FGF21 gene therapy and methods therefor

Abstract

Provided herein are polynucleotides comprising a coding sequence for expression of a fibroblast growth factor 21 (FGF21) pathway activating agent. Also provided are vectors, recombinant viral genomes, and recombinant virus compositions comprising said polynucleotides, as well as associated methods.

Description

FGF21 GENE THERAPY AND METHODS THEREFOR CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application number 63 / 647,136 filed on May 14, 2024, and U.S. Provisional Patent Application number 63 / 761,685 filed on February 21, 2025, each of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] Fibroblast growth factor 21 (FGF21) is a growth factor expressed in numerous tissues, including liver, brown adipose tissue, white adipose tissue and pancreas, and predominantly secreted by the liver. FGF21 stimulates glucose uptake in adipocytes and has been shown to stimulate energy expenditure, fat utilization, and lipid excretion. Overexpression of FGF21 in transgenic mice has been shown to provide protection from diet-induced obesity, and administration to diabetic rodents has been shown to result in lower blood glucose and triglycerides and improved insulin sensitivity. Further, FGF21 has recently been shown to act directly on the cardiovascular system and to play an important role in the prediction, treatment, and improvement of prognosis in cardiovascular diseases.

[0003] While treatment with FGF21 has potential for subjects with various diseases, including diabetes, obesity, liver inflammation, fibrosis, and cancer, native FGF21 polypeptide exhibits poor pharmacokinetic properties, including a short half-life and susceptibility to in vivo proteolytic degradation and in vitro aggregation. Several FGF21 mimetics have been tested in humans. However, such mimetics require multiple administrations and may exhibit a higher risk of immunogenicity in treated patients.

[0004] Accordingly, there is a need in the art for FGF21 therapies that can avoid the unfavorable pharmacokinetic properties of recombinant FGF21 and the negative side effects of existing methods. SUMMARY

[0005] The present disclosure provides a recognition that certain products (e.g., gene products, e.g., polypeptide products) that activate and / or modulate the FGF21 signaling pathway may exhibit advantageous properties in treating a subject suffering from a disease, disorder, or condition. In certain embodiments, an FGF21 pathway activating agent activates the FGF21 signaling pathway. In certain embodiments, an FGF21 pathway activating agent modulates theFGF21 signaling pathway. In certain embodiments, an FGF21 pathway activating agent is or includes an FGF21 polypeptide or variant thereof. In certain embodiments, an FGF21 pathway activating agent is or includes an FGF21 fusion polypeptide or variant thereof. In certain embodiments, an FGF21 pathway activating agent is or includes an antibody or antigen binding fragment.

[0006] The present disclosure also provides for the administration of compositions that results in expression of certain products (e.g., gene products, e.g., polypeptide products) that activate the FGF21 signaling pathway. The present disclosure also provides for the administration of compositions that results in expression of certain products (e.g., gene products, e.g., polypeptide products) that modulate the FGF21 signaling pathway.

[0007] The present disclosure also provides for the administration of compositions that result in expression of certain products (e.g., gene products, e.g., polypeptide products) that can be used in treating a subject suffering from a disease, disorder, or condition.

[0008] The present disclosure also provides for the administration of compositions that results in expression of certain products (e.g., gene products, e.g., polypeptide products) that activate and / or modulate the FGF21 signaling pathway and / or uses in treating a subject suffering from a disease, disorder, or condition.

[0009] In certain embodiments, a disease, a disorder, or a condition is associated with arrythmias. Arrythmias can be associated with a cardiovascular disease, a cardiovascular disorder, or a cardiovascular condition. Arrythmias can be associated with a neurological disease, a neurological disorder, or a neurological condition. Arrythmias can be associated with a neurological disease, a neurological disorder, or a neurological condition and a cardiovascular disease, a cardiovascular disorder, or a cardiovascular condition. In certain embodiments, a disease, a disorder, or a condition can be associated with atrial fibrillation. In certain embodiments, arrythmias can be associated with aberrant calcium signaling. In some embodiments, a disease, a disorder, or a condition can be associated with arrythmias and atrial fibrillation.

[0010] The present disclosure also provides for recombinant adeno-associated virus (rAAV) genomes including a polynucleotide encoding an FGF21 pathway activating agent. The present disclosure also provides for rAAV particles including a polynucleotide encoding an FGF21 pathway activating agent and a capsid including capsid polypeptides. The present disclosure also provides for compositions comprising rAAV genomes described herein and / or rAAV particlesdescribed herein, as well as methods for making and using said rAAV genomes and / or rAAV particles. In certain embodiments, rAAV particles described herein can be useful for delivery of gene products that result in expression of an FGF21 pathway activating agent in a subject. In certain embodiments, an FGF21 pathway activating agent can be involved in the development, function, and / or maintenance of diseases, disorders, or conditions (e.g., diseases, disorders, or conditions associated with arrhythmias, e.g., diseases, disorders, or conditions associated with atrial fibrillation). In certain embodiments, an FGF21 pathway activating agent is or comprises an FGF21 polypeptide or variant thereof. In certain embodiments, an FGF21 pathway activating agent is or comprises an FGF21 fusion polypeptide or variant thereof. In certain embodiments, an FGF21 pathway activating agent is or comprises an antibody or antigen binding fragment.

[0011] In certain embodiments, administration of a polynucleotide described herein, a rAAV genome described herein, a rAAV particle described herein, or a composition described herein may minimize a risk of immunogenicity in a subject as compared to methods that employ recombinant FGF21 therapeutics. In certain embodiments, administration of a polynucleotide described herein, a rAAV genome described herein, a rAAV particle described herein, or a composition described herein may minimize a risk of toxicity in a subject as compared to methods that employ recombinant FGF21 therapeutics.

[0012] Among other things, the present disclosure provides a gene product. In certain embodiments, a gene product is or includes a DNA agent or an RNA agent (e.g., a nucleotide sequence encoding FGF21, e.g., an inhibitory nucleic acid that modulates the FGF21 signaling pathway). In certain embodiments, a gene product encodes an FGF21 pathway activating agent.

[0013] In certain embodiments, the present disclosure provides a polynucleotide including a coding sequence. In certain embodiments, a coding sequence encodes a polypeptide. In certain embodiments, a polypeptide includes an FGF21 pathway activating agent. In certain embodiments, an FGF21 pathway activating agent can be encoded by an FGF21 gene or a characteristic portion thereof (e.g., an FGF21 polypeptide or variant thereof). In certain embodiments, an FGF21 pathway activating agent can be an FGF21 polypeptide or variant thereof. In certain embodiments, an FGF21 pathway activating agent can be an FGF21 fusion polypeptide or variant thereof. In certain embodiments, an FGF21 pathway activating agent can be an antibody or antigen binding fragment. In certain embodiments, an antibody or antigen binding fragment can activate the FGF21pathway. In certain embodiments, an antibody or antigen binding fragment can modulate the FGF21 pathway.

[0014] In certain embodiments, an FGF21 pathway activating agent includes an FGF21 polypeptide or variant thereof. In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more amino acid substitutions relative to a reference polypeptide. In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

[0015] In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more amino acid deletions relative to a reference polypeptide. In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more amino acid deletions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

[0016] In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more amino acid insertions relative to a reference polypeptide. In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more amino acid insertions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

[0017] In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more structural modifications. In certain embodiments, one or more structural modifications include methylation of one or more amino acid residues. In certain embodiments, one or more structural modifications comprise amidation of one or more amino acid residues. In certain embodiments, one or more structural modifications comprise acetylation of one or more amino acid residues. In certain embodiments, one or more structural modifications comprise pegylation of one or more amino acid residues. In certain embodiments, one or more structural modifications comprise glycosylation of one or more amino acid residues. In certain embodiments, one or more structural modifications comprise phosphorylation of one or more amino acid residues. In certain embodiments, one or more structural modifications comprise any combination(s) of methylation, amidation, acetylation, pegylation, glycosylation, and phosphorylation of one or more amino acid residues.

[0018] In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more substitutions that modulate a function of a FGF21 polypeptide or variant thereof.

[0019] In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more amino acid deletions that modulate a function of an FGF21 polypeptide or variant thereof.

[0020] In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more amino acid insertions relative to a reference polypeptide that modulate a function of an FGF21 polypeptide or variant thereof. In certain embodiments, one or more amino acid insertions include one or more tags (e.g., for increased efficacy, e.g., for activation of one or more pathways, e.g., for simultaneous activation of one or more pathways, e.g., for activation of an FGF21 pathway, e.g., for activation of a GLP1 pathway, e.g., for simultaneous activation of an FGF21 pathway and GLP1 pathway). In certain embodiments, one or more tags can increase efficacy. In certain embodiments, one or more tags can activate one or more pathways. In certain embodiments, one or more tags can simultaneously activate one or more pathways. In certain embodiments, one or more tags can activate an FGF21 pathway. In certain embodiments, one or more tags can activate a GLP1 pathway. In certain embodiments, one or more tags can simultaneously activate an FGF21 pathway and GLP1 pathway.

[0021] In certain embodiments, an FGF21 polypeptide or variant thereof includes one or more structural modifications that modulate a function of an FGF21 polypeptide or variant thereof.

[0022] In certain embodiments, modulation of a function includes increasing half-life of a FGF21 polypeptide or variant thereof. In certain embodiments, modulation of a function includes improving physicochemical properties of an FGF21 polypeptide or variant thereof. In certain embodiments, modulation of a function includes reducing aggregation of an FGF21 polypeptide or variant thereof. In certain embodiments, modulation of a function includes reducing proteolysis of an FGF21 polypeptide or variant thereof. In certain embodiments, modulation of a function includes increasing binding of an FGF21 polypeptide or variant thereof to a β-Klotho and / or FGF receptor as compared to a reference (e.g., a reference polypeptide).

[0023] In certain embodiments, a FGF21 polypeptide or variant thereof includes an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 27, 43, 45, or 47.

[0024] In certain embodiments, an FGF21 fusion polypeptide or variant thereof includes a FGF21 polypeptide or variant thereof. In certain embodiments, a FGF21 fusion polypeptide or variant thereof includes an agent associated with an FGF21 polypeptide or variant thereof. In certain embodiments, an agent extends a half-life of the FGF21 polypeptide or variant thereof to a longer length of time relative to a half-life of a reference polypeptide that does not include theagent. In certain embodiments, a half-life is a serum half-life. In certain embodiments, an agent includes an antibody or a fragment thereof. In certain embodiments, an antibody or a fragment thereof includes an IgG constant domain. In certain embodiments, an IgG constant domain includes IgG1. In certain embodiments, an agent includes albumin or a fragment thereof.

[0025] In certain embodiments, an FGF21 pathway activating agent includes an antibody or antigen binding fragment. In certain embodiments, an antibody or antigen binding fragment activates an FGF receptor. In certain embodiments, an antibody or antigen binding fragment activates β-Klotho. In certain embodiments, an antibody or antigen binding fragment activates a β-Klotho and FGF receptor complex.

[0026] In certain embodiments, a polynucleotide includes one or more transcriptional regulatory elements (TREs). In certain embodiments, one or more TREs are operably linked to a coding sequence. In certain embodiments, one or more TREs include one or more promoters. In certain embodiments, one or more TREs include one or more enhancers. In certain embodiments, one or more TREs include one or more introns. In certain embodiments, one or more TREs include or are one or more enhancer elements. In certain embodiments, one or more TREs include or are one or more promoters. In certain embodiments, one or more TREs include or are one or more introns. In certain embodiments, one or more TREs include any combination(s) of (i) one or more enhancer elements, (ii) one or more promoters, and (iii) one or more introns.

[0027] In certain embodiments, one or more TREs are or include one or more constitutively active TREs. In certain embodiments, one or more TREs are or include one or more inducible TREs. In certain embodiments, one or more TREs are or include one or more ubiquitous TREs. In certain embodiments, one or more TREs are or include one or more tissue-specific TREs. In certain embodiments, one or more TREs are or include any combination(s) of one or more constitutively active TREs, one or more inducible TREs, one or more ubiquitous TREs, and one or more tissue-specific TREs. In certain embodiments, one or more TREs are or include one or more tissue-specific TREs. In certain embodiments, one or more tissue-specific TREs are or include one or more liver-specific TREs.

[0028] In certain embodiments, one or more TREs are or include one or more enhancer elements. In certain embodiments, one or more enhancer elements include one or more ApoE enhancer elements. In certain embodiments, one or more enhancer elements include two ApoE enhancer elements. In certain embodiments, one or more enhancer elements include three ApoEenhancer elements. In certain embodiments, two or more ApoE enhancer elements each include a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 28, 40, or 41. In certain embodiments, three or more ApoE enhancer elements each include a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 28, 40, or 41. In certain embodiments, two or more ApoE enhancer elements are not the same. In certain embodiments, two or more ApoE enhancer elements are the same. In certain embodiments, three or more ApoE enhancer elements are not the same. In certain embodiments, three or more ApoE enhancer elements are the same.

[0029] In certain embodiments, one or more TREs are or include one or more promoters. In certain embodiments, one or more promoters include a human alpha 1-antitrypsin (hAAT) promoter. In certain embodiments, a hAAT promoter includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4.

[0030] In certain embodiments, one or more TREs are or include an intron element. In certain embodiments, an intron element includes a β-globin intron. In certain embodiments, an intron element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0031] In certain embodiments, one or more TREs include a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 29, 30, or 31.

[0032] In certain embodiments, one or more TREs include two or more enhancer elements. In certain embodiments, one or more TREs include three or more enhancer elements. In certain embodiments, one or more TREs include a promoter. In certain embodiments, one or more TREsinclude an intron element. In certain embodiments, one or more TREs include (i) two or more enhancer elements, (ii) a promoter; (iii) an intron element; or (iv) any combination thereof. In certain embodiments, one or more TREs include (i) three or more enhancer elements, (ii) a promoter, (iii) an intron element, and / or (iv) any combination thereof.

[0033] In certain embodiments, a polynucleotide includes two or more enhancer elements including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3 , 28 , 40 , or 41. In certain embodiments, the enhancer elements are not the same. In certain embodiments, the enhancer elements are the same In certain embodiments, a polynucleotide includes a promoter including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In certain embodiments, a polynucleotide includes an intron element comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0034] In certain embodiments, a polynucleotide includes (i) two or more enhancer elements comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 28, 40, or 41; (ii) a promoter comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; and (iii) an intron element comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5, and / or (iv) any combination thereof. In certain embodiments, the two or more enhancer elements are not the same. In certain embodiments, the two or more enhancer elements are not same.

[0035] In certain embodiments, a polynucleotide includes one or more post-transcriptional regulatory elements (PREs). In certain embodiments, one or more PREs are operably linked to a coding sequence. In certain embodiments, one or more PREs include a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE). In certain embodiments, a WPRE comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0036] In certain embodiments, one or more PREs include a polyadenylation (pA) sequence. In certain embodiments, a polyadenylation sequence includes a simian virus 40 polyadenylation (SV40pA) sequence or variant thereof. In certain embodiments, a polyadenylation sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 8, 32, or 33.

[0037] In certain embodiments, one or more PREs include a PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 34 or 35.

[0038] In certain embodiments, a polynucleotide includes one or more TREs including (a) one or more ApoE enhancer elements, (b) a human alpha 1-antitrypsin (hAAT) promoter, and (c) an intron element. In certain embodiments, a polynucleotide includes a coding sequence encoding an FGF21 pathway activating agent. In certain embodiments, a polynucleotide includes one or more PREs including (a) a WPRE, and (b) a polyadenylation sequence. Among other things, a polynucleotide includes, from 5’ to 3’: (a) one or more TREs including from 5’ to 3’: (i) one or more ApoE enhancer elements, (ii) a human alpha 1-antitrypsin (hAAT) promoter, and (iii) an intron element; (b) a coding sequence encoding an FGF21 pathway activating agent; and (c) one or more PREs comprising: (i) a WPRE and (i) a polyadenylation sequence.

[0039] In certain embodiments, a polynucleotide includes a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence. In certain embodiments, a polynucleotide includes and a 3’ inverted terminal repeat (3’ ITR) nucleotide sequence. In certain embodiments, a polynucleotide includes a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence and a 3’ inverted terminal repeat (3’ITR) nucleotide sequence. In certain embodiments, a 5’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In certain embodiments, a 3’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0040] Among other things, the present disclosure provides a vector comprising any of the polynucleotides described herein.

[0041] In certain embodiments, a vector is a plasmid. In certain embodiments, a vector is a viral vector. In certain embodiments, a vector is a DNA minimal vector. In certain embodiments, a vector is an expression vector.

[0042] In certain embodiments, a vector is a viral vector including an adenoviral vector, an adeno-associated virus (AAV) vector, or a lentiviral vector. In certain embodiments, a vector is an AAV vector.

[0043] Among other things, the present disclosure provides a recombinant adeno- associated virus (rAAV) genome including any of the polynucleotides described herein.

[0044] In certain embodiments, a rAAV genome includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 18, 20, 22, 24, and 26.

[0045] Among other things, the present disclosure provides a rAAV particle including a capsid comprising an AAV capsid polypeptide and any of the rAAV genomes described herein. In certain embodiments, an AAV capsid polypeptide is derived from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid polypeptide, or an engineered variant thereof. In certain embodiments, an AAV capsid polypeptide includes a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid polypeptide, or an engineered variant thereof. In certain embodiments, an AAV capsid polypeptide is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10,AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid polypeptide, or an engineered variant thereof. In certain embodiments, the AAV capsid polypeptide includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, 38, and / or 39.

[0046] Among other things, the present disclosure provides compositions comprising a fibroblast growth factor 21 (FGF21) pathway activating agent. In certain embodiments, a FGF21 pathway activating agent comprises: (i) an FGF21 polypeptide or variant thereof, (ii) a peptide (iii) an FGF21 fusion polypeptide or variant thereof, or (iv) an antibody or antigen binding fragment. In certain embodiments, a FGF21 pathway activating agent comprises any combination of: (i) an FGF21 polypeptide or variant thereof, (ii) a peptide (iii) an FGF21 fusion polypeptide or variant thereof, and (iv) an antibody or antigen binding fragment.

[0047] In certain embodiments, a composition described herein is or includes a pharmaceutical composition.

[0048] Among other things, the present disclosure provides a packaging system for preparation of an rAAV particle described herein. In certain embodiments, a packaging system includes a first nucleotide sequence encoding one or more AAV Rep polypeptides. In certain embodiments, a packaging system includes a second nucleotide sequence encoding an AAV capsid polypeptide. In certain embodiments, a packaging system includes a third nucleotide sequence including any of the rAAV genomes described herein. In certain embodiments, a packaging system includes (a) a first nucleotide sequence encoding one or more AAV Rep polypeptides, (b) a second nucleotide sequence encoding an AAV capsid polypeptide, and (c) a third nucleotide sequence comprising any of the rAAV genomes described herein.

