifications selected from 5’-O-(1- Thiophosphatej-Adenosine, 5’-O-(1 -ThiophosphateJ-Cytidine, 5’-O-(1-Thiophosphate)-Guanosine, 5’-O-(1- Thiophosphatej-Uridine and 5’-O-(1-Thiophosphate)-Pseudouridine.

[0339] In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP), a lipoplex, or a liposome. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP). In embodiments, the mmRNAs described herein may be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid that can interact with the mRNA anchoring the molecule to the emulsion particle. In embodiments, the mRNAs described herein may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed. Exemplary emulsions can be made by the methods described in PCT International Application Publication Nos. WO 2012 / 006380 and WO 2010 / 87791 , each of which is herein incorporated by reference in its entirety.

[0340] In some embodiments, nucleic acids of the invention (e.g., mRNA) are formulated in a lipid nanoparticle (LNP). Lipid nanoparticles comprise typically comprise ionizable cationic lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the invention can be generated using components, compositions, and methods as are disclosed, e.g., in PCT International Application Publication Nos. WO 2021 / 231854, WO 2021 / 050986, WO 2021 / 055833, WO 2021 / 213924, WO 2021 / 055849, WO 2021 / 214204, WO 2021 / 188969, WO 2021 / 055835, WO 2020 / 061284, WO 2020 / 061295, WO 2017 / 049245, WO 2017 / 031232, WO 2017 / 112865, WO 2017 / 218704, WO 2017 / 218704, WO 2017 / 099823, WO 2017 / 049074, WO 2017 / 117528, WO 2017 / 180917, WO 2017 / 075531 , WO 2017 / 223135, WO 2016 / 118724, WO 2015 / 164674, WO 2015 / 038892, WO 2014 / 152211 , and WO 2013 / 090648, the entire contents of each which are herein incorporated by reference. PEG-lipids selected from an ionizable lipid (e.g., as known in the art, such as those described in U.S. Pat. No. 8,158,601 and PCT International Application Publication Nos. WO 2012 / 099755 and WO 2015 / 130584, which are incorporated herein by reference in their entirety. The ionizable lipid may be selected from, but not limited to, an ionizable lipid described in International Patent Application Publication Nos. WO 2013 / 086354 and WO 2013116126; the contents of each of which are herein incorporated by reference in their entirety. In embodiments, the lipid may be a cleavable lipid such as those described in PCT International Patent Application Publication No. WO 2012 / 170889, herein incorporated by reference in its entirety. In embodiments, the lipid may be synthesized by methods known in the art and / or as described in International Patent Application Publication Nos. WO 2013 / 086354; the contents of each of which are herein incorporated by reference in their entirety. In embodiments, the LNP formulations described herein can additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in U.S. Patent Application Publication No. US 2005 / 0222064, herein incorporated by reference in its entirety.

[0341] In embodiments, the pharmaceutically acceptable carrier is a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric nanoparticle, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP), a lipoplex, or a liposome. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP). In embodiments, the lipid nanoparticle comprises lipids selected from an ionizable lipid (e.g. an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200); a structural lipid (e.g. distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g. a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG- dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2- dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE).

[0342] In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP). In embodiments, the LNP comprises a molar ratio of about 20-60% ionizable amino lipid, about 5-25% phospholipid, about 25-55% structural lipid, and about 0.5-1.5% PEG lipid. In embodiments, the LNP comprises a molar ratio of about 50% ionizable amino lipid, about 8-12% phospholipid, about 37-40% structural lipid, and about 1-2% PEG lipid. In embodiments, the lipid nanoparticle comprises lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2- DMA, DLin-MC3-DMA, 98N12-5, and C12-200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG-diacylglycerol (DAG), a PEG- dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG- dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (C18)); 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE).

[0343] In embodiments, the pharmaceutically acceptable carrier is a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric nanoparticle, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP), a lipoplex, or a liposome. In embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle (LNP).

[0344] In embodiments, the lipid nanoparticle comprises lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG- ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA.

[0345] In any of the embodiments disclosed herein, the pharmaceutical composition is formulated for parenteral administration. In any of the embodiments disclosed herein, the pharmaceutical composition is formulated for topical administration.

[0346] In aspects, the present disclosure provides a pharmaceutical composition comprising the mmRNA of any embodiment disclosed herein, or an LNP comprising an mmRNA of any embodiment disclosed herein. In embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0347] In embodiments, the pharmaceutical composition comprises a modified mRNA (mmRNA) comprising at least one modified nucleotide selected from a modified sugar, a modified base and / or a modified internucleotide linkage, wherein the mmRNA encodes a chimeric protein having an amino acid sequence that has at least about 90%, or at least about 91 %, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with the amino acid sequence of the amino acid sequence of SEQ ID NO: 74 or SEQ ID NO: 113.

[0348] In embodiments, the pharmaceutical composition is formulated for parenteral administration. In embodiments, the pharmaceutical composition is formulated for topical, dermal, intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial or transdermal administration. In embodiments, the pharmaceutical composition is formulated for topical administration.

[0349] In aspects, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier, and the chimeric protein of any one of the embodiments disclosed herein, the isolated polynucleotide of any one of the embodiments disclosed herein, the vector of the embodiments disclosed herein, or the host cell of any of the embodiments disclosed herein. In embodiments, the pharmaceutical composition comprises the nucleic acid, e.g., the mmRNA of any one of the embodiments disclosed herein.

[0350] In aspects, the present disclosure provides a pharmaceutical composition comprising a modified mRNA (mmRNA) comprising at least one modified nucleotide selected from a modified base (without limitation, e.g., 1 -methylpseudouridine, N1 -methylpseudouridine), a modified sugar (without limitation, e.g., 2'fluoro-2'- deoxyribose and 2'-0-methyl-ribose), and / or a modified internucleotide linkage (without limitation, e.g., phosphorothioate), wherein the mmRNA encodes a chimeric protein comprising a general structure of: N terminus - (a) - (b) - (c) - C terminus, wherein: (A) (a) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, wherein the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71, or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71 , (c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), wherein the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73, and (b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge-CH2-CH3 Fc domain; or (B) (a) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), wherein the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73, (c) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, wherein the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71 , or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71 , and (b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2-CH3 Fc domain, wherein the mmRNA comprises one or more nucleoside modifications and wherein the pharmaceutical composition is formulated as a lipid nanoparticle (LNP).

[0351] In embodiments, the lipid nanoparticle comprises lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG- ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA. In embodiments, the lipid nanoparticle comprises (a) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the particle; (b) a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and (c) a conjugated lipid that inhibits aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle. In embodiments, the lipid nanoparticle comprises a lipid selected from SM-102, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12- 5, and C12-200; a cholesterol; and a PEG-lipid.

[0352] In embodiments, the nucleoside modifications are selected from pseudouridine, 1 -methylpseudouridine, N1- methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3- methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2- thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio- cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1- methyl-1-deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2- methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio- adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6- thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and a combination thereof. In embodiments, the mmRNA further comprises one or more of a 5'-cap, a poly A tail, a 5' UTR, optionally selected from SEQ ID NOs: 132-153, and / or a 3’ UTR, optionally selected from SEQ ID NOs: 118- 131.

[0353] In embodiments, the pharmaceutical composition is formulated for parenteral administration. In embodiments, the pharmaceutical composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial or transdermal administration.

[0354] In embodiments, the isolated polynucleotide is conjugated polynucleotide sequence that is introduced into cells by various transfection methods such as, e.g., methods that employ lipid particles. In embodiments, a composition, including a gene transfer construct, comprises a delivery particle. In embodiments, the delivery particle comprises a lipid-based particle (e.g., a lipid nanoparticle (LNP)), cationic lipid, or a biodegradable polymer). Lipid nanoparticle (LNP) delivery of gene transfer construct provides certain advantages, including transient, non-integrating expression to limit potential off-target events and immune responses, and efficient delivery with the capacity to transport large cargos. LNPs have been used for delivery of small interfering RNA (siRNA) and mRNA, and for in vitro and in vivo delivering CRISPR / Cas9 components to hepatocytes and the liver. For example, U.S. Pat. No. 10,195,291 describes the use of LNPs for delivery of RNA interference (RNAi) therapeutic agents.

[0355] In embodiments, the composition in accordance with embodiments of the present disclosure is in the form of a LNP. In embodiments, the LNP comprises one or more lipids selected from 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP); N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine- N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethyleneglycol - 2000 (DMG-PEG 2K), and 1 ,2 distearol-sn-glycerol-3phosphocholine (DSPC).

[0356] In embodiments, the composition can have a lipid and a polymer in various ratios, wherein the lipid can be selected from, e.g., DOTAP, DC-Chol, PC, Triolein, DSPE-PEG, and wherein the polymer can be, e.g., PEI or Poly Lactic-co-Glycolic Acid (PLGA). Any other lipid and polymer can be used additionally or alternatively. In embodiments, the ratio of the lipid and the polymer is about 0.5:1 , or about 1 :1 , or about 1 :1.5, or about 1 :2, or about 1 :2.5, or about 1 :3, or about 3:1, or about 2.5:1 , or about 2:1 , or about 1.5:1 , or about 1 :1 , or about 1 :0.5.

[0357] In embodiments, the LNP comprises a cationic lipid, non-limiting examples of which include N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2- Di Li noleyloxy-N, N-dimethylaminopropane (DLinDMA), 1 , 2-Dili nolenyloxy-N , N-dimethylami nopropane (DLenDMA), 1 ,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-Dili noleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1 ,2- Dili noleoyl-3-dimethyl ami nopropane (DLinDAP), 1 , 2-Dili noleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1 ,2-Dilinoleyloxy-3- trimethylaminopropane chloride salt (DLin-TMA.CI), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1 ,2- propanediol (DLinAP), 3-(N,N-Dioleylamino)-1 ,2-propanedio (DOAP), 1 ,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH- cyclopenta[d][1 ,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (MC3), 1 , T-(2-(4-(2-((2-(bis(2-‘)amino)ethyl)(2 hydroxydodecyl)amino)ethyl) piperazin-1 -yl)ethylazanediyl)didodecan-2-ol (Tech G1), 1 ,2-Dilinoleyloxo-3-(2-N,N-dimethylamino) ethoxypropane (DLin-EG-DMA), or a mixture thereof. In embodiments, the LNP comprises one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc), which are suitable for hepatic delivery. In embodiments, the LNP comprises a hepatic-directed compound as described, e.g., in U.S. Pat. No. 5,985,826, which is incorporated by reference herein in its entirety. GalNAc is known to target Asialoglycoprotein Receptor (ASGPR) expressed on mammalian hepatic cells. See Hu et al. Protein Pept Lett. 2014;21 (10): 1025-30.

[0358] In some examples, the isolated polynucleotide can be formulated or complexed with PEI or a derivative thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.

[0359] In embodiments, the lipid nanoparticle comprises lipids selected from an ionizable lipid (e.g. an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200); a structural lipid (e.g. distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g. a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG- ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)); 1 ,2-dioleoyl-3- trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the nucleic acid, e.g., the mmRNA.