[0049] In certain embodiments, a packaging system comprises (a) a first vector including a first nucleotide sequence a second nucleotide sequence, and (b) a second vector including a third nucleotide sequence. In certain embodiments, a packaging system comprises a fourth nucleotide sequence including one or more helper virus genes. In certain embodiments, a fourth nucleotide sequence is included within a third vector. In certain embodiments, a fourth nucleotide sequence comprises one or more genes from a virus including adenovirus, herpesvirus, vaccinia virus, or cytomegalovirus (CMV).

[0050] In certain embodiments, a first vector is a plasmid. In certain embodiments, a second vector is a plasmid. In certain embodiments, a third vector is a plasmid.

[0051] Among other things, the present disclosure provides methods including any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein. In certain embodiments, a method includes recombinant preparation of a rAAV particle. In certain embodiments, a method includes introducing any of the packaging systems described herein into a cell. In certain embodiments, any of the packaging systems described herein are introduced into a cell under conditions whereby an rAAV particle is produced.

[0052] In certain embodiments, a method includes introducing into a cell any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein.

[0053] In certain embodiments, a method includes expressing an FGF21 coding sequence in a cell. In certain embodiments, said method includes introducing into the cell any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein.

[0054] In certain embodiments, the present disclosure provides any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein for use in medicine. In certain embodiments, the present disclosure provides any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein for use as therapy. In certain embodiments, the present disclosure provides any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein, for use as a gene therapy. In certain embodiments, the present disclosure provides any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein for use as a medicament.

[0055] In certain embodiments, a method includes expressing an FGF21 coding sequence in a subject. In certain embodiments, a method includes administering to a subject a therapeutically effective amount of any of the polynucleotides, vectors, rAAV genomes, rAAV particles, pharmaceutical compositions, or compositions described herein.

[0056] In certain embodiments, a method includes measuring calcium signaling in a subject. In certain embodiments, a method includes administering to a subject a therapeuticallyeffective amount of any of the polynucleotides, vectors, rAAV genomes, rAAV particles, pharmaceutical compositions, or compositions described herein.

[0057] In certain embodiments, a method includes treating a disease or disorder in a subject in need thereof. In certain embodiments, a method includes administering to a subject a therapeutically effective amount of any of the polynucleotides, vectors, rAAV genomes, rAAV particles, pharmaceutical compositions, or compositions described herein. In certain embodiments, a disease or disorder is or includes an arrhythmogenic cardiomyopathy (ACM), a mitral valve disease (MVD), and a familial partial lipodystrophy (FPL). In certain embodiments, ACM comprises arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), or biventricular arrhythmogenic cardiomyopathy. In certain embodiments, the MVD comprises myxomatous mitral valve disease, mitral valve stenosis, mitral valve prolapse, or mitral valve regurgitation. In certain embodiments, the FPL comprises type 1 FPL, type 2 FPL, type 3 FPL, type 4 FPL, type 5 FPL, or type 6 FPL.

[0058] In certain embodiments, a subject is a human, a non-human primate, a canine, a feline, an equine, a bovine, a swine, an avian, or a rodent. In certain embodiments, a subject is human.

[0059] In certain embodiments, a method includes activating an FGF21 pathway. In certain embodiments, a method includes administering to a subject a therapeutically effective amount of any of the polynucleotides, vectors, rAAV genomes, rAAV particles, pharmaceutical compositions, or compositions described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 is a graph showing the level of FGF21 expression in PKP2cKO mice administered AAV8-mFGF21 at a dose of 1.5E13 vg / kg. Wild type mice (WT) and PKP2cKO mice administered vehicle (WT) were used as controls. Means with standard error of the mean are presented.

[0061] FIGs.2A-2C are graphs showing cardiac structure and function of PKP2cKO mice administered AAV8-mFGF21, as measured by left ventricle ejection fraction (LVEF; FIG. 2A), fractional shortening (FS; FIG. 2B) and right ventricle area (RV area; FIG. 2C). Wild type mice (WT) and PKP2cKO mice administered vehicle were used as controls. Means with standard deviation are presented. * Indicates a p value of <0.05 and ** indicates a p value of <0.01 by one- way ANOVA and Dunn’s multiple comparisons test.

[0062] FIG. 3 is a graph showing arrhythmia burden of PKP2cKO mice administered AAV8-mFGF21, measured as the percent of premature ventricular contractions (PVCs) observed in 30 minutes following an isoproterenol challenge. The numbers refer to the number of PVCs observed in 30 minutes following an isoproterenol challenge.

[0063] FIGs. 4A-4B are graphs showing the percentage of collagen in cardiac tissues of PKP2cKO mice administered AAV8-mFGF21, indicative of left ventricle (LV) fibrosis (FIG. 4A), and right ventricle (RV) fibrosis (FIG. 4B). Wild type mice (WT) and PKP2cKO mice administered vehicle (WT) were used as controls. Means with standard deviation are presented.

[0064] FIGs. 5A-5B are graphs showing arrhythmia burden of PKP2cKO mice administered AAV8-mFGF21, measured as the percent of premature ventricular contractions (ectopic beats; FIG. 5A) and percent of premature ventricular contractions (PVCs) measured per 100 heart beats.

[0065] FIGs. 6A-6D are graphs showing calcium transients in cardiomyocytes isolated from control and PKP2cKO mice administered vehicle, and PKP2cKO mice administered AAV8- mFGF21. Depicted are quantifications of decay time constant (FIG. 6A), time to peak (FIG.6B), amplitude (FIG.6C) and ratio of cells with delay calcium transients (DCT; FIG.6D). Cells were paced at 1 Hz. Wild type mice (WT) and PKP2cKO mice administered vehicle (WT) were used as controls. Means with standard deviation are presented.

[0066] FIGs. 7A-7C are graphs of showing in vivo evaluation of left ventricle ejection fraction (LVEF; FIG. 7A), right ventricular area (RV Area; FIG. 7B), and premature ventricular contractions (ectopic beats; FIG. 7C) in WT and PKP2cKO mice administered vehicle, and PKP2cKO mice treated with 1.5 × 1013vg / kg of rAAV8 vectors expressing FGF21, sTGFβR2 or FGF21 + sTGFβR2 (mRJB-01) (n=8 per group). *=p <0.05, **=p <0.01, ***=p <0.001, ****=p <0.0001, ns=not significant.

[0067] FIGs. 8A-8D are graphs showing repetitive nerve stimulation (RNS) of animals administered with (i) control and theophylline (theo) or (ii) FGF21. Testing was conducted at a rate of 3Hz and resulting compound muscle action potential (CMAP) amplitudes are depicted. DETAILED DESCRIPTION

[0068] The present disclosure provides certain products (e.g., gene products, e.g., polypeptide products) that activate and / or modulate an FGF21 signaling pathway. In certain embodiments, such products may exhibit advantageous properties in treating a subject sufferingfrom a disease, disorder, or condition (e.g., a disease, disorder, or condition associated with arrythmias, e.g., a disease, disorder, or condition associated with atrial fibrillation, e.g., a cardiovascular disease, disorder, or condition, e.g., a cardiovascular disease, disorder, or condition associated with arrhythmias, e.g., a neurological disease, disorder, or condition associated with arrythmias, e.g., a disease, disorder, or condition associated with aberrant calcium signaling). Also provided are vectors, recombinant adeno-associated virus (rAAV) genomes comprising said products, and rAAV particles, as well as associated methods for making and using the products and rAAV particles. I. Definitions

[0069] Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

[0070] The articles “a” and “an,” as used herein, should be understood to include the plural referents unless clearly indicated to the contrary. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. In certain embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In certain embodiments, more than one, or all group members are present in, employed in, or otherwise relevant to a given product or process. It is to be understood that the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists (e.g., in Markush group or similar format), it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where embodiments or aspects are referred to as “comprising” particular elements, features, etc., certain embodiments or aspects “consist,” or “consist essentially of,” such elements, features, etc. Forpurposes of simplicity, those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0071] Throughout the specification, whenever a polynucleotide, polyribonucleotide or polypeptide is represented by a sequence of letters (e.g., A, C, G, and T which denote adenosine, cytidine, guanosine, thymidine, and uracil respectively in the case of a polynucleotide or A, C, G, and U which denote adenosine, cytidine, guanosine, and uracil respectively in the case of a polyribonucleotide), such polynucleotides, polyribonucleotide or polypeptides are presented in 5’ to 3’ or N-terminus to C-terminus order, from left to right.

[0072] AAV: As used herein, the term “AAV” is a standard abbreviation for adeno- associated virus.

[0073] Administration: As used herein, the term “administration” typically refers to administration of a composition to a subject or system to achieve delivery of an agent to a subject or system. In certain embodiments, an agent is, or is included in, a composition; In certain embodiments, an agent is generated through metabolism of a composition or one or more components thereof. Those of ordinary skill in the art, reading the present disclosure, will appreciate, for example, that a variety of routes are available for administration of compositions; for example, some compositions may be administered by one or more routes such as ocular, oral, parenteral, topical, etc. For example, in certain embodiments, administration may be systematic or local. In certain embodiments, a systematic administration can be intravenous. In certain embodiments, administration can be local. In certain embodiments, administration may involve only a single dose. In certain embodiments, administration may involve application of a fixed number of doses. In certain embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In certain embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0074] Agent: As used herein, the term “agent,” may refer to a physical entity. In certain embodiments, an agent may be characterized by a particular feature and / or effect. For example, as used herein, the term “therapeutic agent” refers to a physical entity has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In certain embodiments, an agent maybe a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, peptide, nucleic acid, saccharide, lipid, metal, or any combination or complex thereof.

[0075] Amelioration: As used herein, the term “amelioration” refers to prevention, reduction or palliation of a state, or improvement of a state of a subject. Amelioration may include, but does not require, complete recovery or complete prevention of a disease, disorder or condition.

[0076] Associated: As used herein, the term “associated” describes two events or entities as “associated” with one another, if the presence, level and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of and / or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In certain embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In certain embodiments, two or more entities that are physically associated with one another are covalently linked to one another; In certain embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0077] Characteristic portion: As used herein, the term “characteristic portion,” in the broadest sense, refers to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance. In certain embodiments, a characteristic portion of a substance is a portion that is found in a given substance and in related substances that share a particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in certain embodiments, a “characteristic portion” of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. In certain embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a substance (e.g., of a protein, a polypeptide, an antibody, etc.) is one that, in addition to a sequence and / or structural identity specified above, shares at least onefunctional characteristic with the relevant intact substance. In certain embodiments, a characteristic portion may be biologically active.

[0078] Characteristic sequence: As used herein, the term “characteristic sequence” is a sequence that is found in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.

[0079] Characteristic sequence element: As used herein, the phrase “characteristic sequence element” refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In certain embodiments, presence of a characteristic sequence element correlates with presence or level of a particular activity or property of a polymer. In certain embodiments, presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In certain embodiments, a characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., contiguously linked monomers). In certain embodiments, a characteristic sequence element includes at least first and second stretches of contiguous monomers spaced apart by one or more spacer regions whose length may or may not vary across polymers that share a sequence element.

[0080] Coding Sequence: As used herein, the term “coding sequence” refers to a non- AAV nucleic acid sequence that encodes a polypeptide (e.g., a therapeutic polypeptide) or non- coding RNA (e.g., a miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, or RNA aptamer).

[0081] Effective Amount: As used herein, the term “effective amount” in the context of the administration of a polynucleotide, vector, composition, or rAAV to a subject refers to the amount of the polynucleotide, vector, composition, or rAAV that achieves a desired prophylactic or therapeutic effect.

[0082] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode apolypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In certain embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In certain embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a non- coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0083] Expression Vector: As used herein, an “expression vector” refers to a vector comprising transcriptional regulatory elements operably linked to a gene of interest (e.g., a polynucleotide described herein) that facilitate the expression of the gene of interest in a cell and / or a cell free expression system.

[0084] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In certain embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0085] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in certain embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be “increased” relative to that obtained with a comparable reference composition. Alternatively or additionally, In certain embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In certain embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance. In certain embodiments, the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In certain embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In certain embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0086] Inhibitory nucleic acid: As used herein, the term “inhibitory nucleic acid” refers to a nucleic acid sequence that hybridizes specifically to a gene that modulates (e.g., directly or indirectly) activity of the FGF21 pathway. In certain embodiments, an inhibitory nucleic acid activates (e.g., directly or indirectly) activity of the FGF21 pathway. In certain embodiments, an inhibitory nucleic acid reduces (e.g., directly or indirectly) activity of the FGF21 pathway. in certain embodiments, an inhibitory nucleic acid inhibits expression and / or activity of a gene that results in modulated expression of a polypeptide that modulates activity of the FGF21 pathway. Incertain embodiments, modulation of the activity of the FGF21 pathway results in increased expression of an FGF21 polypeptide. In certain embodiments, an inhibitory nucleic acid is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA ( or “miRNA”), an antisense oligonucleotide, a guide RNA (gRNA), or a ribozyme. In certain embodiments, an inhibitory nucleic acid is between about 10 nucleotides to about 30 nucleotides in length (e.g., about 10 nucleotides to about 28 nucleotides, about 10 nucleotides to about 26 nucleotides, about 10 nucleotides to about 24 nucleotides, about 10 nucleotides to about 22 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 18 nucleotides, about 10 nucleotides to about 16 nucleotides, about 10 nucleotides to about 14 nucleotides, about 10 nucleotides to about 12 nucleotides, about 12 nucleotides to about 30 nucleotides, about 12 nucleotides to about 28 nucleotides, about 12 nucleotides to about 26 nucleotides, about 12 nucleotides to about 24 nucleotides, about 12 nucleotides to about 22 nucleotides, about 12 nucleotides to about 20 nucleotides, about 12 nucleotides to about 18 nucleotides, about 12 nucleotides to about 16 nucleotides, about 12 nucleotides to about 14 nucleotides, about 16 nucleotides to about 30 nucleotides, about 16 nucleotides to about 28 nucleotides, about 16 nucleotides to about 26 nucleotides, about 16 nucleotides to about 24 nucleotides, about 16 nucleotides to about 22 nucleotides, about 16 nucleotides to about 20 nucleotides, about 16 nucleotides to about 18 nucleotides, about 18 nucleotides to about 30 nucleotides, about 18 nucleotides to about 28 nucleotides, about 18 nucleotides to about 26 nucleotides, about 18 nucleotides to about 24 nucleotides, about 18 nucleotides to about 22 nucleotides, about 18 nucleotides to about 20 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 28 nucleotides, about 20 nucleotides to about 26 nucleotides, about 20 nucleotides to about 24 nucleotides, about 20 nucleotides to about 22 nucleotides, about 22 nucleotides to about 30 nucleotides, about 22 nucleotides to about 28 nucleotides, about 22 nucleotides to about 26 nucleotides, about 22 nucleotides to about 24 nucleotides, about 24 nucleotides to about 30 nucleotides, about 24 nucleotides to about 28 nucleotides, about 24 nucleotides to about 26 nucleotides, about 26 nucleotides to about 30 nucleotides, about 26 nucleotides to about 28 nucleotides, about 28 nucleotides to about 30 nucleotides, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides).

[0087] Isolated polynucleotide: As used herein, an “isolated polynucleotide” refers to a polynucleotide that has been separated from one or more nucleic acid molecules present in the natural source of the polynucleotide.

[0088] Linker: As used herein, the term “linker” refers to a portion of a polypeptide that connects different regions, portions, or antigens to one another.

[0089] Nucleic acid or polynucleotide: As used herein, in its broadest sense, nucleic acid or polynucleotide refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In certain embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in certain embodiments, "nucleic acid" or "polynucleotide" refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in certain embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In certain embodiments, a "nucleic acid" is or comprises RNA; in certain embodiments, a "nucleic acid" or "polynucleotide" is or comprises DNA. In certain embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In certain embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In certain embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in certain embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively, or additionally, in certain embodiments, a nucleic acid has one or more phosphorothioate and / or 5’-N- phosphoramidite linkages rather than phosphodiester bonds. In certain embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In certain embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 - methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In certain embodiments, a nucleic acid comprisesone or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In certain embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein or polypeptide. In certain embodiments, a nucleic acid includes one or more introns. In certain embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In certain embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In certain embodiments, a nucleic acid is partly or wholly single stranded; in certain embodiments, a nucleic acid is partly or wholly double stranded. In certain embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In certain embodiments, a nucleic acid has enzymatic activity.

[0090] Nucleotide sequence encoding an AAV capsid polypeptide: As used herein, the term “nucleotide sequence encoding an AAV capsid polypeptide” refers to a nucleic acid sequence that encodes a capsid polypeptide. For AAV, the capsid polypeptide may be VP1, VP2, or VP3. VP1, VP2, and / or VP3 capsid polypeptides assemble into a capsid that surrounds the rAAV genome.

[0091] Nucleotide sequence encoding one or more AAV Rep polypeptides: As used herein, the term “nucleotide sequence encoding one or more AAV Rep polypeptides” refers to one or more nucleic acid sequences that encode the non-structural polypeptides (e.g., rep78, rep68, rep52, and rep40) required for the replication and production of an AAV.

[0092] Operably Linked: As used herein, the term “operably linked” is used to describe the connection between a TRE and / or a PRE and a polynucleotide sequence (e.g., a coding sequence described herein) to be transcribed. Typically, gene expression is placed under the control of a TRE comprising one or more promoter and / or enhancer elements and / or a PRE comprising, e.g., a transcription termination sequence. The coding sequence is “operably linked” to the TRE if the transcription of the coding sequence is controlled or influenced by the TRE. The coding sequence is “operably linked” to the PRE if the RNA molecule encoded by the codingsequence is controlled or influenced by the PRE. The elements of the TRE and / or PRE may be in any orientation and / or at any distance from the coding sequence, as long as the desired transcriptional and / or post-transcriptional activity is obtained. In an embodiment, the TRE is upstream from the coding sequence. In an embodiment, the PRE is downstream from the coding sequence.

[0093] Percentage Identity: As used herein, the “percentage identity” between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between the pair of aligned sequences by 100, and dividing by the length of the aligned region, including internal gaps. Identity scoring only counts perfect matches, and does not consider the degree of similarity of amino acids to one another. Note that only internal gaps are included in the length, not gaps at the sequence ends.