[0360] In embodiments, the LNP comprises a molar ratio of about 20-60% ionizable amino lipid, about 5-25% phospholipid, about 25-55% structural lipid, and about 0.5-1.5% PEG lipid. In embodiments, the ionizable amino lipid comprises the following formula:

[0361] In embodiments, the lipid nanoparticle comprises lipids selected from an ionizable lipid; a structural lipid; cholesterol, and a polyethyleneglycol (PEG)-lipid; 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the nucleic acid, e.g., the mmRNA. In embodiments, the ionizable lipid is an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin- MC3-DMA, 98N12-5, and C12-200. In embodiments, the polyethyleneglycol (PEG)-lipid is selected from a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG-dilauryloxypropyl (e.g., C12, a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18)).

[0362] In embodiments, the LNP is a conjugated lipid, non-limiting examples of which include a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (C18).

[0363] In embodiments, the LNP formulations may further contain a phosphate conjugate, which can increase in vivo circulation times and / or increase the targeted delivery of the nanoparticle. Phosphate conjugates can be made by the methods described in, e.g., PCT International Patent Application Publication No. WO 2013 / 033438 or U.S. Pub. No. US 2013 / 0196948. The LNP formulation can also contain a polymer conjugate (e.g., a water-soluble conjugate) as described in, e.g., U.S. Patent Application Publication Nos. US 2013 / 0059360, US 2013 / 0196948, and US 2013 / 0072709, each of the references is herein incorporated by reference in its entirety.

[0364] In embodiments, the LNP formulations may comprise a carbohydrate carrier. As a non-limiting example, the carbohydrate carrier can include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin (e.g., PCT International Patent Application Publication No. WO 2012 / 109121 , herein incorporated by reference in its entirety). In embodiments, the LNP formulations can be coated with a surfactant or polymer to improve the delivery of the particle. In some embodiments, the LNP can be coated with a hydrophilic coating such as, but not limited to, PEG coatings and / or coatings that have a neutral surface charge as described in U.S. Patent Application Publication No. US 2013 / 0183244, herein incorporated by reference in its entirety. In embodiments, the LNP formulations can be engineered to alter the surface properties of particles so that the lipid nanoparticles can penetrate the mucosal barrier as described in U.S. Pat. No. 8,241,670 or PCT International Patent Application Publication No. WO 2013 / 110028, each of which is herein incorporated by reference in its entirety. In embodiments, the mucus penetrating LNP can be a hypotonic formulation comprising a mucosal penetration enhancing coating. The formulation can be hypotonic for the epithelium to which it is being delivered. Non-limiting examples of hypotonic formulations can be found in, e.g., PCT International Patent Application Publication No. WO 2013 / 110028, herein incorporated by reference in its entirety.

[0365] In embodiments, an mmRNA described herein is formulated as a solid lipid nanoparticle (SLN), which can be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. Exemplary SLN can be those as described in PCT International Patent Application Publication No. WO 2013 / 105101 , herein incorporated by reference in its entirety.

[0366] In embodiments, a nanoparticle is a particle having a diameter of less than about 1000 nm. In embodiments, nanoparticles of the present disclosure have a greatest dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In embodiments, nanoparticles of the present disclosure have a greatest dimension ranging between about 50 nm and about 150 nm, or between about 70 nm and about 130 nm, or between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In embodiments, the nanoparticles of the present disclosure have a greatest dimension (e.g., a diameter) of about 100 nm.

[0367] In embodiments, the chimeric protein or the therapeutic nanoparticle comprising mRNA can be formulated for sustained release, which, as used herein, refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. In embodiments, the period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle of the mRNAs described herein can be formulated as disclosed in PCT International Patent Application Publication No. WO 2010 / 075072 and U.S. Patent Application Publication Nos. US 2010 / 0216804, US 2011 / 0217377, US 2012 / 0201859 and US 2013 / 0150295, each of which is herein incorporated by reference in their entirety.

[0368] In embodiments, the chimeric protein or the isolated polynucleotide or mmRNA (and / or additional agents) are included various formulations. Any chimeric protein, or the isolated polynucleotide or mmRNA (and / or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. DNA or RNA constructs encoding the protein sequences may also be used. In embodiments, the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated herein by reference.

[0369] In embodiments, the present disclosure provides an expression vector, comprising a nucleic acid encoding the chimeric protein described herein. In embodiments, the expression vector comprises DNA or RNA. In embodiments, the expression vector is a mammalian expression vector. Both prokaryotic and eukaryotic vectors can be used for expression of the chimeric protein. Prokaryotic vectors include constructs based on E. coll sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512- 538). Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 and APL. Non-limiting examples of prokaryotic expression vectors may include the Agt vector series such as Agt11 (Huynh et al., in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vector systems cannot perform much of the post-translational processing of mammalian cells, however. Thus, eukaryotic host-vector systems may be particularly useful. A variety of regulatory regions can be used for expression of the chimeric proteins in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the 0- interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the chimeric proteins in recombinant host cells.

[0370] In embodiments, expression vectors of the disclosure comprise a nucleic acid encoding the chimeric proteins (and / or additional agents), or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell. The expression control region is capable of driving expression of the operably linked blocking and / or stimulating agent encoding nucleic acid such that the blocking and / or stimulating agent is produced in a human cell transformed with the expression vector.

[0371] Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, which influence expression of an operably linked nucleic acid. An expression control region of an expression vector of the disclosure is capable of expressing operably linked encoding nucleic acid in a human cell. In embodiments, the cell is a tumor cell. In embodiments, the cell is a non-tumor cell. In embodiments, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

[0372] In embodiments, the present disclosure contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue. For example, when in the proximity of a tumor cell, a cell transformed with an expression vector for the chimeric protein (and / or additional agents) comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue. Illustrative inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound. Particular examples can be found, for example, in U.S. Patent Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941 , each of which is incorporated herein by reference in its entirety.

[0373] Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function. As used herein, the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).

[0374] As used herein, “operable linkage” refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. Typically, an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid ( / .e., “upstream”). Expression control regions can also be located at the 3’ end of the transcribed sequence ( / .e., “downstream”) or within the transcript (e.g., in an intron). Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.

[0375] Expression systems functional in human cells are well known in the art and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

[0376] Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3’ terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs.

[0377] There are a variety of techniques available for introducing nucleic acids into viable cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc. For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction. In some situations, it is desirable to provide a targeting agent, such as an antibody or ligand specific for a tumor cell surface membrane protein. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and / orto facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins thattarget intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).

[0378] Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacterio!., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667- 678, 2004), sleeping beauty, transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the chimeric proteins including CRISPR / CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.

[0379] In aspects, the disclosure provides expression vectors for the expression of the chimeric proteins (and / or additional agents) that are viral vectors. Many viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol., 21 : 1 17, 122, 2003. Illustrative viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and a viruses, though other viral vectors may also be used. For in vivo uses, viral vectors that do not integrate into the host genome are suitable for use, such as a viruses and adenoviruses. Illustrative types of a viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and Antiviruses. In embodiments, the disclosure provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the disclosure.

[0380] In embodiments, the present disclosure provides a host cell, comprising the expression vector comprising the chimeric protein described herein.

[0381] Expression vectors can be introduced into host cells for producing the present chimeric proteins. Cells may be cultured in vitro or genetically engineered, for example. Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler in “Gene Transfer and Expression: A Laboratory Manual,” 1990, New York, Freeman & Co.). These include monkey kidney cell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); human embryonic kidney lines (e.g., 293, 293- EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sei USA 1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells (Mather, Biol Reprod ] 980, 23:243-251); mouse fibroblast cells (e.g., NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidney cells, (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells (e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51). Illustrative cancer cell types for expressing the chimeric proteins described herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, EG7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.

[0382] Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.

[0383] Cells that can be used for production of the present chimeric proteins in vitro, ex vivo, and / or in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, fetal liver, etc. The choice of cell type depends on the type of tumor or infectious disease being treated or prevented, and can be determined by one of skill in the art.

[0384] Where necessary, the formulations comprising the chimeric protein, or the isolated polynucleotide (and / or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.

[0385] The formulations comprising the chimeric protein (and / or additional agents) of the present disclosure may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art) In embodiments, any chimeric protein, or the isolated polynucleotide or mmRNA (and / or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.

[0386] Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In embodiments, the administering is effected orally or by parenteral injection. In some instances, administration results in the release of any agent described herein into the bloodstream, or alternatively, the agent is administered directly to the site of active disease.

[0387] Any chimeric protein, or the isolated polynucleotide (and / or additional agents) described herein can be administered orally. Such chimeric proteins (and / or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer.

[0388] In embodiments, the pharmaceutical composition is formulated for parenteral administration. In embodiments, the pharmaceutical composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial or transdermal administration.

[0389] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

[0390] The dosage of any chimeric protein, or the isolated polynucleotide or mmRNA (and / or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject’s general health, and the administering physician’s discretion. Any chimeric protein described herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof. In embodiments any chimeric protein and additional agent described herein are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days part, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.

[0391] The dosage of any chimeric protein, or the isolated polynucleotide or mmRNA (and / or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Fu...

Claims

CLAIMSCLAIMSWhat is claimed is1 . A chimeric protein comprising a general structure of:N terminus - (a) - (b) - (c) - C terminus, wherein:(A)(a) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist,(c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), and(b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge-CH2-CH3 Fc domain; or(B)(a) is a second domain comprising a portion of activin receptor type-2B (ACVR2B),(c) is a first domain comprising a glucagon-like peptide-1 (GLP-1) receptor agonist, and(b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2-CH3 Fc domain.

2. The chimeric protein of claim 1 , wherein the GLP-1 receptor agonist is selected from GLP-1 , a DPP4 degradation resistant derivative of GLP-1, exenatide, lixisenatide, albiglutide, dulaglutide, or a variant thereof having one or more amino acid mutations, independently selected from substitutions, insertions, deletions, and truncations.

3. The chimeric protein of claim 2, wherein the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71 , or a variant having about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71.

4. The chimeric protein of claim 3, wherein the GLP-1 receptor agonist is a DPP4 degradation resistant GLP-1.

5. The chimeric protein of any one of claims 1 to 4, wherein the GLP-1 receptor agonist is capable of binding a GLP-1 receptor.

6. The chimeric protein of claim 5, wherein the GLP-1 receptor agonist is capable of activate the GLP- 1 receptor.

7. The chimeric protein of any one of claims 1 to 6, wherein the GLP-1 receptor agonist is capable of stimulating and / or increasing insulin secretion, and / or inhibiting and / or decreasing glucagon secretion.

8. The chimeric protein of any one of claims 1 to 7, wherein the portion of ACVR2B comprises substantially the entire extracellular domain of ACVR2B.

9. The chimeric protein of any one of claims 1 to 8, wherein the portion of ACVR2B comprises the entire extracellular domain of ACVR2B.

10. The chimeric protein of any one of claims 1 to 9, wherein the portion of ACVR2B is capable of binding activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11).11 . The chimeric protein of claim 10, wherein the portion of ACVR2B is capable of reducing or inhibiting the binding of one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11) to one or more of their receptors.

12. The chimeric protein of claim 10 or claim 11 , wherein the portion of ACVR2B is capable of sequestering one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11).