[0094] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In certain embodiments, a polypeptide has an amino acid sequence that occurs in nature. In certain embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In certain embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In certain embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In certain embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In certain embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In certain embodiments, a polypeptide may comprise only D-amino acids. In certain embodiments, a polypeptide may comprise only L-amino acids. In certain embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In certain embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In certain embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In certain embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In certain embodiments, a polypeptide is linear. In certain embodiments, a polypeptide may be or comprise a stapled polypeptide. In certain embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activityor structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides exemplary polypeptides within the class whose amino acid sequences and / or functions are known; In certain embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In certain embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (In certain embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; In certain embodiments with all polypeptides within the class). For example, In certain embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and / or includes at least one region (e.g., a conserved region that may In certain embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; In certain embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In certain embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0095] Post-transcriptional regulatory element: As used herein, the term “post- transcriptional regulatory element” or “PRE” refers to a cis-acting nucleotide sequence, for example, a DNA sequence, that regulates (e.g., controls, increases, or reduces) the production, stability, processing, or levels of an RNA molecule (e.g., an RNA molecule that is being transcribed or has been transcribed from a coding sequence). A post-transcriptional regulatory element may be, e.g., any sequence that effectively terminates transcription. It is an insight of the present disclosure that post-transcriptional regulatory element sequences can be isolated from any genomic loci that are associated with expression of genes in the cell in which transcription of the coding sequence is desired.

[0096] rAAV genome: As used herein, the term “rAAV genome” refers to a nucleic acid molecule (e.g., DNA and / or RNA) comprising the genome sequence of an rAAV. It is an insight of the present disclosure that where an rAAV genome comprises a coding sequence (e.g., apolypeptide encoding a therapeutic polypeptide operably linked to a transcriptional regulatory element (i.e., payload)), the rAAV genome can be in the sense or antisense orientation relative to the direction of transcription of the coding sequence.

[0097] Recombinant adeno-associated virus: As used herein interchangeably, the terms “recombinant adeno-associated virus” or “rAAV” refer to an AAV comprising a genome lacking functional rep and cap genes.

[0098] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in certain embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In certain embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In certain embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0099] Treat, Treating, or Treatment: As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration of a polynucleotide to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0100] Variant: As used herein, the term “variant” refers to a molecule that shows significant structural (e.g., primary or secondary) identity with a reference molecule but differs structurally from the reference molecule. For example, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide ornucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In certain embodiments, a variant polypeptide comprises a characteristic portion of a reference polypeptide.

[0101] Vector: As used herein, a “vector” refers to a nucleic acid molecule that is a vehicle for introducing a nucleic acid molecule (e.g., a polynucleotide described herein) into a cell. II. FGF21 Signaling Pathway

[0102] Fibroblast growth factor 21 (FGF21) belongs to the fibroblast growth factor (FGF) family (see, e.g., Tan H., et al., Int J Biol Sci.2023 Jan 1;19(1):66-88, which is hereby incorporated by reference herein in its entirety). The human FGF family includes 22 members, which are divided into seven subfamilies. Subfamily 19 comprises three endocrine factors: FGF21, FGF19, and FGF23. Conventionally, FGFs promote fibroblast proliferation and growth, while FGF21, FGF19, and FGF23 primarily regulate metabolism (see, e.g., Dolegowska K., et al., Journal Physiology Biochem. 2019 Jun;75(2):229-240, which is hereby incorporated by reference herein in its entirety).

[0103] Circulating FGF21 binds to fibroblast growth factor receptor 1 (FGFR1; also referred to as e.g., basic fibroblast growth factor receptor 1 [BFGFR], fms-related tyrosine kinase- 2 / Pfeiffer syndrome, and CD331) via the C-terminus with β-Klotho serving as an essential co- receptor binding at the N-terminus (see, e.g., Suzuki M. et al., Mol Endocrinol. 2008 Apr;22(4):1006-14, which is hereby incorporated by reference herein in its entirety). The binding of FGF21 initiates the formation of the FGFR1 / β-Klotho complex through heterodimerization, resulting in activation of the receptor tyrosine kinase activity. The activated tyrosine kinase phosphorylates the adaptor protein FRS2α, triggering the activation of cytoplasmic signal transduction pathways, several which can impact e.g., calcium and sodium handling, inflammation and fibrosis, as well as mitochondrial energetics (see, e.g., Kharitonenkov A. and Shanafelt AB., BioDrugs. 2008;22(1):37-44.; and Suzuki M. et al., Mol Endocrinol. 2008 Apr;22(4):1006-14, which are hereby incorporated by reference herein in their entirety).

[0104] FGF21 can be expressed after being induced by various stress conditions, such as cold exposure, nutritional stresses, exercise, and some pathologic conditions (see, e.g., Tan H., et al., Int J Biol Sci. 2023 Jan 1;19(1):66-88, which is hereby incorporated by reference herein in its entirety). Tissues expressing FGF21 include liver, brown adipose tissue (BAT), white adipose tissue (WAT), muscle, pancreas, heart, and brain (see, e.g., Fisher F. and Maratos-Flier E., et al., Annu Rev Physiol. 2016:78:223-41, which is hereby incorporated by reference herein in itsentirety). The liver is a primary source of serum FGF21 under physiological conditions, and different FGF21 transcription factors (including peroxisome proliferator-activated receptor [PPAR] α, activating transcription factor 4 [ATF4], carbohydrate response element-binding protein [ChREBP], and CCR4-NOT transcription complex subunit 6-like [CNOT6 L]) can be regulated under different states to promote FGF21 expression (see, e.g., Tan H., et al., Int J Biol Sci.2023 Jan 1;19(1):66-88, which is hereby incorporated by reference herein in its entirety).

[0105] Target tissues of FGF21 include liver, adipose tissues, brain, pancreas, heart, and kidney, and FGF21 can mediate biological functions of target tissues with the assistance of β- Klotho and FGFR (see, e.g., Yan C., et al., PLoS One. 2012;7(3):e33870, which is hereby incorporated by reference herein in its entirety). For example, myocytic FGF21 can act on WAT to induce the browning of WAT, on skeletal muscle to protect against insulin resistance, and on the heart to protect against cardiac hypertrophy (see, e.g., Tan H., et al., Int J Biol Sci. 2023 Jan 1;19(1):66-88, which is hereby incorporated by reference herein in its entirety).

[0106] The present disclosure provides a recognition that certain products (e.g., gene products, e.g., polypeptide products) that activate and / or modulate an FGF21 signaling pathway may exhibit advantageous properties in treating a subject suffering from a disease, disorder, or condition (e.g., a disease, disorder, or condition associated with arrythmias, e.g., a disease, disorder, or condition associated with atrial fibrillation, e.g., a cardiovascular disease, a cardiovascular disorder, or a cardiovascular condition, e.g., a neurological disease, a neurological disorder, or a neurological condition, e.g., a disease, disorder, or condition associated with atrial fibrillation, e.g., a disease, a disorder, or a condition is associated with aberrant calcium signaling). Without wishing to be bound by any particular theory, fine-tuned activation and / or modulation of an FGF21 signaling pathway (e.g., increased expression of FGF21 activity) may reduce and / or ameliorate one or more symptoms in a subject suffering from a disease, disorder, or condition (e.g., a disease, a disorder, or a condition associated with arrythmias, e.g., a disease, a disorder, or a condition associated with atrial fibrillation, e.g., a cardiovascular disease, a cardiovascular disorder, or a cardiovascular condition, e.g., a cardiovascular disease, a cardiovascular disorder, or a cardiovascular condition, e.g., a disease, disorder, or condition associated with atrial fibrillation, e.g., disease, disorder, or condition is associated with aberrant calcium signaling).

[0107] In certain embodiments, the present disclosure provides a gene product comprising an FGF21 pathway activating agent. In certain embodiments, an FGF21 pathway activating agentis encoded by an FGF21 gene or a characteristic portion thereof as described herein. In certain embodiments, an FGF21 pathway activating agent is an FGF21 polypeptide or variant thereof as described herein. In certain embodiments, an FGF21 pathway activating agent is an antibody or antigen binding fragment that activates the FGF21 pathway as described herein. In certain embodiments, an FGF21 pathway activating agent is an FGF21 fusion polypeptide or variant thereof as described herein.

[0108] In certain embodiments, the present disclosure provides a polypeptide product comprising an FGF21 pathway activating agent. In certain embodiments, an FGF21 pathway activating agent can be an FGF21 polypeptide or variant thereof as described herein. In certain embodiments, an FGF21 pathway activating agent can be an antibody or antigen binding fragment that activates the FGF21 pathway as described herein. In certain embodiments, an FGF21 pathway activating agent can be an FGF21 fusion polypeptide or variant thereof as described herein.

[0109] In certain embodiments, an FGF21 pathway activating agent is a peptide or variant thereof as described herein. In certain embodiments, an FGF21 pathway activating agent is a peptide or variant thereof that activates the FGF21 pathway as described herein.

[0110] In certain embodiments, an FGF21 pathway activating agent increases an expression of FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent increases an expression of circulating FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can increase an expression of one or more factors (e.g., transcription factor) involved in regulating (e.g., positive regulation) circulating FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can increase an expression of one or more factors (e.g., transcription factor) involved in positive regulation of circulating FGF21 in a subject. In certain embodiments, positive regulation of circulating FGF21 may include increasing a level of a factor (e.g., transcription factor) directly and / or indirectly involved in increasing expression of FGF21 (e.g., FGF21 transcript and / or FGF21 polypeptide).

[0111] In certain embodiments, an FGF21 pathway activating agent can reduce an expression of one or more factors (e.g., transcription factor) involved in regulating (e.g., negative regulation) FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can reduce an expression of one or more factors (e.g., transcription factor) involved in regulating (e.g., negative regulation) circulating FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can reduce an expression of one or more factors (e.g., transcription factor)involved in negative regulation of circulating FGF21 in a subject. In certain embodiments, negative regulation of circulating FGF21 may include reducing a level of a factor directly and / or indirectly involved in reducing expression of FGF21 (e.g., FGF21 transcript and / or FGF21 polypeptide).

[0112] In certain embodiments, an FGF21 pathway activating agent increases an expression of serum FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can increase an expression of one or more factors (e.g., transcription factor) involved in regulating (e.g., positive regulation) serum FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can increase an expression of one or more factors (e.g., transcription factor) involved in positive regulation of serum FGF21 in a subject. In certain embodiments, positive regulation of serum FGF21 may include increasing a level of a factor (e.g., transcription factor) directly and / or indirectly involved in increasing expression of FGF21 (e.g., FGF21 transcript and / or FGF21 polypeptide).

[0113] In certain embodiments, an FGF21 pathway activating agent can reduce an expression of one or more factors (e.g., transcription factor) involved in regulating (e.g., negative regulation) serum FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can reduce an expression of one or more factors (e.g., transcription factor) involved in negative regulation of serum FGF21 in a subject. In certain embodiments, negative regulation of serum FGF21 may include reducing a level of a factor directly and / or indirectly involved in reducing expression of FGF21 (e.g., FGF21 transcript and / or FGF21 polypeptide).

[0114] In certain embodiments, an FGF21 pathway activating agent increases localized expression (e.g., expression localized to a tissue and / or cell) of FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can increase an expression of one or more factors (e.g., transcription factor) involved in regulating (e.g., positive regulation) localized expression (e.g., expression localized to a tissue and / or cell) of FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can increase an expression of one or more factors (e.g., transcription factor) involved in positive regulation of localized expression (e.g., expression localized to a tissue and / or cell) of FGF21 in a subject. In certain embodiments, positive regulation of localized expression (e.g., expression localized to a tissue and / or cell) of FGF21 may include increasing a level of a factor (e.g., transcription factor) directly and / or indirectly involved in increasing localized expression (e.g., expression localized to a tissue and / or cell) of FGF21 (e.g., FGF21 transcript and / or FGF21 polypeptide).

[0115] In certain embodiments, an FGF21 pathway activating agent can reduce an expression of one or more factors (e.g., transcription factor) involved in regulating (e.g., negative regulation) localized expression (e.g., expression localized to a tissue and / or cell) of FGF21 in a subject. In certain embodiments, an FGF21 pathway activating agent can reduce an expression of one or more factors (e.g., transcription factor) involved in negative regulation of localized expression (e.g., expression localized to a tissue and / or cell) of FGF21 in a subject. In certain embodiments, negative regulation of localized expression (e.g., expression localized to a tissue and / or cell) of FGF21 may include reducing a level of a factor directly and / or indirectly involved in reducing expression of FGF21 (e.g., FGF21 transcript and / or FGF21 polypeptide).

[0116] In certain embodiments, an FGF21 pathway activating agent activates a FGF receptor. In certain embodiments, an FGF21 pathway activating agent activates β-Klotho. In certain embodiments, an FGF21 pathway activating agent activates a β-Klotho and FGF receptor complex. In certain embodiments, an FGF21 pathway activating agent facilitates heterodimerization of a β-Klotho and FGF receptor complex.

[0117] In certain embodiments, an FGF21 pathway activating agent activates FGFR1. In certain embodiments, an FGF21 pathway activating agent activates a β-Klotho and FGFR1 receptor complex. In certain embodiments, an FGF21 pathway activating agent facilitates heterodimerization of a β-Klotho and FGFR1 receptor complex.

[0118] In certain embodiments, the present disclosure provides a composition comprising a certain product (e.g., a gene product, e.g., a polypeptide product) that activates and / or modulates an FGF21 signaling pathway. In certain embodiments, a composition comprises one or more gene products comprising an FGF21 pathway activating agent. In certain embodiments, an FGF21 pathway activating agent is encoded by an FGF21 gene or a characteristic portion thereof.

[0119] In certain embodiments, a composition comprises one or more polypeptide products comprising an FGF21 pathway activating agent as described herein. In certain embodiments, a composition comprises an FGF21 polypeptide or variant thereof as described herein. In certain embodiments, a composition comprises an antigen binding fragment that activates the FGF21 pathway as described herein. In certain embodiments, a composition comprises an FGF21 fusion polypeptide or variant thereof as described herein.

[0120] In certain embodiments, a composition comprises a peptide or variant thereof as described herein. In certain embodiments, a composition comprises a peptide or variant thereof that activates the FGF21 pathway as described herein. III. Polynucleotides, Vectors, and Compositions Coding Sequence

[0121] Among other things, the present disclosure provides polynucleotides or nucleic acid sequences (e.g., isolated polynucleotides, vectors, etc.) comprising a coding sequence encoding an FGF21 pathway activating agent. In certain embodiments, a polynucleotide sequence described herein comprises an FGF21 polypeptide or variant thereof coding sequence. In certain embodiments, a polynucleotide sequence described herein comprises an FGF21 fusion polypeptide or variant thereof coding sequence. In certain embodiments, a polynucleotide sequence described herein comprises an antibody or antigen binding fragment coding sequence.

[0122] In certain embodiments, polynucleotides or nucleic acid sequences (e.g., isolated polynucleotides, vectors, etc.) comprise an expression cassette comprising a coding sequence encoding an FGF21 pathway activating agent. In certain embodiments, a polynucleotide sequence described herein comprises an expression cassette comprising an FGF21 polypeptide or variant thereof coding sequence. In certain embodiments, a polynucleotide sequence described herein comprises an expression cassette comprising an FGF21 fusion polypeptide or variant thereof coding sequence. In certain embodiments, a polynucleotide sequence described herein comprises an expression cassette comprising an antibody or antigen binding fragment coding sequence.

[0123] In certain embodiments, polynucleotides or nucleic acid sequences (e.g., isolated polynucleotides, vectors, etc.) comprise an expression cassette useful for expressing a coding sequence of interest (e.g., a coding sequence encoding fibroblast growth factor 21 [FGF21]) in a cell.

[0124] The present disclosure recognizes that certain changes to a polynucleotide sequence will not impact its expression, or a protein encoded by said polynucleotide. In certain embodiments, a polynucleotide sequence comprises a coding sequence encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence) having one or more silent mutations. In certain embodiments, a polynucleotide sequence comprises a coding sequence encoding an FGF21 pathway activatingagent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence) having one or more amino acid substitutions. In certain embodiments, a polynucleotide sequence comprises a coding sequence encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence) having one or more amino acid deletions.

[0125] In certain embodiments, a polynucleotide sequence comprises a coding sequence encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence) having one or more acid structural modifications. In certain embodiments, one or more structural modifications comprise methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, or any combination thereof, of one or more amino acid residues.

[0126] In certain embodiments, a polynucleotide comprises a coding sequence encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence) having one or more substitutions, one or more amino acid deletions, and / or one or more structural modifications that modulates a function of the encoded FGF21 pathway activating agent. In certain embodiments, modulation of a function comprises increasing half-life, improving physicochemical properties, reducing aggregation and / or reducing proteolysis of an encoded FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof, e.g., an FGF21 fusion polypeptide or variant thereof, e.g., an antibody or antigen binding fragment). In certain embodiments, modulation of a function comprises increasing binding of an encoded FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof, e.g., an FGF21 fusion polypeptide or variant thereof, e.g., an antibody or antigen binding fragment) to a β-Klotho and / or FGF receptor as compared to a reference.

[0127] In certain embodiments, a polynucleotide sequence comprises a coding sequence encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., anantibody or antigen binding fragment coding sequence) having one or more amino acid insertions. In certain embodiments, the one or more amino acid insertions comprises one or more tags. In certain embodiments, a tag may increase efficacy of an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof, e.g., an FGF21 fusion polypeptide or variant thereof, e.g., an antibody or antigen binding fragment). In certain embodiments, a tag may facilitate activation of one or more pathways. In certain embodiments, a tag may facilitate simultaneous activation of one or more pathway. In certain embodiments, a tag may facilitate activation of an FGF21 signaling pathway. In certain embodiments, a tag may facilitate activation of a GLP1 signaling pathway. In certain embodiments, a tag may facilitate simultaneous activation of an FGF21 signaling pathway and a GLP1 signaling pathway.

[0128] A polynucleotide sequence as described herein can be optimized (e.g., codon optimized) to achieve increased or optimal expression in an animal, e.g., a mammal, e.g., a human. In certain embodiments, a polynucleotide sequence described herein is optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and / or elimination of mRNA instability elements. Methods to generate optimized polynucleotides for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos.5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly, all of which are herein incorporated by reference in their entireties. For example, potential splice sites and instability elements (e.g., A / T or A / U rich elements) within the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequences to increase stability of the RNA for recombinant expression. The alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid. In certain embodiments, it can be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid with similar chemical structure and properties and / or function as the original amino acid. Such methods can increase expression of the encoded capsid polypeptide relative to the expression of the capsid encoded by polynucleotides that have not been optimized.

[0129] Embodiments of a polynucleotide sequence described herein are non-limiting configurations of coding sequences and additional elements (e.g., linear arrangement of the coding sequences and additional elements within the provided nucleic acid sequences, sense / antisense orientation of coding sequences and / or additional elements relative to each other, etc.).

[0130] In certain embodiments, a polynucleotide comprises one or more coding sequences. In certain embodiments, the one or more coding sequences are the same. In certain embodiments, the one or more coding sequences are different.