13. The chimeric protein of ant one of claims 10 to 12, wherein the portion of ACVR2B is capable of reducing or inhibiting ACVR2B signaling induced by one or more of activin A, GDF-5 (BMP-14), GDF-8 (myostatin), and / or GDF-11 (BMP-11).

14. The chimeric protein of any one of claims 1 to 13, wherein the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73.

15. The chimeric protein of any one of claims 1 to 14, wherein the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence.

16. The chimeric protein of any one of claims 1 to 15, wherein the hinge-CH2-CH3 Fc domain is derived from lgG1.

17. The chimeric protein of claim 16, wherein the lgG1 is human lgG1.

18. The chimeric protein of any one of claims 1 to 17, wherein the hinge-CH2-CH3 Fc domain is derived from I gG4.

19. The chimeric protein of claim 18, wherein the lgG4 is human lgG4.

20. The chimeric protein of any one of claims 1 to 19, wherein the hinge-CH2-CH3 Fc domain comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 53.

21. The chimeric protein of any one of claims 1 to 20, wherein the linker further comprises the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 4 to 52.

22. The chimeric protein of claim 21 , wherein the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 4 to 52; wherein one joining linker is N terminal to the hinge- CH2-CH3-Fc domain and another joining linker is C terminal to the hinge-CH2-CH3-Fc domain.

23. The chimeric protein of any one of claims 1 to 22, wherein the chimeric protein is a recombinant fusion protein.

24. The chimeric protein of any one of claims 1 to 23, wherein the chimeric protein is capable of inducing insulin production by pancreatic islet beta cells.

25. The chimeric protein of any one of claims 1 to 24, wherein the chimeric protein is capable of reducing one or more of subcutaneous white adipose tissue (sWAT), peri-renal adipose tissue, mesenteric adipose tissue, epididymal white adipose tissue (eWAT), cervical adipose tissue, visceral adipose tissue, and subcutaneous adipose tissue.

26. The chimeric protein of any one of claims 1 to 25, wherein the chimeric protein is capable of reducing or inhibiting adipocyte progenitor proliferation.

27. The chimeric protein of any one of claims 1 to 26, wherein the chimeric protein is capable of promoting adipocyte progenitor differentiation.

28. The chimeric protein of any one of claims 1 to 27, wherein the chimeric protein is capable of reducing fat mass.

29. The chimeric protein of any one of claims 1 to 28, wherein the chimeric protein is capable of inducing skeletal muscle hypertrophy.

30. The chimeric protein of any one of claims 1 to 29, wherein the chimeric protein is capable of promoting muscle growth.

31. The chimeric protein of any one of claims 1 to 30, wherein the chimeric protein is capable of maintaining while retaining muscle size, muscle striation and / or muscle mass.

32. The chimeric protein of any one of claims 1 to 30, wherein the chimeric protein is capable of reducing connective tissue within muscle, phagocytising necrotic muscle fibers and / or fragmented sarcoplasm.

33. The chimeric protein of any one of claims 1 to 32, wherein the chimeric protein is capable of reducing hepatic glucose production.

34. The chimeric protein of any one of claims 1 to 33, wherein the chimeric protein is capable of increasing lean tissue.

35. The chimeric protein of any one of claims 1 to 34, wherein the chimeric protein is capable of preventing diet-induced obesity and insulin resistance.

36. The chimeric protein of any one of claims 1 to 35, wherein the chimeric protein is capable of reducing liver weight.

37. The chimeric protein of any one of claims 1 to 36, wherein the chimeric protein is capable of reducing liver steatosis, hepatocellular ballooning, and / or liver fibrosis.

38. An isolated polynucleotide encoding the chimeric protein of any one of claims 1 to 37.

39. The isolated polynucleotide of claim 38, wherein the polynucleotide is DNA.

40. The isolated polynucleotide of claim 38, wherein the polynucleotide is selected from mRNA, circular RNA (circRNA) and self-amplifying RNA (saRNA), optionally wherein the polynucleotide comprises at least one modified nucleotide selected from a modified sugar, a modified base and / or a modified internucleotide linkage.41 . The isolated polynucleotide of claim 40, wherein the polynucleotide is an mmRNA.

42. The isolated polynucleotide of claim 40 or claim 41 , wherein the mmRNA comprises one or more nucleoside modifications.

43. The isolated polynucleotide of claim 42, wherein the nucleoside modifications are selected from pseudouridine, 1 -methylpseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4- thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1 - methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2- thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl- pseudoisocytidine, 4-thio-1-methyl-1 -deaza-pseudoisocytidine, 1 -methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy- cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl- pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza- 2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine, inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6- thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7- methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and a combination thereof.

44. The isolated polynucleotide of claim 43, wherein the mmRNA further comprises one or more of a 5'- cap, a poly A tail, a 5' UTR, optionally selected from SEQ ID NOs: 132-153, and / or a 3’ UTR, optionally selected from SEQ ID NOs: 118-131.

45. The isolated polynucleotide of any one of claims 38 to 44, wherein the polynucleotide comprises a liver, skin and / or muscle-specific control element.

46. The isolated polynucleotide of claim 45, wherein the liver-specific control element is a liver-specific promoter selected from albumin promoter, thyroxine-binding globulin (TBG) promoter, hybrid liver-specific promoter (HLP), human a 1 -antitrypsin promoter, LP1 promoter, and hemopexin promoter.

47. The isolated polynucleotide of any one of claims 38 to 45, wherein the polynucleotide is codon optimized.

48. The isolated polynucleotide of claim 47, wherein the polynucleotide is codon optimized for expression in a mammalian cell.

49. The isolated polynucleotide of claim 48, wherein the polynucleotide is codon optimized for expression in a human cell.