[0131] In certain embodiments, a polynucleotide sequence comprises at least two coding sequences. In certain embodiments, the at least two coding sequences are the same. In certain embodiments, the at least two coding sequences are different.

[0132] In certain embodiments, a coding sequence encodes one or more polypeptides, or a fragment or fragments thereof. Such coding sequences can comprise the complete coding sequence of a polypeptide, or only a fragment of a coding sequence of a polypeptide. In certain embodiments, the coding sequence encodes a polypeptide that is useful to treat a disease or disorder in a subject.

[0133] In certain embodiments, a coding sequence is linked to a signal peptide. The signal peptide can be derived from the same polypeptide it is linked to, or it can be heterologous to the polypeptide it is linked to. Polypeptides

[0134] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence encoding an FGF21 polypeptide or variant thereof.

[0135] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence comprising a canine fgf21 nucleotide sequence. In certain embodiments, a coding sequence comprises a nucleotide sequence set forth in NCBI Reference Sequence XM_022424196.2. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a nucleotide sequence set forth in NCBI Reference Sequence XM_022424196.2. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequence set forth in SEQ ID NO: 42.

[0136] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence comprising a murine fgf21 nucleotide sequence. In certain embodiments, a coding sequence comprises a nucleotide sequence set forth in NCBI Reference Sequence NM_020013.4. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a nucleotide sequence set forth in NCBI Reference Sequence NM_020013.4. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequence set forth in SEQ ID NO: 44.

[0137] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence comprising a feline fgf21 nucleotide sequence. In certain embodiments, a coding sequence comprises nucleotide sequence set forth in NCBI Reference Sequence XM_003997528.4. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to nucleotide sequence set forth in NCBI Reference Sequence XM_003997528.4. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequence set forth in SEQ ID NO: 48.

[0138] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence comprising a human FGF21 nucleotide sequence. In certain embodiments, a coding sequence comprises a nucleotide sequence set forth in NCBI Reference Sequence NM_019113.4. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequence set forth in SEQ ID NOs: 46. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequence set forth in SEQ ID NO: 6.

[0139] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence encoding a canine FGF21 polypeptide or variant thereof. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof comprising an amino acid sequence set forth in NCBI Reference Sequence XP_022279904.1. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to an amino acid sequence set forth in NCBI Reference Sequence XP_022279904.1. In certain embodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 43.

[0140] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence encoding a murine FGF21 polypeptide or variant thereof. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof comprising an amino acid sequence set forth in NCBI Reference Sequence NP_064397.1. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to an amino acid sequence set forth in NCBI Reference Sequence NP_064397.1. In certainembodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 45.

[0141] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence encoding a feline FGF21 polypeptide or variant thereof. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof comprising an amino acid sequence set forth in NCBI Reference Sequence XP_003997577.3. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof having at least 80% sequence (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to an amino acid sequence set forth in NCBI Reference Sequence XP_003997577.3. In certain embodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 47.

[0142] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence encoding a human FGF21 polypeptide or variant thereof. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof comprising an amino acid sequence set forth in NCBI Reference Sequence NP_061986.1. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to an amino acid sequence set forth in NCBI Reference Sequence NP_061986.1. In certain embodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence set forth in SEQ ID NO: 27.

[0143] In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof comprising one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 27, 43, or 45. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof comprising one or more amino acid deletions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47. In certain embodiments, a coding sequence encodes an FGF21 polypeptide or variant thereof comprising one or more amino acid insertions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

[0144] In certain embodiments, an FGF21 polypeptide or variant thereof comprises an additional disulfide bond through Leu118Cys and Ala134Cys (e.g., to reduce aggregation). In certain embodiments, an FGF21 polypeptide or variant thereof comprises an Ser167Ala (e.g., for elimination of O-glycosylation site). In certain embodiments, an FGF21 polypeptide or variant thereof comprises a deletion of four amino-terminal amino acid residues His-Pro-Ile-Pro.

[0145] In certain embodiments, an FGF21 polypeptide or variant thereof comprises LY2405319. In certain embodiments, an FGF21 polypeptide or variant thereof comprises Pegbelfermin. In certain embodiments, an FGF21 polypeptide or variant thereof comprises PEG30-Q108. In certain embodiments, an FGF21 polypeptide or variant thereof is pegylated.

[0146] In certain embodiments, an FGF21 polypeptide or variant thereof comprises ZT003 (Beijing QL Biopharmaceutical).

[0147] In certain embodiments, an FGF21 polypeptide or variant thereof comprises one or more modifications and / or mutations described herein. Fusion Polypeptides

[0148] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence encoding an FGF21 fusion polypeptide or variant thereof.

[0149] In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a canine FGF21 polypeptide or variant thereof. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises an amino acid sequence set forth in NCBI Reference Sequence XP_022279904.1.

[0150] In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a murine FGF21 polypeptide or variant thereof. In certain embodiments, an FGF21 fusionpolypeptide or variant thereof comprises an amino acid sequence set forth in NCBI Reference Sequence NP_064397.1.

[0151] In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a human FGF21 polypeptide or variant thereof. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises an amino acid sequence set forth in NCBI Reference Sequence NP_061986.1

[0152] In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises an FGF21 polypeptide or variant thereof comprising one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 27, 43, or 45. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises an FGF21 polypeptide or variant thereof comprising one or more amino acid deletions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises an FGF21 polypeptide or variant thereof comprising one or more amino acid insertions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

[0153] In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a mutation at position 98. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a mutation at position 171. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a mutation at position 180. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises glycoPEGylation at position 173. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a S173T mutation. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a R176A mutation. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises methionine residue at position 1. In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises two molecules of modified hFGF21 (ΔHis1, Ala129Cys) conjugated to Fab of a scaffold antibody (CVX-2000). In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises substituting a thermally labile and low receptor affinity core of FGF21 with a heparin sulfate-binding-deficient core derived from a stable and high receptor affinity paracrine FGF1 (FGF1∆HBS) (e.g., to overcome the inherent instability and weak FGFR-binding affinity of FGF21). In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises conjugation to an Fc region of human IgG1 via a linker peptide (e.g., Leu98Arg and Pro171Gly).

[0154] In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises efruxifermin (Amgen). In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises BOS-580 (Novartis). In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises BOS-580 (Novartis). In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises PF-05231023 (Pfizer). In some embodiments, an FGF21 fusion polypeptide or variant thereof comprises pegozafermin (89 Bio).

[0155] In certain embodiments, an FGF21 fusion polypeptide or variant thereof further comprises one or more agents associated with an FGF21 polypeptide or variant thereof described herein. In certain embodiments, the one or more agents extends a half-life of an FGF21 polypeptide or variant thereof described herein to a longer length of time relative to a half-life of a reference polypeptide that does not include the one or more agents.

[0156] In certain embodiments, the one or more agents comprise albumin or a fragment thereof.

[0157] In certain embodiments, the one or more agents comprise an antibody or a fragment thereof. In certain embodiments, the antibody or a fragment thereof comprises an IgG constant domain. In certain embodiments, the antibody or a fragment thereof comprises an IgG constant domain. In certain embodiments, the IgG constant domain comprises IgG1.

[0158] In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises a mutation and / or modification described herein. Antibodies or Antigen Binding Fragments

[0159] In certain embodiments, a polynucleotide sequence described herein comprises a coding sequence encoding an antibody or antigen binding fragment. In certain embodiments, an antibody or antigen binding fragment activates an FGF receptor. In certain embodiments, an antibody or antigen binding fragment the antibody or antigen binding fragment activates β-Klotho. In certain embodiments, an antibody or antigen binding fragment activates a β-Klotho and FGF receptor complex. In certain embodiments, an antibody or antigen binding fragment facilitates heterodimerization of a β-Klotho and FGF receptor complex.

[0160] In certain embodiments, an antibody or antigen binding fragment activates FGFR1. In certain embodiments, an antibody or antigen binding fragment activates a β-Klotho and FGFR1 receptor complex. In certain embodiments, an antibody or antigen binding fragment facilitates heterodimerization of a β-Klotho and FGFR1 receptor complex.

[0161] In certain embodiments, an antibody or antigen binding fragment comprises biparatopic agonistic mAb for an FGFR1–β-klotho complex that has been constructed by grafting two variable heavy (VH) domains specific to different epitopes of β-klotho onto the VH and variable light (VL) positions of an IgG scaffold, yielding a tetravalent binder with a close and a distant geometry between the two antigen-binding sites, as described by Shi et al., J Biol Chem. 2018 Feb 26;293(16):5909–5919, which is hereby incorporated by reference herein in its entirety. In certain embodiments, an antibody or antigen binding fragment is conjugated to human serum albumin. In certain embodiments, an antibody or antigen binding fragment, one arm of the antibody binds to β-klotho with the other arm binds to FGFR1c.

[0162] In certain embodiments, an antibody or antigen binding fragment comprises MK- 3655 (Merck). In certain embodiments, an FGF21 fusion polypeptide or variant thereof comprises YH25724 (Boehringer Ingelheim). In certain embodiments, an antibody or antigen binding fragment comprises thereof comprises R1MAb-1 (Genentech). In certain embodiments, an antibody or antigen binding fragment comprises thereof comprises bFKB1 (Genentech).

[0163] In certain embodiments, an antibody or antigen binding fragment comprises one or more modifications and / or mutations described herein. Transcriptional Regulatory Element (TRE)

[0164] In certain embodiments, a polynucleotide described herein comprises a transcriptional regulatory element (TRE). In certain embodiments, a polynucleotide sequence described herein comprise an expression cassette comprising a TRE. In certain embodiments, a TRE is operably linked to one or more coding sequences, i.e., to control expression of an RNA or polypeptide encoded by the coding sequence.

[0165] In certain embodiments, any one or more of the TREs described herein can be combined in any order. In certain embodiments, any one or more of the TREs described herein can be combined in any order to drive efficient transcription.

[0166] In certain embodiments, a TRE can be active in any mammalian cell (e.g., any human cell). In certain embodiments, a TRE is active in a broad range of human cells. Such TREs may comprise constitutive promoter and / or enhancer elements. Promoters

[0167] In certain embodiments, a TRE comprises a promoter. In certain embodiments, a TRE comprises one or more promoters. In certain embodiments, a TRE comprises a constitutivepromoter. In certain embodiments, a TRE comprises an inducible promoter. In certain embodiments, a TRE may be a tissue-specific TRE, i.e., it is active in specific tissue(s) and / or organ(s). A tissue-specific TRE comprises one or more tissue-specific promoter and / or enhancer elements, and optionally one or more constitutive promoter and / or enhancer elements It is an insight of the present disclosure that tissue-specific promoter and / or enhancer elements can be isolated from genes specifically expressed in the tissue by methods well known in the art. Suitable promoters include, e.g., cytomegalovirus promoter (CMV) (Stinski et al. (1985) Journal of Virology 55(2): 431-441); CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG) (Miyazaki et al. (1989) Gene 79(2): 269-277); CBSB(Jacobson et al. (2006) Molecular Therapy 13(6): 1074-1084); human elongation factor 1α promoter (EF1α) (Kim et al. (1990) Gene 91 (2): 217-223); human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417); mitochondrial heavy-strand promoter (Lodeiro et al. (2012) PNAS 109(17): 6513-6518); and ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101- 105). In certain embodiments, a TRE comprises a cytomegalovirus (CMV) promoter / enhancer, an SV40 promoter, a chicken beta actin (CBA) promoter, an smCBA promoter, a human elongation factor 1 alpha (EF1α) promoter, a minute virus of mouse (MVM) intron which comprises transcription factor binding sites, a human phosphoglycerate kinase (PGK1) promoter, a human ubiquitin C (Ubc) promoter, a human beta actin promoter, a human neuron-specific enolase (ENO2) promoter, a human beta-glucuronidase (GUSB) promoter, a rabbit beta-globin element, a human calmodulin 1 (CALM1) promoter, a human ApoE / C-I hepatic control region (HCR1), an ApoE enhancer, a human α1-antitrypsin (hAAT) promoter, an extended HCR1, a HS-CRM8 element of an hAAT promoter, a human transthyretin (TTR) promoter, a human AFP enhancer, and / or a human Methyl-CpG Binding Protein 2 (MeCP2) promoter.

[0168] In certain embodiments, a TRE is liver-specific. Exemplary liver-specific TREs may comprise one or more elements from, without limitation, an ApoA-I promoter, an ApoA-II promoter, an ApoA-IV promoter, an ApoB promoter, an ApoC-I promoter, an ApoC-II promoter, an ApoC-III promoter, an ApoE promoter, an albumin promoter (e.g., the human albumin promoter), an α-fetoprotein promoter, a phosphoenolpyruvate carboxykinase 1 (PCK1) promoter, a phosphoenolpyruvate carboxykinase 2 (PCK2) promoter, a transthyretin (TTR) promoter, an α1- antitrypsin promoter (e.g., the human α1-antitrypsin (AAT or SERPINA1) promoter), a TK (thymidine kinase) promoter, a hemopexin promoter, an alcohol dehydrogenase 6 promoter, acholesterol 7alpha-hydroxylase promoter, a factor IX promoter, an α-microglobulin promoter, a SV40 promoter, a CMV promoter, a Rous Sarcoma Virus-LTR promoter and a HBV promoter.

[0169] In certain embodiments, a TRE is muscle-specific. Exemplary muscle-specific TREs may comprise one or more elements from, without limitation, a human skeletal muscle α- actin (HSA) promoter, a muscle creatine kinase (MCK) promoter, a MHCK7 promoter, a dMCK promoter, a tMCK promoter, a CK6 promoter, a CK8 promoter, a CK8e promoter, a human desmin (DES) promoter or variant thereof, a cardiac troponin T (cTnT) promoter, a myosin light-chain (MLC2v) promoter, a human α-myosin heavy chain gene (αMHC) promoter, a MLC promoter, a human troponin I (TNNI1) promoter, a ΔUSEx3 promoter, a SPcΔ5-12 promoter, a SP-301 promoter, a MH promoter, and a Sk-CRM4 / DES promoter.

[0170] In certain embodiments, a native promoter for the coding sequence may be used. A native promoter may be preferred when it is desired that expression of the coding sequence should mimic the native expression. A native promoter may be used when expression of a coding sequence must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0171] In certain embodiments, a polynucleotide sequence described herein comprise a human alpha 1-antitrypsin (hAAT) promoter. In certain embodiments, a hAAT promoter comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 4. Enhancers and 5’ Cap

[0172] In certain embodiments, a polynucleotide sequence described herein comprise one or more ApoE enhancer elements. In certain embodiments, polynucleotide or nucleic acid sequences comprise one ApoE enhancer element. In certain embodiments, a polynucleotide or nucleic acid sequences comprise two ApoE enhancer elements. In certain embodiments, polynucleotide or nucleic acid sequences comprise three ApoE enhancer elements. In certain embodiments, polynucleotide or nucleic acid sequences comprise more than three ApoE enhancer elements. In certain embodiments, one or more ApoE enhancer elements each comprise anucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 3. In certain embodiments, three ApoE enhancer elements comprise a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 28.

[0173] In certain embodiments, a polynucleotide sequence described herein comprise a 5’ cap. As described herein, a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m.sup.7G cap) is a modified guanine nucleotide that has been added to a “front” or 5’ end of a eukaryotic messenger RNA shortly after a start of transcription. In certain embodiments, a 5’ cap consists of a terminal group which is linked to a first transcribed nucleotide. Its presence is critical for recognition by a ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after start of transcription, a 5’ end of an mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes a chemical reaction that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. A capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation. Introns

[0174] In certain embodiments, polynucleotide or nucleic acid sequences described herein comprise one or more intron elements. In certain embodiments, polynucleotide or nucleic acid sequences comprise an intron element 3’ of a promoter. In certain embodiments, polynucleotide or nucleic acid sequences comprise one intron element. In certain embodiments, polynucleotide or nucleic acid sequences comprise two intron elements. In certain embodiments, polynucleotide or nucleic acid sequences comprise more than two intron elements. In certain embodiments, one or more intron elements each comprise a β-globin intron element.

[0175] In certain embodiments, polynucleotide or nucleic acid sequences described herein comprise a β-globin intron element. In certain embodiments, the β-globin intron element comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5. In certain embodiments, the β-globin intron element is 3’ of a promoter (e.g., a hAAT promoter).

[0176] In certain embodiments, polynucleotide or nucleic acid sequences described herein comprise a TRE comprising a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 3, 4, 5, 28, 29, 30, and 31. Post-transcriptional Regulatory Element

[0177] In certain embodiments, a polynucleotide sequence described herein comprises a post-transcriptional regulatory element (PRE). In certain embodiments, polynucleotide or nucleic acid sequences described herein comprise an expression cassette comprising a PRE.

[0178] In certain embodiments, a PRE is operably linked to one or more coding sequence. In certain embodiments, a PRE is operably linked to one or more coding sequences, i.e., to control expression of an RNA or polypeptide encoded by the coding sequence.

[0179] In certain embodiments, a PRE is identical or substantially identical to a native PRE for the coding sequence. In certain embodiments, a PRE is an exogenous PRE.

[0180] In certain embodiments, any one or more of the PREs described herein can be combined in any order. In certain embodiments, any one or more of the PREs described herein can be combined in any order to control expression of an RNA or polypeptide encoded by ae coding sequence described herein. Polyadenylation Sequences

[0181] In certain embodiments, a PRE comprises a polyadenylation signal (poly(A)) sequence. In certain embodiments, a polynucleotide sequence described herein may comprise at least one poly(A) sequence. Most nascent eukaryotic mRNA possesses a poly(A) tail at its 3’ end which is added during a complex process that includes cleavage of a primary transcript and a coupled polyadenylation reaction (see, e.g., Proudfoot et al., Cell 108:501-512, 2002, which is hereby incorporated herein by reference in its entirety). A poly(A) tail can confer RNA stabilityand transferability (see, e.g., Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994). In certain embodiments, a poly(A) sequence is positioned 3’ to a coding sequence.

[0182] In certain embodiments, polyadenylation refers to a covalent linkage of a polyadenylyl moiety, or its modified variant, to an RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at a 3’ end. In certain embodiments, a 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to pre-mRNA through enzymatic action, polyadenylate polymerase. In higher eukaryotes, a poly(A) tail is added onto transcripts that contain a specific sequence, a polyadenylation signal. In certain embodiments, a poly(A) tail and a protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of mRNA from a cell’s nucleus, and translation. Polyadenylation occurs in a cell nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, an mRNA chain is cleaved through action of an endonuclease complex associated with RNA polymerase. A cleavage site is usually characterized by the presence of a base sequence AAUAAA near a given cleavage site. After an mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site.

[0183] In certain embodiments, a poly(A) signal sequence is a sequence that triggers endonuclease cleavage of an mRNA and addition of a series of adenosines to the 3’ end of a cleaved mRNA. A “poly(A)” portion refers to a series of adenosines attached by polyadenylation to an mRNA. In certain embodiments of the present disclosure, such as, e.g., transient expression, a polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.

[0184] There are several poly(A) signal sequences that can be used, including those derived from bovine growth hormone (bgh) (Woychik et al., Proc. Natl. Acad. Sci. U.S.A. 81(13):3944-3948, 1984; U.S. Patent No.5,122,458; Yew et al., Human Gene Ther.8(5):575-584, 1997; Xu et al., Human Gene Ther.12(5):563-573, 2001; Xu et al., Gene Ther.8:1323-1332, 2001; Wu et al., Mol. Ther. 16(2):280-289, 2008; Gray et al., Human Gene Ther. 22:1143-1153, 2011; Choi et al., Mol. Brain 7:17, 2014, each of which is incorporated in its entirety herein by reference),mouse-β-globin, mouse-α-globin (Orkin et al., EMBO J. 4(2):453-456, 1985; Thein et al., Blood 71(2):313-319, 1988, each which is incorporated in its entirety herein by reference), human collagen, polyoma virus (Batt et al., Mol. Cell Biol. 15(9):4783-4790, 1995, each of which is incorporated in its entirety herein by reference), Herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy-chain gene polyadenylation signal (US 2006 / 0040354, which is incorporated in its entirety herein by reference), human growth hormone (hGH) (Szymanski et al., Mol. Therapy 15(7):1340-1347, 2007; Ostegaard et al., Proc. Natl. Acad. Sci. U.S.A. 102(8):2952-2957, 2005, each of which is incorporated in its entirety herein by reference), synthetic polyA (Levitt et al., Genes Dev.3(7):1019-1025, 1989; Yew et al., Human Gene Ther.8(5):575-584, 1997; Ostegaard et al., Proc. Natl. Acad. Sci. U.S.A. 102(8):2952-2957, 2005; Choi et al., Mol. Brain 7:17, 2014, each of which is incorporated in its entirety herein by reference), HIV-1 upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6):1167-1173, 2007, each of which is incorporated in its entirety herein by reference), adenovirus (L3) upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6):1167-1173, 2007, which is incorporated in its entirety herein by reference), hTHGB upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6):1167-1173, 2007), hC2 upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6):1167-1173, 2007), a SV40 poly(A) signal sequence, such as the SV40 late and early poly(A) signal sequence (Schek et al., Mol. Cell Biol. 12(12):5386-5393, 1992; Choi et al., Mol. Brain 7:17, 2014; Schambach et al., Mol. Ther. 15(6):1167-1173, 2007, each of which is incorporated in its entirety herein by reference).

[0185] In certain embodiments, a poly(A) signal sequence can be the sequence AATAAA. In certain embodiments, an AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA which are capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e.g., WO 06 / 12414, which is incorporated in its entirety herein by reference).

[0186] In certain embodiments, a poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., the pCl-neo expression construct of Promega which is based on Levitt el al, Genes Dev. 3(7):1019-1025, 1989, which is incorporated in its entirety herein by reference). In certain embodiments, a poly(A) signal sequence is a polyadenylation signal ofsoluble neuropilin-1 (sNRP) (see, e.g., WO 05 / 073384, which is incorporated in its entirety herein by reference). In certain embodiments, a poly(A) sequence is a bovine growth hormone poly(A) sequence. Additional examples of poly(A) signal sequences are known in the art.

[0187] In certain embodiments, a polynucleotide sequence described herein comprises a simian virus 40 polyadenylation (SV40pA) sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 8, 32, and 33. Other Regulatory Sequences

[0188] In certain embodiments, a PRE comprises a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE). In certain embodiments, a polynucleotide sequence described herein comprises one or more WPRE sequences.

[0189] In certain embodiments, polynucleotide sequences described herein comprise a WPRE having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7. Kozak Sequences

[0190] In certain embodiments, a PRE comprises a Kozak consensus sequence. In certain embodiments, a polynucleotide sequence described herein comprises one or more Kozak sequences. In certain embodiments, a natural 5’ UTR includes a sequence that plays a role in translation initiation. For example, in certain embodiments, a natural 5’ UTR harbors signatures like Kozak sequences, which are involved in a process by which a ribosome initiates translation of many genes. Kozak sequences generally have a consensus sequence CCR(A / G)CCAUGG, where R is a purine (A or G) three bases upstream of a start codon (AUG), which is followed by another “G”. In certain embodiments, Kozak sequences may be included in synthetic or additional sequence elements, such as cloning sites.

[0191] In certain embodiments, a polynucleotide sequence described herein comprises a Kozak consensus sequence. In certain embodiments, a Kozak consensus sequence comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the nucleotide sequence GCCACCATG. In certain embodiments, a Kozak consensus sequence is 5’ of a coding sequence. Polycistronic Element

[0192] In certain embodiments, a PRE comprises a polycistronic element. In certain embodiments, a polynucleotide sequence described herein comprises one or more polycistronic element.

[0193] In certain embodiments, a polycistronic element comprises a nucleotide sequence that encodes for an internal ribosome entry site (IRES). An IRES is an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a polypeptide coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites, including, without limitation, an IRES obtainable from viral or cellular mRNA sources, e.g., immunoglobulin heavy-chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0194] In certain embodiments, a polycistronic element comprises a nucleotide sequence that encodes for a 2A sequence or element. A 2A sequence refers to an oligopeptide that allow multiple polypeptides to be encoded as polypeptides, which dissociate into component polypeptides upon translation. Various 2A sequences, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO), Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses can be used in accordance with embodiments of the present disclosure. 2A sequences derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.

[0195] In certain embodiments, a polynucleotide sequence described herein comprises one or more PREs comprising a nucleotide sequence having at least 70% sequence identity (e.g., atleast 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 7, 8, 32, 33, 34, and 35. Untranslated Regions (UTRs)

[0196] In certain embodiments, a polynucleotide sequence described herein can include one or more untranslated regions. In certain embodiments, a polynucleotide sequence can include a 5’ UTR and / or a 3’ UTR. In certain embodiments, if more than one UTR is present, UTRs may come from a single gene or more than one gene.

[0197] An untranslated region (UTR) of a gene is transcribed but not translated. In certain embodiments, a 5’ UTR starts at a transcription start site and continues to a start codon but does not include that start codon. In certain embodiments, a 3’ UTR starts immediately following a stop codon and continues until a transcriptional termination signal. In certain embodiments, regulatory features of a UTR can be incorporated into any technologies (e.g., a polynucleotide sequence, a composition, a kit, and a method) as described herein to, e.g., enhance stability of an encoded polypeptide.

[0198] For example, in certain embodiments, a 5’ UTR is included in any a polynucleotide sequence described herein. Non-limiting examples of 5’ UTRs including those from the following genes: albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as a mRNA. In certain embodiments, 5’ UTRs can form secondary structures that are involved in elongation factor binding.

[0199] In certain embodiments, a 5’ UTR from an mRNA that is transcribed by a cell can be included in any technologies (e.g., a polynucleotide sequence, a composition, a kit, and / or a method) described herein.

[0200] Generally, 3’ UTRs have stretches of adenosines and uridines embedded in them. These AU-rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, AU-rich elements (AREs) can be separated into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995, each of which is incorporated in its entirety herein by reference): Class IAREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A) (U / A) nonamers. GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs. Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif. Two well-studied examples of this class are c-Jun and myogenin mRNAs.

[0201] Most proteins binding to AREs destabilize a messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase stability of mRNA. HuR binds to AREs of all three classes. Engineering HuR specific binding sites into a 3’ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of a message in vivo.

[0202] In certain embodiments, introduction, removal, or modification of 3’ UTR AREs can be used to modulate stability of an mRNA encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence). In other embodiments, AREs can be removed or mutated to increase intracellular stability and thus increase translation and production of a polypeptide. Stuffer Sequence

[0203] In certain embodiments, a polynucleotide sequence described herein comprise one or more stuffer sequences. In certain embodiments, a polynucleotide sequence described herein comprise an expression cassette and further comprise one or more stuffer sequences. Stuffer sequences may be employed to maintain the size of a nucleic acid (e.g., a nucleic acid comprised within a vector) within appropriate limits for efficient DNA packaging (e.g., viral packaging of a vector), and as such may be employed to increase the efficiency of DNA packaging.

[0204] For example, AAV preferentially packages genomes that are approximately the same size of a native AAV genome. However, some expression cassettes intended to be packaged within AAV can be substantially smaller than the size of a native AAV genome, and may result in the packaging of unwanted nucleic acid sequences. Hence, to increase the efficiency of AAV packaging, stuffer sequences may be employed, linked to the expression cassette intended to be packaged, to result in a vector genome that is closer to the packaging capacity of AAV.

[0205] Without wishing to be bound by any particular theory, a stuffer sequence may have an effect on the function of the nucleic acid. Further, stuffer sequences can be designed to minimize any adverse effects in the context of gene therapy, e.g., by selecting stuffer sequences from regionsof a genome that have minimal impact if integration occurs, and regions of a genome that have minimal risk of initiating unexpected transcription. In certain embodiments, stuffer sequences are from regions of the human genome. Stuffer sequences can be derived from, for example, a natural non-coding sequence (e.g., an intron sequence, an intergenic sequence, etc.), a synthetic non- coding sequence, and fragments and combinations thereof.

[0206] In certain embodiments, a stuffer sequence comprises an intron sequence comprising human albumin or a fragment thereof. In certain embodiments, a stuffer sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14.

[0207] In certain embodiments, a stuffer sequence is located 5’ to a coding sequence described herein. In certain embodiments, a stuffer sequence is located 5’ to a polynucleotide sequence comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and / or a coding sequence described herein. In certain embodiments, a stuffer sequence is located 5’ to an expression cassette described herein (e.g., an expression cassette comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and a coding sequence described herein).

[0208] In certain embodiments, a stuffer sequence is located 3’ to a coding sequence described herein. In certain embodiments, a stuffer sequence is located 3’ to a sequence comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and / or a coding sequence described herein. In certain embodiments, a stuffer sequence is located 3’ to an expression cassette described herein (e.g., an expression cassette comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and a coding sequence described herein).

[0209] In certain embodiments, a first stuffer sequence is located 5’ to a coding sequence described herein, and a second stuffer sequence is located 3’ to a coding sequence described herein. In certain embodiments, a first stuffer sequence is located 5’ to a polynucleotide sequence comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and / or a coding sequence described herein, and a second stuffer sequence is located 3’ to a polynucleotide sequence comprising a transcriptional regulatory element, a post-transcriptional regulatoryelement, and / or a coding sequence described herein. In certain embodiments, a first stuffer sequence is located 5’ to an expression cassette described herein (e.g., an expression cassette comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and a coding sequence described herein), and a second stuffer sequence is located 3’ to an expression cassette (e.g., an expression cassette comprising a transcriptional regulatory element, a post- transcriptional regulatory element, and a coding sequence described herein).

[0210] In certain embodiments, a 5’ stuffer sequence is positioned 5’ to one or more TREs. In certain embodiments, a 3’ stuffer sequence positioned 3’ to one or more PREs. In certain embodiments, a 5’ stuffer sequence positioned 5’ to one or more TREs and a 3’ stuffer sequence positioned 3’ to one or more PREs.

[0211] In certain embodiments, a stuffer sequence is located within a 5’ and 3’ ITR nucleotide sequence of a vector genome. In certain embodiments, a stuffer sequence is located 3’ to a 5’ ITR nucleotide sequence and 5’ to a 3’ ITR nucleotide sequence of the vector genome.

[0212] In certain embodiments, a stuffer sequence is located outside the 5’ and 3’ ITR nucleotide sequence of a vector genome. Where a stuffer sequence is located outside the 5’ and 3’ ITR nucleotide sequence of a vector genome, this may allow for enhanced AAV purity of packaging. In such embodiments, a stuffer sequence outside of the 5’ and 3’ ITR nucleotide sequence of the vector genome may enhance viral purity and reduce off-target incorporation of plasmid backbone sequences, for example, by making such plasmid backbone sequences larger than the packaging capacity of AAV (i.e., larger than 4.7 kbp). In certain embodiments, a stuffer sequence is located 5’ to a 5’ ITR nucleotide sequence and / or 3’ to a 3’ ITR nucleotide sequence of a vector genome. Exemplary Polynucleotides

[0213] Exemplary embodiments of a polynucleotide sequence described herein are described below and in the Examples. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleotide sequence having at least 80% sequence identity e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 40, and 41.

[0214] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising, from 5’ to 3’: one or more TREs comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31; a coding sequence comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6; and one or more PREs comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 35. In certain embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 36.

[0215] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising, from 5’ to 3’: a 5’ stuffer comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14; one or more TREs comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31; a coding sequence comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%) to SEQ ID NO: 6; and one or more PREs comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 35. In certain embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 15 or 19.

[0216] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising, from 5’ to 3’: one or more TREs comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31; a coding sequence comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6; one or more PREs comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 35; and a 3’ stuffer comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14. In certain embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17 or 21.

[0217] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising, from 5’ to 3’: a 5’ stuffer comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14; one or more TREs comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31; a coding sequence comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6; one or more PREs comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 35; and a 3’ stuffer comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14. In certain embodiments, the polynucleotide comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23 or 25.

[0218] In certain embodiments, the present disclosure provides a polynucleotide that is the reverse complement of any of the nucleic acid sequences described herein. Vectors

[0219] The present disclosure also provides a vector comprising a nucleic acid sequence (e.g., a polynucleotide) described herein. Suitable vectors, include, without limitation, plasmids,viruses, cosmids, artificial chromosomes, linear DNA, and mRNA. In certain embodiments, a vector is a plasmid, a viral vector or a DNA minimal vector. In certain embodiments, a vector is an expression vector. In certain embodiments, a viral vector is an adenoviral vector, an adeno- associated virus (AAV) vector, or a lentiviral vector. In certain embodiments, a viral vector is an AAV vector.

[0220] Vectors (e.g., expression vectors) can be introduced into cells (using any techniques known in the art) for propagation of the vector and / or for expression of a polypeptide encoded by the vector. Accordingly, in certain embodiments, the instant disclosure provides a recombinant cell comprising a nucleic acid sequence (e.g., a polynucleotide) or a vector (e.g., an expression vector) described herein.

[0221] Suitable vectors, include, without limitation, plasmids, minimal vectors (e.g., minicircles, Nanoplasmids™, doggybones, MIDGE vectors, and the like), viruses, cosmids, artificial chromosomes, linear DNA, and mRNA. In certain embodiments, the vector is a DNA plasmid or a DNA minimal vector. Any DNA plasmid or DNA minimal vector that can accommodate the necessary vector elements can be used for the vector. Suitable DNA minimal vectors include, without limitation, linear covalently closed DNA (e.g., ministring DNA), linear covalently closed dumbbell shaped DNA (e.g., doggybone DNA, dumbbell DNA), minicircles, Nanoplasmids™, and minimalistic immunologically defined gene expression (MIDGE) vectors. DNA minimal vectors and their methods of production are described in, e.g., U.S. Patent Application Nos. 20100233814, 20120282283, 20130216562, 20150218565, 20150218586, 20160008488, 20160215296, 20160355827, 20190185924, 20200277624, and 20210010021, all of which are herein incorporated by reference in their entireties.

[0222] A variety of host cells and expression vector systems can be utilized. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with, e.g., recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing capsid polypeptide coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with, e.g., recombinant yeast expression vectors containing capsid polypeptide coding sequences; insect cell systems infected with, e.g., recombinant virus expression vectors (e.g., baculovirus) containing capsid polypeptide coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with, e.g., recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with, e.g.,recombinant plasmid expression vectors (e.g., Ti plasmid) containing capsid polypeptide coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20 and BMT10 cells) harboring, e.g., recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In certain embodiments, suitable cells are human cells, e.g., human cell lines. In certain embodiments, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), are suitable. For example, mammalian cells such as CHO or HEK293 cells, together with a vector containing a major intermediate early gene promoter element from human cytomegalovirus is an effective expression system that can be used in conjunction with the nucleic acid sequences described herein.

[0223] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended. For example, when a large quantity of polypeptide is to be produced, vectors which direct the expression of high levels of fusion polypeptide products that are readily purified can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794); pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509); and the like, all of which are herein incorporated by reference in their entireties. For example, pGEX vectors can also be used to express foreign polypeptides as fusion polypeptides with glutathione 5-transferase (GST). In general, such fusion polypeptides are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0224] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[0225] In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the capsid polypeptide molecule in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12): 3655-9, which is herein incorporated by reference in its entirety). Specific initiation signals can also be required for efficient translation of inserted capsid polypeptide coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al. (1987) Methods Enzymol. 153: 516-544, which is herein incorporated by reference in its entirety).

[0226] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of polypeptide products can be important for the function of the polypeptide. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of polypeptides and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign polypeptide expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells.

[0227] For long-term, high-yield production of recombinant polypeptides, stable expression cells can be generated. For example, cell lines which stably express a capsid polypeptide described herein can be engineered.

[0228] In certain embodiments, rather than using expression vectors which contain viral origins of replication, host cells can be transformed with a polynucleotide (e.g., DNA or RNA) controlled by appropriate transcriptional regulatory elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of polynucleotide, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express a capsid polypeptide described herein or a fragment thereof.

[0229] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11(1): 223-32); hypoxanthineguanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034); and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22(3): 817-23) genes in tk-, hgprt- or aprt-cells, respectively, all of which are herein incorporated by reference in their entireties. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al. (1980) PNAS 77(6): 3567-70; O’Hare K et al. (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al. (1984) Gene 30(1-3): 147- 56), all of which are herein incorporated by reference in their entireties. Methods related to recombinant DNA technology can be applied to select the desired recombinant clone and such methods are described, for example, in Ausubel FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al.(eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al. (1981) J Mol Biol 150: 1-14, all of which are herein incorporated by reference in their entireties. IV. Recombinant Adeno-Associated Virus (rAAV) Genomes and Compositions

[0230] Among other things, the present disclosure provides recombinant adeno-associated virus (rAAV) genomes comprising a nucleic acid sequence (e.g., a polynucleotide) described herein.

[0231] In certain embodiments, an rAAV genome further comprises a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence 5’ of the polynucleotide, and / or a 3’ inverted terminal repeat (3’ ITR) nucleotide sequence 3’ of the polynucleotide. ITR sequences from any AAV serotype or variant thereof can be used in the rAAV genomes described herein. A 5’ and 3’ ITR can be from an AAV of the same serotype or from AAVs of different serotypes.

[0232] In certain embodiments, a 5’ ITR nucleotide sequence has at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 1. In certain embodiments, a 3’ ITR nucleotide sequence has at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9.

[0233] In certain embodiments, an rAAV genome is a single-stranded rAAV genome. In certain embodiments, an rAAV genome is a self-complementary rAAV genome.