50. The isolated polynucleotide of claim 48, wherein the human cell is a liver cell, skin cell and / or muscle cell.51 . An expression vector comprising the isolated polynucleotide of any one of claims 38 to 50.

52. The expression vector of claim 51 , wherein the expression vector is a mammalian expression vector.

53. A host cell comprising the isolated polynucleotide of any one of claims 38 to 50 or the expression vector of claim 51 or claim 52.

54. The host cell of claim 53, wherein the host cell is located in vivo.

55. A pharmaceutical composition comprising the chimeric protein of any one of claims 1 to 37, the isolated polynucleotide of any one of claims 38 to 50, or the expression vector of claim 51 or claim 52, the host cell of claim 53, and a pharmaceutically acceptable carrier.

56. A pharmaceutical composition comprising the mmRNA of any one of claims 41 to 50, and a pharmaceutically acceptable carrier.

57. The pharmaceutical composition of claim 55 or claim 56, wherein the carrier is a lipidoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric nanoparticle, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate.

58. The pharmaceutical composition of claim 57, wherein the pharmaceutical composition is formulated as a lipid nanoparticle (LNP), a lipoplex, or a liposome.

59. The pharmaceutical composition of claim 58, wherein the pharmaceutical composition is formulated as a lipid nanoparticle (LNP).

60. The pharmaceutical composition of claim 59, wherein the lipid nanoparticle comprises lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG-diacylglycerol (DAG), a PEG- dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG- dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (C18)); 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA.61 . The pharmaceutical composition of claim 60, wherein the lipid nanoparticle comprises (a) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the particle; (b) a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and (c) a conjugated lipid that inhibits aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle.

62. The pharmaceutical composition of claim 60 or claim 61 , wherein the lipid nanoparticle comprises a lipid selected from SM-102, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200; a cholesterol; and a PEG-lipid.

63. A pharmaceutical composition comprising a modified mRNA (mmRNA) comprising at least one modified nucleotide selected from a modified sugar, a modified base and / or a modified internucleotide linkage, wherein the mmRNA encodes a chimeric protein comprising a general structure of:N terminus - (a) - (b) - (c) - C terminus, wherein:(A)(a) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, wherein the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71, or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71 ,(c) is a second domain comprising a portion of activin receptor type-2B (ACVR2B), wherein the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73, and(b) is a linker adjoining the first domain and a second domain, optionally wherein the linker comprises a hinge-CH2-CH3 Fc domain; or(B)(a) is a second domain comprising a portion of activin receptor type-213 (ACVR2B), wherein the portion of ACVR2B comprises an amino acid sequence that is at least about 90%, or at least about 95% identical to the amino acid sequence of SEQ ID NO: 73,(c) is a first domain comprising a glucagon-like peptide- 1 (GLP-1) receptor agonist, wherein the GLP-1 receptor agonist has an amino acid sequence of any one of SEQ ID NOs: 61 to 71, or a variant having about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid mutations with respect to an amino acid sequence selected from the amino acid sequence of SEQ ID NOs: 61 to 71 , and(b) is a linker adjoining the first domain and a second domain, optionally a hinge-CH2-CH3 Fc domain, wherein the mmRNA comprises one or more nucleoside modifications and wherein the pharmaceutical composition is formulated as a lipid nanoparticle (LNP).

64. The pharmaceutical composition of claim 63, wherein the lipid nanoparticle comprises lipids selected from an ionizable lipid (e.g., an ionizable cationic lipid selected from DLin-DMA, DLin-K-DMA, DLin-KC2- DMA, DLin-MC3-DMA, 98N12-5, and C12-200); a structural lipid (e.g., distearoylphosphatidylcholine (DSPC)); cholesterol, and a polyethyleneglycol (PEG)-lipid (e.g., a PEG-diacylglycerol (DAG), a PEG- dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof, or a PEG- dilauryloxypropyl (C12, a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG- distearyloxypropyl (C18)); 1 ,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoylphosphatidylethanolamine (DOPE); and the mmRNA.

65. The pharmaceutical composition of claim 64, wherein the lipid nanoparticle comprises (a) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the particle; (b) a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and (c) a conjugated lipid that inhibits aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle.

66. The pharmaceutical composition of claim 64 or claim 65, wherein the lipid nanoparticle comprises a lipid selected from SM-102, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12- 200; a cholesterol; and a PEG-lipid.

67. The pharmaceutical composition of any one of claims 63 to 66, wherein the nucleoside modifications are selected from pseudouridine, 1 -methylpseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1- taurinomethyl-pseudouridine, 5-tau ri nomethyl-2-th io-uri di ne, 1 -taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1 - deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4- acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy- adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6- thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7- methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and a combination thereof.

68. The pharmaceutical composition of any one of claims 63 to 67, wherein the mmRNA further comprises one or more of a 5'-cap, a poly A tail, a 5' UTR, optionally selected from SEQ ID NOs: 132-153, and / or a 3’ UTR, optionally selected from SEQ ID NOs: 118-131.

69. The pharmaceutical composition of any one of claims 63 to 68, wherein the mmRNA comprises a tissue-specific control element.

70. The pharmaceutical composition of claim 69, wherein the tissue-specific control element is a liver, skin and / or muscle-specific control element.

71. The pharmaceutical composition of claim 70, wherein the liver-specific control element is a liverspecific promoter selected from albumin promoter, thyroxine-binding globulin (TBG) promoter, hybrid liverspecific promoter (HLP), human a 1 -antitrypsin promoter, LP1 promoter, and hemopexin promoter.