[0234] In certain embodiments, an rAAV genome comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 16, 18, 20, 22, 24, and 26.

[0235] In certain embodiments, a present disclosure provides an rAAV genome comprising, from 5’ to 3’: a 5’ ITR comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 1; a 5’ stuffer comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14; a TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31; a coding sequence comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6; a PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 35; and a 3’ ITR comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9.

[0236] In certain embodiments, the present disclosure provides an rAAV genome comprising, from 5’ to 3’: a 5’ ITR comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 1; a TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31; a coding sequence comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6; a PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 35; a 3’ stuffer comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14; and a 3’ ITR comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:9.

[0237] In certain embodiments, the present disclosure provides an rAAV genome comprising, from 5’ to 3’: a 5’ ITR comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 1; a 5’ stuffer comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14; a TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31; a coding sequence comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6; a PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 35; a 3’ stuffer comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14; and a 3’ ITR comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9.

[0238] In certain embodiments, the present disclosure provides an rAAV genome comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 15-26.

[0239] Among other things, the present disclosure provides an rAAV particle comprising a capsid comprising an AAV capsid polypeptide and an rAAV genome as described herein.

[0240] A capsid polypeptide can, include, without limitation, a capsid polypeptide from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. A capsid polypeptide can be from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid polypeptide, or an engineered variant thereof.

[0241] In certain embodiments, a capsid polypeptide comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 37-39.

[0242] Among other things, the present disclosure provides compositions, e.g., pharmaceutical compositions, e.g., comprising a nucleic acid, polynucleotide, vector, rAAV genome, and / or rAAV particle as described herein. In certain embodiments, compositions described herein comprise a pharmaceutically acceptable excipient, adjuvant, diluent, vehicle or carrier, or a combination thereof. A “pharmaceutically acceptable carrier” includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and without causing disruptive physiological reactions, such as an unintended immune reaction. Pharmaceutically acceptable carriers include water, phosphate buffered saline, emulsions such as oil / water emulsion, and wetting agents. Compositions comprising such carriers are formulated by conventional methods such as those set forth in Remington’s Pharmaceutical Sciences, current Ed., Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., 3rd ed. Amer. Pharmaceutical Assoc. V. Adeno-Associated Virus Packaging Systems

[0243] Among other things, the instant disclosure provides packaging systems for recombinant preparation of a recombinant adeno-associated virus (rAAV) particle. In certain embodiments, such packaging systems generally comprise: a first nucleotide encoding one or more AAV Rep polypeptides; a second nucleotide encoding an AAV capsid polypeptide as described herein; and a third nucleotide sequence comprising any of the rAAV genome sequences as described herein. In certain embodiments, such packaging systems generally comprise: a first nucleotide encoding one or more AAV Rep polypeptides; a second nucleotide encoding an AAV capsid polypeptide as described herein; and a third nucleotide sequence comprising any of the rAAV genome sequences as described herein, wherein the packaging system is operative in a cell for enclosing the transfer genome in the capsid to form the AAV.

[0244] In certain embodiments, a packaging system comprises a first vector comprising the first nucleotide sequence encoding the one or more AAV Rep polypeptides and the second nucleotide sequence encoding the AAV capsid polypeptide, and a second vector comprising the third nucleotide sequence comprising the rAAV genome. As used in the context of a packaging system as described herein, a “vector” refers to a nucleic acid molecule that is a vehicle forintroducing nucleic acids into a cell (e.g., a plasmid, a virus, a cosmid, an artificial chromosome, etc.). In certain embodiments of a packaging system, the packaging system further comprises a fourth nucleotide sequence comprising one or more helper virus genes. In certain embodiments, a fourth nucleotide sequence comprises adenoviral E2, E4, and VA genes. In certain embodiments of a packaging system, the packaging system further comprises a third vector (e.g., a helper virus vector), comprising the fourth nucleotide sequence. The third vector may be an independent third vector, integral with a first vector (i.e., the third vector and the first vector are part of the same vector), or integral with a second vector (i.e., the third vector and the second vector are part of the same vector).

[0245] In certain embodiments, a packaging system comprises a first vector comprising the first nucleotide sequence encoding one or more AAV Rep polypeptides, a second nucleotide sequence encoding one or more recombinant AAV capsid polypeptide, and a third nucleotide sequence comprising any of the rAAV genome sequences as described herein, wherein a packaging system is operative in a cell for enclosing the transfer genome in a capsid to form an AAV particle. In certain embodiments of a packaging system, the packaging system further comprises a fourth nucleotide sequence comprising one or more helper virus genes. In certain embodiments, a fourth nucleotide sequence comprises adenoviral E2, E4, and VA genes. In certain embodiments of a packaging system, the packaging system further comprises a second vector (e.g., a helper virus vector), comprising the fourth nucleotide sequence. The second vector may be an independent second vector, integral with the first vector.

[0246] Any AAV Rep polypeptide can be employed in packaging systems described herein. In certain embodiments of a packaging system, the Rep nucleotide sequence encodes an AAV2 Rep polypeptide. Suitable AAV2 Rep polypeptides may include, without limitation, Rep 78 / 68 or Rep 68 / 52.

[0247] In certain embodiments of a packaging system, a helper virus comprises an adenovirus, a herpes virus (including herpes simplex virus (HSV)), a poxvirus (such as vaccinia virus), a cytomegalovirus (CMV), or a baculovirus. In certain embodiments of a packaging system, a helper virus is adenovirus, the adenovirus genome comprises one or more adenovirus RNA genes comprising El, E2, E4, VA, or any combination thereof. In certain embodiments of a packaging system, where a helper virus is adenovirus, the adenovirus genome comprises one or more adenovirus RNA genes comprising E2, E4, VA, or any combination thereof. In certainembodiments of the packaging system, where the helper virus is HSV, the HSV genome comprises one or more of HSV genes comprising UL5 / 8 / 52, ICPO, ICP4, ICP22, UL30 / UL42, or any combination thereof.

[0248] In certain embodiments of a packaging system, vectors (e.g., first, second, and / or third vectors) are contained within one or more plasmids.

[0249] In certain embodiments of a packaging system, first, second, and / or third vectors are contained within one or more recombinant helper viruses. In certain embodiments, a first vector and a third vector are contained within a recombinant helper virus. In certain embodiments, a second vector and a third vector are contained within a recombinant helper virus.

[0250] Among other things, the disclosure provides a method for recombinant preparation of an AAV as described herein, wherein the method comprises transfecting or transducing a cell with a packaging system as described herein under conditions operative for enclosing an rAAV genome in the capsid to form an rAAV particle as described herein. Exemplary methods for recombinant preparation of an rAAV particle include transient transfection (e.g., with one or more transfection plasmids containing a first, and a second, and optionally a third vector as described herein), viral infection (e.g. with one or more recombinant helper viruses, such as a adenovirus, poxvirus (such as vaccinia virus), herpes virus (including HSV, cytomegalovirus, or baculovirus, containing a first, and a second, and optionally a third vector as described herein)), and stable producer cell line transfection or infection (e.g., with a stable producer cell, such as a mammalian or insect cell, containing a Rep nucleotide sequence encoding one or more AAV Rep polypeptides and / or a Cap nucleotide sequence encoding one or more AAV capsid polypeptides, and with a rAAV genome as described herein being delivered in the form of a plasmid or a recombinant helper virus).

[0251] Accordingly, the instant disclosure provides a packaging system for preparation of an rAAV particle, wherein the packaging system comprises: a first nucleotide sequence encoding one or more AAV Rep polypeptides; a second nucleotide sequence encoding a capsid polypeptide of any one of the AAVs described herein; a third nucleotide sequence comprising an rAAV genome sequence of any one of the AAVs described herein; and optionally a fourth nucleotide sequence comprising one or more helper virus genes (e.g., adenoviral E2, E4, and VA genes).VI. Methods

[0252] Among other things, the present disclosure provides a method comprising introducing into a cell a polynucleotide as described herein, a vector as described herein, a rAAV genome as described herein, a rAAV particle as described herein, or a pharmaceutical composition as described herein. In certain embodiments, the method comprises transducing a cell with an rAAV as described herein.

[0253] Accordingly, the present disclosure provides methods for transducing a cell. The methods generally comprise contacting the cell with an rAAV particle described herein under conditions whereby the cell is transduced. A rAAV particle described herein can be used to transduce cells in vitro, in vivo and ex vivo.

[0254] Among other things, the present disclosure provides methods for delivering a coding sequence, e.g., an FGF21 coding sequence, into a cell. The methods generally comprise contacting the cell with a polynucleotide as described herein, a vector as described herein, a rAAV genome as described herein, a rAAV particle as described herein, or a pharmaceutical composition as described herein, under conditions whereby the cell is transduced and the coding sequence is expressed.

[0255] A rAAV particle described herein can comprise a coding sequence under the control of a TRE. Accordingly, in certain embodiments, the instant disclosure provides methods for expressing a coding sequence in a cell, the method generally comprising contacting the cell with such an rAAV particle under conditions whereby the cell is transduced and the coding sequence is expressed. The coding sequence can encode a polypeptide and / or an RNA molecule, as described herein. Accordingly, in certain embodiments, the instant disclosure provides methods for producing a polypeptide and / or an RNA molecule in a cell, the method generally comprising contacting the cell with such an rAAV particle under conditions whereby the cell is transduced and the polypeptide and / or an RNA molecule is produced. In certain embodiments, the method comprises expressing a first coding sequence and a second coding sequence in a cell, comprising introducing into the cell a polynucleotide as described herein, a vector as described herein, a rAAV genome as described herein, or a rAAV particle as described herein.

[0256] In certain embodiments, the cell is in a subject and the polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein is administered to the subject. In certain embodiments, methods described herein comprise expressing a codingsequence in a subject, comprising administering to the subject an effective amount of a polynucleotide as described herein, a vector as described herein, a rAAV genome as described herein, a rAAV particle as described herein, or a pharmaceutical composition as described herein. A polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein can be administered to a subject by all suitable routes, including, without limitation, intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally.

[0257] In certain embodiments, a subject is a member of any mammalian or non- mammalian species. Suitable subjects include, without limitation, humans, non-human primates, canines, felines, ungulates (e.g., equine, bovine, swine (e.g., pig)), avians, rodents (e.g., rats, mice), and other subjects. In certain embodiments, the subject is human. In certain embodiments, a subject is canine. In certain embodiments, a subject is feline. In certain embodiments, a subject is equine.

[0258] Among other things, the present disclosure provides a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition as described herein for use in medicine. Among other things, the present disclosure provides a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition as described herein for use as therapy. Among other things, the present disclosure provides a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition as described herein for use as a medicament. The present disclosure also provides a polynucleotide, vector, rAAV genome, rAAV particle, and pharmaceutical composition described herein for use as a gene therapy in the treatment of various diseases and / or disorders.

[0259] In certain embodiments, the present disclosure provides a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition as described herein for use in a method of treating a disease, disorder and / or condition. In certain embodiments, a disease, disorder, or condition is a cardiac disease, disorder, or condition. In certain embodiments, a disease, disorder, or condition is a cardiac disease, disorder, or condition associated with arrhythmias.

[0260] Without wishing to be bound by any particular theory, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may modify expression of proteins and receptors in a subject that maintain the balance of calcium, sodium and potassium ions, which are pathways that impact arrhythmogenesis. In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositionsdescribed herein may influence ion channel dynamics (e.g., calcium channel, sodium channel). In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may influence calcium dynamics (e.g., calcium signaling). In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may improve calcium dynamics (e.g., calcium signaling) as compared to a relevant reference. In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may influence calcium dynamics (e.g., calcium signaling) by downregulating expression (e.g., gene, transcript, or polypeptide) of L-type Ca2+channels (LTCC). In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may influence calcium dynamics (e.g., calcium signaling) by downregulating expression e.g., gene, transcript, or polypeptide) of L-type Ca2+channels (LTCC) on the cell surface via PI3Kα.

[0261] In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may protect the heart (e.g., from apoptosis). In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may protect the heart from apoptosis. In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and / or pharmaceutical compositions described herein may protect the heart from apoptosis via adiponectin signaling.

[0262] In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may produce exogenous FGF21 and improve cardiac structure and / or function in a subject. In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may produce exogenous FGF21 and improve cardiac structure and / or function (e.g., by decreasing arrhythmias, apoptosis, inflammation, and fibrosis) in a subject.

[0263] In certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may exhibit anti-arrhythmogenic properties as compared to a relevant reference. In certain embodiments, administration of polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may result in a reduction in arrhythmia in the subject as compared to a relevant reference. In certain embodiments, administration of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein may result in a reduction in arrhythmia in the subjectat a duration after administration of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein, as compared to a relevant reference. In certain embodiments, treatment results in a reduction in arrhythmia in a subject at a duration after administration of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein, as compared to the degree of arrhythmia in the subject prior to administration of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein. The duration in which a subject exhibits a reduction in the degree of arrhythmia compared to the subject’s base level of arrhythmia can be 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer.

[0264] In certain embodiments, a disease or disorder is a neurological diseases, disorders or conditions. In certain embodiments, a disease or disorder is atrial fibrillation. In certain embodiments, a subject suffers from a neurological diseases, disorders or conditions and one or more arrhythmias. In certain embodiments, a disease, disorder, or condition is associated with aberrant calcium signaling. Without wishing to be bound to any theory, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may impact physiological, physical, and / or behavioral aspects of the nervous system: the brain, the spinal cord, and peripheral nerves. For example, disorders of the nervous system can cause physical, cognitive, emotional, and / or behavioral symptoms (e.g., coordination difficulties). There are hundreds of neurological conditions, diseases, or disorders (e.g., neurodegenerative disorders), including neuromuscular conditions (e.g., Amyotrophic Lateral Sclerosis). Neurological conditions may be associated with genetic changes, congenital conditions, injury, infection, and / or tumor. By way of specific example, in certain embodiments, polynucleotides, vectors, rAAV genomes, rAAV particles, and pharmaceutical compositions described herein may be used to treat Amyotrophic Lateral Sclerosis. In certain embodiments, treatment comprises reduction of signs and / or symptoms associated with Amyotrophic Lateral Sclerosis. Arrhythmogenic Cardiomyopathy (ACM)

[0265] Among other things, the present disclosure provides methods of treating arrhythmogenic cardiomyopathy (ACM). Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder characterized by redistribution of junctional polypeptides, arrhythmias, andprogressive myocardial injury. Pathological features of ACM include loss of myocytes and fibrofatty replacement of right ventricular myocardium. Biventricular involvement is often observed. Arrhythmogenic cardiomyopathy is a cell junction cardiomyopathy, typically caused by genetic abnormalities of cardiac desmosomes, which results in the detachment of myocytes and affects intracellular signal transduction.

[0266] Arrhythmogenic cardiomyopathy is a genetically heterogeneous disorder, for which there are several genes in which mutations can be causative. Genes in which mutations have been reported to cause ACM include: junction plakoglobin (JUP); desmoplakin (DSP); plakophilin-2 (PKP2); desmoglein-2 (DSG2); desmocollin-2 (DSC2); transmembrane protein 43 (TMEM43); lamin A / C (LMNA); desmin (DES); alpha-T-catenin (CTNNA3); phospholamban (PLN); transforming growth factor 3 (TGFB3); titin (TTN); sodium voltage-gated channel alpha subunit 5 (SCN5A; Nav1.5); and cadherin C (CDH2).

[0267] While the original ACM disease phenotype was characterized by predominant right ventricle (RV) involvement (ARVC), with minor and late left ventricle (LV) disease, clinical variants characterized by early and greater LV involvement, which may parallel (e.g., biventricular ACM) or exceed (e.g., left-dominant ACM, or ALVC) the severity of RV involvement, have been increasingly reported.

[0268] There is not a single gold standard method for the diagnosis of ACM, and as such, the best strategy consists of combining multiple sources of clinical information, such as genetic, electrocardiographic, arrhythmic, morpho-functional, and histopathological findings. Current international criteria for ARVC and ALVC diagnosis include assessing morpho-functional ventricular abnormalities (i.e., global and regional ventricular dilatation and systolic dysfunction), structural myocardial abnormalities (i.e., for existence of fibrous or fibro-fatty myocardial replacement), echocardiographic abnormalities (e.g., depolarization and repolarization abnormalities), ventricular arrhythmias, and / or family history and molecular genetics. Phenotypic variants of ACM such as ARVC, biventricular ACM, and ALVC, are diagnosed based on a scoring system for fulfilling the above-mentioned criteria.

[0269] In certain embodiments, methods of treating ACM in a subject described herein comprise administering to the subject a gene therapy comprising an effective amount of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein. In certain embodiments, a polynucleotide, a vector, an rAAV genome, an rAAV particle,or a pharmaceutical composition comprises a coding sequence encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence). In certain embodiments, a polynucleotide, a vector, an rAAV genome, an rAAV particle, or a pharmaceutical composition comprises a coding sequence encoding a fibroblast growth factor 21 (FGF21). In certain embodiments, a polynucleotide, a vector, an rAAV genome, an rAAV particle, or a pharmaceutical composition comprises a coding sequence encoding a fibroblast growth factor 21 (FGF21) polypeptide or variant thereof.

[0270] In certain embodiments, an ACM comprises an arrhythmogenic right ventricular cardiomyopathy (ARVC), an arrhythmogenic a left ventricular cardiomyopathy (ALVC), or a biventricular arrhythmogenic cardiomyopathy. In certain embodiments, an ACM is an ARVC. In certain embodiments, a subject is a mammal. In certain embodiments, a subject is a human. In certain embodiments, a subject is a canine. In certain embodiments, a subject is a feline.

[0271] In certain embodiments, a method for treating an ACM in a subject further comprises administering to the subject an effective amount of one or more additional therapeutics to treat an ACM (e.g., a non-gene therapy therapeutic to treat an ACM). Such additional therapeutics to treat an ACM may include, without limitation, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, anti-arrhythmia agents, beta blockers, blood thinners, and diuretics. In certain embodiments, the one or more additional therapeutics to treat an ACM is administered at the same time as the gene therapy. In certain embodiments, the one or more additional therapeutics to treat an ACM is administered at a different time as the gene therapy. In certain embodiments, the ACM is ACM associated with a loss of function in the gene encoding plakophilin-2 (PKP2). In certain embodiments, the ACM is a PKP2 mutation-associated ACM. In certain embodiments, the ACM is ACM associated with a loss of function in the gene encoding desmoglein-2 (DSG2). In certain embodiments, the ACM is a DSG2 mutation-associated ACM. In certain embodiments, the ACM is ACM associated with a loss of function in the gene encoding desmoplakin (DSP). In certain embodiments, the ACM is a DSP mutation-associated ACM. In certain embodiments, the ACM is ACM associated with a loss of function in a gene encoding a desmocollin (DSC), optionally in the gene encoding desmocollin-2 (DSC2). In certain embodiments, the ACM is a DSC mutation-associated ACM. In certain embodiments, the ACM is a DSC2 mutation-associated ACM. In certain embodiments, the ACM is a genetically elusiveACM, i.e., an ACM with no known genetic cause. In certain embodiments, the ACM is ACM associated with a loss of function in one or more genes comprising a PKP2 gene, a DSG2 gene, a DSP gene, a DSC gene, or any combination thereof. In certain embodiments, the ACM is ACM associated with a loss of function in one or more genes comprising a PKP2 gene, a DSG2 gene, a DSP gene, a DSC2 gene, or any combination thereof. In certain embodiments, the ACM is a PKP2 mutation-associated ACM, a DSG2 mutation-associated ACM, a DSP mutation-associated ACM, a DSC mutation-associated ACM, and / or a DSC2 mutation-associated ACM.