72. The pharmaceutical composition of any one of claims 63 to 71 , wherein the mmRNA is codon optimized.

73. The mmRNA of claim 72, wherein the mmRNA is codon optimized for expression in a human cell.

74. The mmRNA of claim 73, wherein the human cell is a liver cell, skin cell and / or muscle cell.

75. The pharmaceutical composition of any one of claims 63 to 74, wherein the pharmaceutical composition is formulated for parenteral administration.

76. The pharmaceutical composition of claim 75, wherein the pharmaceutical composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial or transdermal administration.

77. A method of preventing and / or treating hyperglycemia, diabetes, obesity, metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, or for reducing blood glucose compared to a pre-treatment blood glucose, or for reducing fed and fasting blood glucose compared to a pre-treatment fed and fasting blood glucose, or for reducing cardiovascular risk compared to a pre-treatment cardiovascular risk, or for decreasing body weight compared to a pre-treatment body weight, for decreasing food intake compared to a pre-treatment food intake, for decreasing blood glucose compared to a pre-treatment blood glucose, for decreasing liver adiposity compared to a pre-treatment liver adiposity, for decreasing liver fibrosis compared to a pre-treatment liver fibrosis, for decreasing liver weight compared to a pre-treatment liver weight, for decreasing an amount of subcutaneous white adipose tissue (sWAT) compared to a pre-treatment amount of sWAT, for decreasing an amount of epididymal white adipose tissue (eWAT) compared to a pre-treatment amount of eWAT, for decreasing liver steatosis compared to a pretreatment liver steatosis, for decreasing hepatocellular ballooning compared to a pre-treatment hepatocellular ballooning, and / or for increasing glucose tolerance compared to a pre-treatment glucose tolerance in a subject in need thereof, the method comprising administering to the subject the chimeric protein of any oneof claims 1 to 37, the isolated polynucleotide of any one of claims 38 to 50, or the expression vector of claim51 or claim 52, the host cell of claim 53, or the pharmaceutical composition of any one of claims 55 to 76.

78. The method of claim 77, wherein the method decreases an amount of connective tissue within muscle of the subject compared to a pre-treatment amount of connective tissue within muscle in the subject.

79. The method of claim 77 or claim 78, wherein the method decreases an amount of phagocytising necrotic muscle fibers of the subject compared to a pre-treatment amount of phagocytising necrotic muscle fibers in the subject.

80. The method of any one of claims 77 to 79, wherein the method decreases an amount of fragmented sarcoplasm of the subject compared to a pre-treatment amount of fragmented sarcoplasm in the subject.81 . The method of any one of claims 77 to 80, wherein the method retains or increases muscle size in the subject compared to a pre-treatment muscle size in the subject.

82. The method of any one of claims 77 to 81 , wherein the method retains or increases muscle striation in the subject compared to a pre-treatment muscle striation in the subject.

83. The method of any one of claims 77 to 81 , wherein the method retains or increases muscle mass and / or muscle density in the subject compared to a pre-treatment muscle mass and / or muscle density in the subject.

84. The method of any one of claims 77 to 83, wherein the method prevents and / or treats sarcopenia, muscle weakness and / or muscle loss in the subject.

85. The method of any one of claims 77 to 84, wherein the method improves one or more of muscle strength, physical frailty, aerobic capacity, physical performance, limitations in mobility, and physical function in the subject compared to pre-treatment level of one or more of muscle strength, physical frailty, aerobic capacity, physical performance, limitations in mobility, and physical function.

86. A method of preventing and / or treating sarcopenia, physical frailty, aerobic resistance, muscle weakness, muscle loss, limitations in mobility and / or physical function, of decreasing a level of connective tissue within muscle, phagocytising necrotic muscle fibers, and / or fragmented sarcoplasm compared to a pre-treatment levels of connective tissue within muscle, phagocytising necrotic muscle fibers, and / or fragmented sarcoplasm, or of retaining or increasing muscle size, muscle striation, muscle mass, muscle density, diminished muscle strength, and / or physical performance compared to pre-treatment level of muscle size, muscle striation, muscle mass, muscle density, diminished muscle strength, or physical performance ina subject in need thereof, the method comprising administering to the subject the chimeric protein of any one of claims 1 to 37, the isolated polynucleotide of any one of claims 38 to 50, or the expression vector of claim 51 or claim 52, the host cell of claim 53, or the pharmaceutical composition of any one of claims 55 to 76.

87. The method of claim 86, wherein the method prevents, decreases or treats: one or more of hyperglycemia, diabetes, obesity, metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) in the subject; and / or one or more of increased liver weight, liver adiposity, liver fibrosis, liver steatosis, hepatocellular ballooning, and / or liver fibrosis compared to pre-treatment levels of liver weight, liver adiposity, liver fibrosis, liver steatosis, hepatocellular ballooning, and / or liver fibrosis in the subject.

88. The method of claim 86 or claim 87, wherein the method reduces or treats one or more of the cardiovascular risk, body weight, food intake, subcutaneous white adipose tissue (sWAT), and epididymal white adipose tissue (eWAT) compared to pre-treatment elevated cardiovascular risk, body weight, food intake, sWAT and eWAT in the subject.