[0272] Ejection fraction refers to how well a heart pumps blood and is calculated by measuring the amount of blood pumped out of a heart’s ventricles each time it contracts. LVEF refers to how much blood is pumped out of the left ventricle to most of the body’s organs with each contraction. LVEF helps determine the severity of dysfunction on the left side of the heart. Fractional shortening (FS) refers to the reduction of the length of the end-diastolic diameter that occurs by the end of systole. Both ejection fraction and FS are measures of the heart’s muscular contractility and health. In certain embodiments, a method of treating an ACM in a subject further comprises determining a base level of fractional shortening (FS) and / or left ventricular ejection fraction (LVEF) in the subject prior to administration of the gene therapy. In certain embodiments, the gene therapy results in a reduction in the level of FS and / or LVEF in the subject at a duration after administration of the gene therapy, as compared to the level of FS and / or LVEF in the subject. The duration in which a subject exhibits a reduction in the level of FS and / or LVEF compared to the subject’s base level of FS and / or LVEF, can be 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer.

[0273] In certain embodiments, a subject having an ACM has arrhythmia. In certain embodiments, a method of treating an ACM in a subject further comprises determining a base level of arrhythmia in the subject prior to administration of the gene therapy. In certain embodiments, a method of treating an ACM in a subject further comprises determining a base level of NF-kB activation in the subject prior to administration of the gene therapy. In certain embodiments, the treatment results in a reduction in arrhythmia and / or NF-kB activation in the subject at a duration after administration of the gene therapy, as compared to the degree of arrhythmia and / or NF-kB in the subject prior to administration of the gene therapy. The durationin which a subject exhibits a reduction in the degree of arrhythmia and / or NF-kB activation compared to the subject’s base level of arrhythmia and / or NF-kB activation, can be 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or longer. Mitral Valve Disease (MVD)

[0274] Among other things, the present disclosure provides methods for treating a mitral valve disease (MVD) in a subject. Various methods for diagnosing MVD are known in the art, and include, without limitation, via echocardiograms, electrocardiograms, chest x-rays, cardiac magnetic resonance imaging, exercise tests, stress tests, and / or cardiac catheterization.

[0275] In human, a normal mitral valve allows for unrestricted antegrade diastolic flow from the left atrium (LA) to the left ventricle (LV) and effectively seals off the atrium from the ventricle during LV systole, allowing atrial refilling from the pulmonary veins at low pressures. Abnormal systolic retrograde flow of blood from the LV to the LA is referred to as mitral regurgitation. Accordingly, the present disclosure provides methods for treating mitral regurgitation in a subject. In certain embodiments, mitral regurgitation is a primary mitral regurgitation which includes, without limitation, myxomatous changes (mitral valve prolapse), rheumatic heart disease (RHD), infective endocarditis, collagen vascular disease, papillary muscle dysfunction, mitral annular calcification (MAC), spontaneous chordal rupture, and trauma. In certain embodiments, mitral regurgitation is a secondary mitral regurgitation which includes, without limitation, ischemic cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, or left atrial dilation.

[0276] In certain subjects, e.g., canine subjects, a staging system for MVD generally refers to four basic groups: Stage A – at risk: risk factors for MVD are present; Stage B – progressive: MVD is mild or moderate, and there are no heart valve symptoms; Stage C – asymptomatic severe: MVD is severe, and there are no heart valve symptoms; and Stage D – symptomatic severe: MVD is severe and is causing symptoms. Stage B can be further subdivided into Stage B1 and Stage B2; Stage B1 is diagnosed when a heart murmur is detected but there is no radiographic or echocardiographic evidence of cardiac remodeling or remodeling that is not severe enough to meet current clinical trial criteria for treatment; Stage B2 is diagnosed when a heart murmur is detectedand there is radiographic or echocardiographic evidence of cardiac remodeling that is severe enough to meet current clinical trial criteria for treatment.

[0277] In certain embodiments, methods of treating MVD in a subject described herein comprise administering to the subject an effective amount of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein. In certain embodiments, a polynucleotide, vector, rAAV genome, rAAV particle, or a pharmaceutical composition comprises a coding sequence encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence). In certain embodiments, a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a coding sequence encoding a fibroblast growth factor 21 (FGF21). In certain embodiments, a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a coding sequence encoding a fibroblast growth factor 21 (FGF21) polypeptide or variant thereof.

[0278] In certain embodiments, a method for treating a MVD in a subject further comprises administering to the subject an effective amount of one or more additional therapeutics to treat MVD. Such additional therapeutics to treat MVD may include, without limitation, diuretics, blood thinners (i.e., anticoagulants), and blood pressure medications. For example, in dogs, pimobendan is used in the management of heart failure due to MVD. Pimobendan is often used in conjunction with an ACE inhibitor such as enalapril or benazepril. In certain embodiments, a method for treating MVD in a subject further comprises administering to the subject an effective amount of pimobendan. Other additional therapeutics for treating MVD include furosemide, spironolactone (i.e., an aldosterone antagonist), and an angiotensin-converting enzyme (ACE) inhibitor. In certain embodiments, an additional therapeutic to treat MVD (e.g., pimobendan) is administered at the same time as the gene therapy. In certain embodiments, an additional therapeutic to treat MVD (e.g., pimobendan) is administered at a different time as the gene therapy.

[0279] In certain embodiments, mitral valve disease comprises one or more disease, disorder, or condition comprising a myxomatous mitral valve disease, a mitral valve stenosis, a mitral valve prolapse, a mitral valve regurgitation, or any combination thereof.Familial Partial Lipodystrophy (FPL)

[0280] Among other things, the present disclosure provides methods for treating familial partial lipodystrophy (FPL) in a subject. Familial partial lipodystrophy (FPL) is a rare genetic disorder that results in selective, progressive loss of body fat from various areas of the body. Individuals with FPL typically have reduced subcutaneous fat in the arms and legs, and they may or may not experience loss of body fat in the head and trunk regions. Individuals with FPL may also experience excess subcutaneous fat in other regions of the body, in particular, the neck, face, and intra-abdominal regions. The prevalence of FPL is estimated to be one in a million people; however, many cases may go misdiagnosed or undiagnosed.

[0281] FPL encompasses several subtypes differentiated by the underlying genetic mutation. The specific symptoms, severity, and prognosis can vary greatly between each type of FPL. FPL is caused by mutations of specific genes, and the various subtypes are characterized by the underlying genetic mutation.

[0282] The most common form of FPL is FPL type 2 (also known as Dunnigan lipodystrophy), in which affected individuals experience progressive loss of body fat in the arms, legs, and trunk, at around the time of puberty. Fat may accumulate in other areas of the body including the face, neck, and upper back between the shoulder blades. Individuals with FPL experience insulin resistance which may be associated with acanthosis nigricans, hepatomegaly (enlarged liver) in the form of steatosis which may lead to cirrhosis and liver dysfunction, glucose intolerance, hypertriglyceridemia which may result in pancreatitis, diabetes, coronary artery disease and other types of atherosclerotic vascular disease, and muscular dystrophy. Some women with FPL may develop polycystic ovary syndrome. In rare cases, individuals with FPL have a specific mutation in the LMNA gene; such individuals have increased risk of developing cardiomyopathy which can result in congestive heart failure and cardiac arrhythmias.

[0283] Other types of FPL include FPL type 1 (also known as Kobberling lidodystrophy), characterized by fat loss that is generally confined to the arms and legs; FPL type 3, caused by a genetic mutation in PPARG, is generally milder than FPL type 2; FPL type 4, caused by a genetic mutation in PLIN1, is characterized by lipodystrophy most prominent in the lower limbs and buttocks; FPL type 5, caused by a genetic mutation in AKT2, is characterized by lipodystrophy most prominently affecting arms and legs; and autosomal recessive FPL (also known as FPL type6), caused by a genetic mutation in CIDEC. FPL has also been associated with a genetic mutation in AGPAT2.

[0284] Diagnosis of FPL is based upon the identification of characteristic symptoms coupled with clinical testing, and molecular genetic testing to detect mutations in genes that cause FPL.

[0285] In certain embodiments, methods of treating FPL in a subject described herein comprise administering to the subject an effective amount of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein. In certain embodiments, a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a coding sequence encoding a fibroblast growth factor 21 (FGF21)

[0286] In certain embodiments, a method for treating FPL in a subject further comprises administering to the subject an effective amount of one or more additional therapeutics to treat FPL (e.g., a non-gene therapy therapeutic to treat FPL). Such additional therapeutics to treat FPL may include, without limitation, fibric acid and derivatives thereof, statins, n-3 polyunsaturated fatty acids, hyperglycemic drugs such as metformin and sulfonylureas, insulin, anti-hypertensives, and / or metreleptin. In certain embodiments, the one or more additional therapeutics to treat FPL is administered at the same time as the gene therapy. In certain embodiments, the one or more additional therapeutics to treat FPL is administered at a different time as the gene therapy.

[0287] In certain embodiments, a subject is diagnosed with autosomal recessive FPL (also known as FPL type 6), FPL type 1 (also known as Kobberling lipodystrophy), FPL type 2 (also known as Dunnigan lipodystrophy), FPL type 3, FPL type 4, or FPL type 5. In certain embodiments, the subject comprises a genetic mutation in LMNA, PPARG, PLIN1, AKT2, CIDEC, and / or AGPAT2. In certain embodiments, a subject is diagnosed with FPL type 2 and / or comprises a genetic mutation in LMNA. In certain embodiments, a subject is diagnosed with FPL type 2 and comprises a genetic mutation in LMNA. EXEMPLIFICATION

[0288] The following examples are offered by way of illustration, and not by way of limitation.Example 1: FGF21 Recombinant AAV Vectors

[0289] This example provides FGF21 recombinant adeno-associated virus (rAAV) vectors for expression of FGF21 in a cell (e.g., a liver cell) to which the vectors are transduced. The genetic elements in the vectors are set forth in Table 1, below. SEQ ID NOs for each element are shown.

[0290] The p402-1 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element (SEQ ID NO: 1); a 5’ stuffer (SEQ ID NO: 2); a TRE comprising 3 ApoE enhancer elements (SEQ ID NO: 28), a human AAT promoter (SEQ ID NO: 4), and an intron element comprising a β-globin intron (SEQ ID NO: 5); a coding sequence encoding a human FGF21 polypeptide (SEQ ID NO: 6); a PRE comprising a WPRE sequence (SEQ ID NO: 7) and a SV40 polyadenylation sequence (SEQ ID NO: 8); and a 3’ ITR element (SEQ ID NO: 9). The sequence of the p402-1 vector excluding the ITR elements is given in SEQ ID NO: 15. The sequence of the p402-1 vector including the ITR elements is given in SEQ ID NO: 21.

[0291] The p402-2 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element (SEQ ID NO: 1); a TRE comprising 3 ApoE enhancer elements (SEQ ID NO: 28), a human AAT promoter (SEQ ID NO: 4), and an intron element comprising a β-globin intron (SEQ ID NO: 5); a coding sequence encoding a human FGF21 polypeptide (SEQ ID NO: 6); a PRE comprising a WPRE sequence (SEQ ID NO: 7) and a SV40 polyadenylation sequence (SEQ IDNO: 8); a 3’ stuffer (SEQ ID NO: 2); and a 3’ ITR element (SEQ ID NO: 9). The sequence of the p402-2 vector excluding the ITR elements is given in SEQ ID NO: 16. The sequence of the p402- 2 vector including the ITR elements is given in SEQ ID NO: 22.

[0292] The p402-3 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element (SEQ ID NO: 1); a 5’ stuffer (SEQ ID NO: 10); a TRE comprising 3 ApoE enhancer elements (SEQ ID NO: 28), a human AAT promoter (SEQ ID NO: 4), and an intron element comprising a β-globin intron (SEQ ID NO: 5); a coding sequence encoding a human FGF21 polypeptide (SEQ ID NO: 6); a PRE comprising a WPRE sequence (SEQ ID NO: 7) and a SV40 polyadenylation sequence (SEQ ID NO: 8); and a 3’ ITR element (SEQ ID NO: 9). The sequence of the p402-3 vector excluding the ITR elements is given in SEQ ID NO: 17. The sequence of the p402-3 vector including the ITR elements is given in SEQ ID NO: 23.

[0293] The p402-4 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element (SEQ ID NO: 1); a TRE comprising 3 ApoE enhancer elements (SEQ ID NO: 28), a human AAT promoter (SEQ ID NO: 4), and an intron element comprising a β-globin intron (SEQ ID NO: 5); a coding sequence encoding a human FGF21 polypeptide (SEQ ID NO: 6); a PRE comprising a WPRE sequence (SEQ ID NO: 7) and a SV40 polyadenylation sequence (SEQ ID NO: 8); a 3’ stuffer (SEQ ID NO: 10); and a 3’ ITR element (SEQ ID NO: 9). The sequence of the p402-4 vector excluding the ITR elements is given in SEQ ID NO: 18. The sequence of the p402- 4 vector including the ITR elements is given in SEQ ID NO: 24.

[0294] The p402-5 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element (SEQ ID NO: 1); a 5’ stuffer (SEQ ID NO: 11); a TRE comprising 3 ApoE enhancer elements (SEQ ID NO: 28), a human AAT promoter (SEQ ID NO: 4), and an intron element comprising a β-globin intron (SEQ ID NO: 5); a coding sequence encoding a human FGF21 polypeptide (SEQ ID NO: 6); a PRE comprising a WPRE sequence (SEQ ID NO: 7) and a SV40 polyadenylation sequence (SEQ ID NO: 8); a 3’ stuffer (SEQ ID NO: 12); and a 3’ ITR element (SEQ ID NO: 9). The sequence of the p402-5 vector excluding the ITR elements is given in SEQ ID NO: 19. The sequence of the p402-5 vector including the ITR elements is given in SEQ ID NO: 25.

[0295] The p402-6 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element (SEQ ID NO: 1); a 5’ stuffer (SEQ ID NO: 13); a TRE comprising 3 ApoE enhancer elements (SEQ ID NO: 28), a human AAT promoter (SEQ ID NO: 4), and an intron elementcomprising a β-globin intron (SEQ ID NO: 5); a coding sequence encoding a human FGF21 polypeptide (SEQ ID NO: 6); a PRE comprising a WPRE sequence (SEQ ID NO: 7) and a SV40 polyadenylation sequence (SEQ ID NO: 8); a 3’ stuffer (SEQ ID NO: 14); and a 3’ ITR element (SEQ ID NO: 9). The sequence of the p402-6 vector excluding the ITR elements is given in SEQ ID NO: 20. The sequence of the p402-6 vector including the ITR elements is given in SEQ ID NO: 26.

[0296] The rAAV vectors described herein can be packaged in an AAV capsid, such as, without limitation, an AAV8 capsid. In general, viral particles are generated using standard triple transfection of HEK293T cells and affinity and anion purification. See, e.g., Davidsohn et al. (2019) Proc. Natl. Acad. Sci. 116(47): 23505-23511; and Nass et al. (2018) Mol. Ther. Methods Clin. Dev. 9: 33–46. The packaged viral particles can be administered to a wild-type animal, or a subject suffering from arrhythmogenic right ventricular cardiomyopathy (ARVC).

[0297] In an initial batch, rAAV vectors packaged in AAV8 capsid were generated and titering was performed using droplet digital PCR (ddPCR) with a probe and primers targeting the ITR sequences, under standard conditions and protocols. Titer determinations are shown in Table 2, below. Table 2. Titers of rAAV vectors packaged in AAV8 capsid.Example 2: Expression of FGF21 in Mouse Model of ARVC

[0298] PKP2cKO mice were generated as described in Cerrone et al. (2017) Nature Communications 8: 106. PKP2cKO mice have cardiomyocyte-specific, tamoxifen-induced knockout of the desmosomal polypeptide plakophilin-2 (PKP2). PKP2 knockout induced PKP2cKO mice have previously been shown to develop progressive right-ventricular dominant arrhythmogenic cardiomyopathy. The general study design in this Example for using PKP2cKO mice to investigate the efficacy of FGF21 gene therapy was to administer the gene therapy at 1.5E13 vg / kg to PKP2cKO mice (day -7), followed by tamoxifen-induced knockout seven dayslater (day 0). 21 days after knockout induction, the mice were subjected to isoproterenol (ISO) challenge and electrocardiography. 28 days after knockout induction, the mice were subjected to echocardiography and were then sacrificed, and terminal blood was collected.

[0299] In a separate experiment, four-week-old mice were administered, via single intravenous retro-orbital or tail vein injection, an rAAV vector containing mFGF21 coding sequence packaged in AAV8 (AAV8-mFGF21). AAV8-mFGF21 was administered at a dose of 1.5E13 vg / kg. Control wild type (WT) and PKP2cKO animals were administered vehicle only. mFGF21 expression was measured by ELISAs using antibodies to mouse FGF21. As shown in FIG.1, the expression level of mFGF21 was confirmed in PKP2cKO mice AAV8-mFGF21.

[0300] Cardiac structure and function of PKP2cKO mice treated with AAV8-mFGF21 was assessed. Fractional shortening (FS) and ejection fraction was measured by standard M-mode echocardiography. As shown in FIGs. 2A-2C, administration of AAV8-mFGF21 resulted in enhanced preservation of left ventricle systolic function measures as a function of left ventricle ejection fraction (LVEF; FIG.2A), enhanced preservation of fractional shortening (FS; FIG.2B), and significant reduction in right ventricle dilatation (FIG.2C). Taken together, the administration of AAV8-mFGF21 significantly improved cardiac structure and function of PKP2cKO mice.