89. A method of preventing and / or treatinghyperglycemia, diabetes, obesity, metabolic syndrome, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, muscle weakness and / or muscle loss, or for reducing blood glucose compared to a pre-treatment blood glucose, or for reducing fed and fasting blood glucose compared to a pre-treatment fed and fasting blood glucose, or for reducing cardiovascular risk compared to a pre-treatment cardiovascular risk, or for decreasing body weight compared to a pre-treatment body weight, for decreasing food intake compared to a pre-treatment food intake, for decreasing blood glucose compared to a pre-treatment blood glucose, for decreasing liver adiposity compared to a pre-treatment liver adiposity, for decreasing liver fibrosis compared to a pretreatment liver fibrosis, for decreasing liver weight compared to a pre-treatment liver weight, for decreasing an amount of subcutaneous white adipose tissue (sWAT) compared to a pre-treatment amount of sWAT, for decreasing an amount of epididymal white adipose tissue (eWAT) compared to a pre-treatment amount of eWAT, for decreasing liver steatosis compared to a pre-treatment liver steatosis, for decreasing hepatocellular ballooning compared to a pre-treatment hepatocellular ballooning, decreasing connective tissue within muscle, phagocytising necrotic muscle fibers and / or fragmented sarcoplasm compared to pretreatment connective tissue within muscle, phagocytising necrotic muscle fibers and / or fragmented sarcoplasm, and / or for increasing glucose tolerance compared to a pre-treatment glucose tolerance, retaining or increasing muscle size, muscle striation, and / or muscle mass compared to pre-treatment muscle size,muscle striation, and / or muscle mass in a subject in need thereof, the method comprising administering to the subject the chimeric protein of any one of claims 1 to 37, the isolated polynucleotide of any one of claims 38 to 50, or the expression vector of claim 51 or claim 52, the host cell of claim 53, or the pharmaceutical composition of any one of claims 55 to 76.

90. The method of any one of claims 77 to 89, wherein the method reduces fat mass in the subject compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

91. The method of any one of claims 77 to 90, wherein the method induces skeletal muscle hypertrophy compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

92. The method of any one of claims 77 to 91 , wherein the method reduces body weight compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

93. The method of any one of claims 77 to 92, wherein the method reduces glucose tolerance compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

94. The method of any one of claims 77 to 93, wherein the method increases insulin secretion compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

95. The method of any one of claims 77 to 94, wherein the method promotes muscle growth compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

96. The method of any one of claims 77 to 95, wherein the method reduces hepatic glucose production compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

97. The method of any one of claims 77 to 96, wherein the method increases lean tissue compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

98. The method of any one of claims 77 to 96, wherein the method prevents diet-induced obesity and insulin resistance compared to another subject that has been treated only with a glucagon-like peptide-1 (GLP-1) receptor agonist.

99. The method of any one of claims 77 to 98, wherein the method increases muscle weight compared to another subject that has been treated with an activin receptor type-2B (ACVR2B) antagonist.

100. The method of any one of claims 77 to 99, wherein the method reduces p hagocytisi ng necrotic fibers and / or fragmented sarcoplasm compared to another subject that has been treated with an activin receptor type-2B (ACVR2B) antagonist.

101. The method of any one of claims 77 to 100, wherein the method reduces body weight compared to another subject that has been treated with a glucagon-like peptide-1 (GLP-1) receptor agonist, an activin receptor type-2B (ACVR2B) antagonist or a combination thereof.

102. A polypeptide comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113.

103. The polypeptide of claim 102, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 74 or 113.

104. An isolated polynucleotide encoding the polypeptide of claim 102 or claim 103.

105. The isolated polynucleotide of claim 104, wherein the isolated polynucleotide is selected from DNA or mRNA.

106. The isolated polynucleotide of claim 104, wherein the isolated polynucleotide is selected from DNA, modified mRNA, circular RNA (circRNA) and self-amplifying RNA (saRNA), wherein the isolated polynucleotide comprises at least one modified nucleotide selected from a modified sugar, a modified base and / or a modified internucleotide linkage.

107. An expression vector comprising the isolated polynucleotide of claim 104 or claim 105.

108. The expression vector of claim 107, wherein the expression vector is a mammalian expression vector.

109. A host cell comprising the isolated polynucleotide of any one of claims 104 to 106, or the expression vector of claim 107 or claim 108.

110. The host cell of claim 109, wherein the host cell is located in vivo.

111. A pharmaceutical composition comprising the chimeric protein of claim 102 or claim 103, the isolated polynucleotide of any one of claims 104 to 106, or the expression vector of claim 107 or claim 108, the host cell of claim 109, and a pharmaceutically acceptable carrier.

112. An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least about 90%, or at least about 92%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 74 or 113.

113. The isolated polynucleotide of claim 112, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 74 or 113.

114. The isolated polynucleotide of claim 112 or claim 113, wherein the isolated polynucleotide is selected from DNA, modified mRNA, circular RNA (circRNA) and self-amplifying RNA (saRNA), wherein the isolated polynucleotide comprises at least one modified nucleotide selected from a modified sugar, a modified base and / or a modified internucleotide linkage.

115. A pharmaceutical composition comprising the isolated polynucleotide of any one of claims 112 to 114, and a pharmaceutically acceptable carrier.

116. A method of preventing and / or treating sarcopenia, physical frailty, aerobic resistance, muscle weakness, muscle loss, limitations in mobility and / or physical function, of decreasing a level of connective tissue within muscle, phagocytising necrotic muscle fibers, and / or fragmented sarcoplasm compared to a pre-treatment levels of connective tissue within muscle, phagocytising necrotic muscle fibers, and / or fragmented sarcoplasm, or of retaining or increasing muscle size, muscle striation, muscle mass, muscle density, diminished muscle strength, and / or physical performance compared to pre-treatment level of muscle size, muscle striation, muscle mass, muscle density, diminished muscle strength, or physical performance in a subject in need thereof, the method comprising administering to the subject the chimeric protein of claim 102 or claim 103, the isolated polynucleotide of any one of claims 104 to 106 or 112 to 114, or the expression vector of claim 107 or claim 108, the host cell of claim 109, or the pharmaceutical composition of claim 111 or 115.

Images