[0301] Arrhythmia burden was also assessed in PKP2cKO mice treated with AAV8- mFGF21. Arrhythmia burden, assessed by number of premature ventricular contractions (PVCs), is a clinically meaningful measurement in arrhythmogenic cardiomyopathy. It is a predictor of survival (correlates with mortality and life-threatening ventricular arrhythmias in arrhythmogenic right ventricular cardiomyopathy), it is a key driver of symptoms, and it is directly linked to quality of life – even single PVCs can trigger anxiety and post-traumatic stress disorder in patients. As shown in FIG.3, PKP2cKO mice administered AAV8-mFGF21 were observed to have a reduction in the percentage of cases with PVCs greater than 100 in 30 minutes following ISO challenge. FIGs.5A-5B depicts a reduction in the percentage of cases with PVCs.

[0302] In order to investigate the progression of cardiac fibrosis, hearts of mice were obtained after sacrifice (28 days after induction of knockout), fixed, embedded, and sectioned according to standard techniques. Sections were then stained with Masson’s Trichrome Staining and analyzed for cardiac fibrosis by assessing the percentage of collagen present, indicative of fibrosis. FIGs. 4A and 4B show the level of fibrosis detected in the left ventricle (LV) and right ventricle (RV) of PKP2cKO hearts of mice that received AAV8-mFGF21 or vehicle as control.Example 3: Treatment of Patients with Arrhythmogenic Right Ventricular Cardiomyopathy

[0303] Arrhythmogenic right ventricular cardiomyopathy (ARVC) – previously referred to as arrhythmogenic right ventricular dysplasia (ARVD), is an inherited, rare, myocardial disorder affecting the right ventricle and, in some cases, also the left ventricle. ARVD / C is caused by mutations in genes that encode desmosomal proteins. These proteins are involved with cell-to-cell adhesion, and disruptions in these proteins account for 17% of all sudden cardiac deaths in young populations.

[0304] This prophetic Example describes a method of treating human patients with ARVC using a non-replicating, recombinant adeno-associated virus (rAAV) vector as described herein. Any of the p402-1, p402-2, p402-3, p402-4, p402-5, or p402-6 vectors will be packaged in AAV8 capsid (AAV8-hFGF21) for administration into human patients, and is expected to promote expression of hFGF21 in patient cells.

[0305] Without being bound by any particular theory, treatment with the AAV8-hFGF21 is expected to delay progression of complications, symptoms, and cardiac tissue changes in ARVC patients. The number and severity of arrhythmias should decrease, the inflammatory environment will be reduced leading to less structural changes and a decrease in fibrofatty tissue replacement of cardiac tissue. Also, heart function is expected to remain normal and not deteriorate.

[0306] Selection of patients will include diagnosis of ARVC according to standard diagnostic procedures. Standard diagnosis is based on a scoring system taking into account right ventricle structural and functional abnormalities (dilatation, akinesia, dyskinesia, aneurysms) detected by echocardiography, MRI and angiography; electrocardiographic features (inverted T waves in right precordial leads, epsilon waves and late potentials by signal averaged ECG (SAECG), left bundle branch block ventricular tachycardia, >500 ventricular extrasystoles per 24 h); tissue characterization at endomyocardial biopsy (fibro-fatty replacement of myocardium); and family history. Contrast enhanced MRI substantially enhances the diagnostic sensitivity, particularly in left ventricle variants, while electroanatomic mapping is superior in detecting early RV involvement. Differential diagnosis includes idiopathic RV outflow tract tachycardia, myocarditis, sarcoidosis and congenital heart diseases (see these terms). Although prenatal diagnosis through amniocentesis is feasible, it is subject to ethical and legal considerations.

[0307] Production of AAV8-hFGF21 for administration to patients will be done by standard transient triple plasmid transfection methods. The plasmids used for transfection will contain rep / cap genes, adenovirus-derived helper plasmid suppling genes needed for replication, and the recombinant AAV plasmid containing the genes of interest (GOI; e.g., hFGF21).

[0308] AAV8-hFGF21 will be administered to patients as a single dose intravenous injection for systemic delivery. Example 4: Evaluation of FGF21 on calcium handling

[0309] The present Example demonstrates that rAAV vectors described herein packaged in an AAV capsid may improve calcium handling in a Mouse Model of ACM.

[0310] Adult Female mice were administered, via single intravenous retro-orbital or tail vein injection, an rAAV vector containing mFGF21 coding sequence packaged in AAV8 (AAV8- mFGF21). AAV8-mFGF21 was administered at a dose of 1.5E13 vg / kg. Control wild type (WT) and PKP2cKO animals were administered vehicle only.

[0311] Ventricular myocytes were obtained by enzymatic dissociation. Briefly, PKP2cKO mice were injected with 0.1 ml heparin (500 IU / ml intraperitoneally) 10 min before heart excision and anaesthetized by inhalation of 100% CO2. When deep anesthesia was confirmed, mice were sacrificed by cervical dislocation and the heart was surgically removed from the chest and placed in a Langendorff column. For cell dissociation, the isolated hearts were perfused sequentially with low calcium, and an enzyme (collagenase, Worthington) solution maintained at 37°C. After digestion, ventricles were cut into small pieces, and gently minced with a Pasteur pipette. Calcium concentration increased gradually to 1.0 mM. Cardiomyocytes were kept in Tyrode’s solution containing (in mM): 148 NaCl, 5.4 KCl, 1.0 MgCl2, 1.0 CaCl2, 0.4 NaH2PO4, 15 HEPES and 5.5 glucose, pH 7.40. Cells were used within 3h after isolation.

[0312] 50 ng / ml recombinant mouse FGF21 (rmFGF21) protein (Catalog #: 8409-FG; RD systems; Minneapolis, MN, USA) or formula buffer (FB) were added to all the solutions for cardiomyocyte dissociation and calcium transient experiments.50 ng / ml rmFGF21 were also used for 1 hour incubation with the isolated cardiomyocytes to test the acute effect of FGF21 on intracellular calcium transients.

[0313] Isolated mouse ventricular myocytes were loaded for 12 min with Fluo-8 / AM (Invitrogen Inc., Eugene, OR, USA) in Tyrode’s solution containing (in mM): 140 NaCl, 4 KCl, 2.0 CaCl2, 1 MgCl2, 10 Hepes and 5.6 glucose followed by a 30 min wash in Tyrode’s solution.Fluorescent signals were acquired with IonOptix system (IonOptix Corp., Milton, MA, USA). Cells were paced at 1 Hz to achieve a steady state, then paced for 15 seconds at different pacing rates (0.5-1-3 Hz) with 15 seconds resting interval between different rates. Following background subtractions, data were calculated as a ratio of Fluo-8 fluorescence intensity over baseline (F / F0). Calcium transient traces at 1 Hz were analyzed using IonWizard (IonOptix) to determine a time to peak, decay time constants, and calcium transient peak amplitude. A ratio of delay calcium transients (DCTs) in one heart was calculated by dividing cell number with DCTs during the 15 second rest period after 15 seconds pacing with 3 Hz by a total cell number recorded in this heart dissociation. A total ratio of DCTs in one group was calculated by dividing a total cell number with DCTs by a total cell number recorded in the same group.

[0314] As shown in FIGs. 6A and 6D, ventricular myocytes isolated from mice administered AAV8-mFGF21 exhibited (i) a reduction in the time to restore calcium localization after a depolarization event and (ii) a percentage of cells with delayed (spontaneous) calcium transient (DCT), as compared to ventricular myocytes isolated from mice administered vehicle.

[0315] Without wishing to be bound by any theory, administration of a polynucleotide comprising a nucleic acid sequence comprising a coding sequence encoding an FGF21 pathway activating agent (e.g., an FGF21 polypeptide or variant thereof coding sequence, e.g., an FGF21 fusion polypeptide or variant thereof coding sequence, e.g., an antibody or antigen binding fragment coding sequence) may restore calcium handling, as compared to a relevant control, in subject suffering for a cardiac disease or disorder (e.g., ACM). Example 5: Evaluation of FGF21 and / or sTGFβR2 in a Mouse Model of ACM

[0316] FGF21 and sTGFBR2, alone or in combination, were evaluated in a PKP2cKO mouse model. A bigenic rAAV8 vector expressing both transgenes under the control of liver- specific promoters was constructed.

[0317] 12-week-old PKP2cKO (Cre +) mice were dosed at 1.5 × 1013vg / kg on Day -7 followed by induction of PKP2 knockout via tamoxifen injection for 4 consecutive days starting on Day 0. As shown in Table 3, the described study included a dual gene construct expressing both murine FGF21 and murine sTGFβR2 as well as an FGF21 only arm and sTGFβR2 only arm. Untreated PKP2cKO mice and wild-type (WT) (Cre - PKP2cKO mice) mice were included as controls. Electrocardiography (ECG) was performed on Day 21 for 30 minutes following an isoproterenol challenge to evaluate premature ventricular contractions (PVC)s. On Day 28, cardiacstructure and function was measured by echocardiogram followed by sacrifice and terminal evaluation of protein expression levels and heart histology. Table 3. In Vivo Study Design

[0318] As shown in FIGs. 7A-7C, all measures were significantly different for WT compared to PKP2cKO mice. Cardiac function (LVEF) was significantly improved in all treatment groups compared to untreated PKP2cKO mice. While cardiac function improved in the sTGFβR2 group, it was not as efficacious as the FGF21 group, and no added benefit was observed in the FGF21 + sTGFβR2 group. Cardiac structure was significantly improved, as measured by RV area, in the FGF21 and FGF21 + sTGFβR2 groups.

[0319] Without wishing to be bound by any theory, efficacy measured in the FGF21 + sTGFβR2 group (Group 3) may be driven by FGF21 given the lack of efficacy in the sTGFβR2 only arm (Group 5). Only the FGF21 arm significantly reduced the number of ectopic beats from premature ventricular contractions. No significant changes in fibrosis were observed (data not shown). Example 6: Provided Technologies can improve neuromuscular function

[0320] As demonstrated herein, provided technologies, among other things, can improve neuromuscular function in a subject (e.g., an animal model). Specifically, provided technologies may improve a CMAP reading (e.g., as determined using nerve stimulation e.g., repetitive nerve stimulation) as compared to a relevant control and / or reference. Thus, provided technologies may be useful in a treatment for a neurological condition, disease and / or disorder (e.g., ALS).

[0321] The neuromuscular junction (NMJ) is a point of communication between the nervous system and skeletal muscle. Repetitive nerve stimulation (RNS) is an electrophysiological study that evaluates the integrity of the NMJ. RNS utilizes an active electrode over a target muscle and a reference electrode over the distal target tendon. RNS repeatedly stimulates the target muscle at a specified rate and measures the resulting compound muscle action potential (CMAP) amplitudes. The CMAP is the sum of action potentials from several muscle fibers. A decrement of >10% between the first and fourth CMAPs can be consider abnormal (see, e.g., Datta N, Hoke A. Repetitive Nerve Stimulation. [Updated 2023 Jul 15]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025).

[0322] Animals were administered with either (i) control and theophylline (“theo”) or (ii) FGF21 prior to testing. Selected muscle (e.g., tibialis anterior) was tested in the animals and selected nerves (e.g., sciatic nerve) were stimulated. Motor responses are recorded from an intramuscular electrode in the selected muscle (e.g., tibialis anterior). The stimulus intensity was increased until the compound muscle action potential (CMAP) was maximized. The amplitude of the CMAP and corresponding stimulation were determined (Figure not shown). For RNS, stimulation intensity is increased at 120% of the intensity required to record the CMAP. Trains (e.g., 4 trains) of at least about 10 stimuli are administered at various rates (e.g., 3 Hz), while the muscle action potentials are recorded. Decrement is expressed at the percentage of decrease of the max amplitude from baseline between, the 1st and the lowest response of each train. The percent change from the initial measurement was calculated.

[0323] As shown in FIGs. 8A-8D, animals administered with an agent that increases FGF21 demonstrated a resistance to decreasing CMAP amplitudes as compared to animals administered with control and theo. Without wishing to be bound to any particular theory, improvements in CMAP and RNS readings may indicate improvements in neuromuscular function. * * *

[0324] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0325] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.SEQUENCE LISTING The following table provides a listing of the nucleic acid and amino acid sequences represented by the various SEQ ID NOs used throughout the specification.EXEMPLARY EMBODIMENTS Embodiment 1. An isolated polynucleotide comprising an FGF21 coding sequence having at least 80% sequence identity to SEQ ID NO: 6. Embodiment 2. The polynucleotide of embodiment 1, wherein the FGF21 coding sequence encodes an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 27. Embodiment 3. The polynucleotide of embodiment 1 or 2, comprising a transcriptional regulatory element (TRE) operably linked to the FGF21 coding sequence. Embodiment 4. The polynucleotide of embodiment 3, wherein the TRE is a constitutively active or an inducible TRE. Embodiment 5. The polynucleotide of embodiment 3 or 4, wherein the TRE is a ubiquitous or tissue-specific TRE. Embodiment 6. The polynucleotide of any one of embodiments 3-5, wherein the TRE is a liver-specific TRE. Embodiment 7. The polynucleotide of any one of embodiments 3-6, wherein the TRE comprises an enhancer element. Embodiment 8. The polynucleotide of any one of embodiments 3-7, wherein the TRE comprises an ApoE enhancer element. Embodiment 9. The polynucleotide of any one of embodiments 3-8, wherein the TRE comprises two or more ApoE enhancer elements, optionally three ApoE enhancer elements. Embodiment 10. The polynucleotide of any one of embodiments 3-9, wherein the TRE comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 28, 40, and 41. Embodiment 11. The polynucleotide of any one of embodiments 3-10, wherein the TRE comprises a promoter. Embodiment 12. The polynucleotide of any one of embodiments 3-11, wherein the TRE comprises a human alpha 1-antitrypsin (hAAT) promoter. Embodiment 13. The polynucleotide of embodiment 12, wherein the hAAT promoter comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. Embodiment 14. The polynucleotide of any one of embodiments 3-13, wherein the TRE comprises an intron element. Embodiment 15. The polynucleotide of embodiment 14, wherein the intron element comprises a β-globin intron element. Embodiment 16. The polynucleotide of embodiment 14 or 15, wherein the intron element comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5. Embodiment 17. The polynucleotide of any one of embodiments 3-16, wherein the TRE comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 29, 30, and 31. Embodiment 18. The polynucleotide of any one of the preceding embodiments, comprising a post-transcriptional regulatory element (PRE) operably linked to the FGF21 coding sequence.Embodiment 19. The polynucleotide of embodiment 18, wherein the PRE comprises a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE). Embodiment 20. The polynucleotide of embodiment 19, wherein the WPRE comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 7. Embodiment 21. The polynucleotide of any one of embodiments 18-20, wherein the PRE comprises a polyadenylation sequence. Embodiment 22. The polynucleotide of embodiment 21, wherein the polyadenylation sequence comprises a simian virus 40 polyadenylation (SV40pA) sequence. Embodiment 23. The polynucleotide of embodiment 21 or 22, wherein the polyadenylation sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 8, 32, and 33. Embodiment 24. The polynucleotide of any one of embodiments 18-23, wherein the PRE comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 34 and 35. Embodiment 25. An isolated polynucleotide comprising, from 5’ to 3’: (a) a TRE comprising from 5’ to 3’: an ApoE enhancer element, a human alpha 1-antitrypsin (hAAT) promoter, and an intron element; (b) an FGF21 coding sequence; and (c) a PRE comprising from 5’ to 3’: a WPRE and a polyadenylation sequence.Embodiment 26. The polynucleotide of embodiment 25, wherein the FGF21 coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 6, 42, 44, and 46. Embodiment 27. The polynucleotide of embodiment 25 or 26, wherein the FGF21 coding sequence encodes a polypeptide comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 27, 43, and 45. Embodiment 28. The polynucleotide of any one of embodiments 25-27, wherein the TRE comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 4, 5, 28, 29, 30, 31, 40, and 41. Embodiment 29. The polynucleotide of any one of embodiments 25-28, wherein the PRE comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 7, 8, 32, 33, 34, and 35. Embodiment 30. The polynucleotide of any one of the preceding embodiments, further comprising a Kozak consensus sequence 5’ to the FGF21 coding sequence. Embodiment 31. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 36. Embodiment 32. The polynucleotide of any one of embodiments 3-31, further comprising a 5’ stuffer sequence 5’ to the TRE.Embodiment 33. The polynucleotide of any one of embodiments 18-31, further comprising a 3’ stuffer sequence 3’ to the PRE. Embodiment 34. The polynucleotide of any one of embodiments 18-31, further comprising a 5’ stuffer sequence 5’ to the TRE and a 3’ stuffer sequence 3’ to the PRE. Embodiment 35. The polynucleotide of any one of embodiments 32-34, wherein the 5’ stuffer sequence and / or the 3’ stuffer sequence comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 10, 11, 12, 13, and 14. Embodiment 36. The polynucleotide of any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 15, 17, 19, 21, 23, and 25. Embodiment 37. A polynucleotide that is the complement of the polynucleotide of any one of the preceding embodiments. Embodiment 38. A polynucleotide that is the reverse complement of the polynucleotide of any one of the preceding embodiments. Embodiment 39. A vector comprising the polynucleotide of any one of the preceding embodiments. Embodiment 40. The vector of embodiment 39, which is a plasmid, a viral vector, or a DNA minimal vector. Embodiment 41. The vector of embodiment 39 or 40, which is an expression vector.Embodiment 42. The vector of embodiment 40 or 41, wherein the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated virus (AAV) vector, and a lentiviral vector. Embodiment 43. The vector of any one of embodiments 39-42, wherein the vector is an AAV vector. Embodiment 44. A recombinant adeno-associated virus (rAAV) genome comprising the polynucleotide of any one of embodiments 1-38. Embodiment 45. The rAAV genome of embodiment 44, wherein the polynucleotide further comprises a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence and a 3’ inverted terminal repeat (3’ ITR) nucleotide sequence. Embodiment 46. The rAAV genome of embodiment 45, wherein the 5’ ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1, and / or the 3’ ITR nucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9. Embodiment 47. The rAAV genome of any one of embodiments 44-46, comprising a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 18, 20, 22, 24, and 26. Embodiment 48. A recombinant AAV comprising an AAV capsid and the rAAV genome of any one of embodiments 44-47. Embodiment 49. The rAAV of embodiment 48, wherein the AAV capsid polypeptide is derived from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I,AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid polypeptide, or an engineered variant thereof. Embodiment 50. The rAAV of embodiment 48 or 49, wherein the AAV capsid polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 37, 38, and / or 39. Embodiment 51. A pharmaceutical composition comprising a polynucleotide of any one of embodiments 1-38, a vector of any one of embodiments 39-43, a rAAV genome of any one of embodiments 44-47, or an rAAV of any one of embodiments 48-50. Embodiment 52. The polynucleotide of any one of embodiments 1-38, the vector of any one of embodiments 39-43, the rAAV genome of any one of embodiments 44-47, the rAAV of any one of embodiments 48-50, or the pharmaceutical composition of embodiment 51, for use in medicine, for use as therapy, optionally gene therapy, or for use as a medicament. Embodiment 53. A packaging system for preparation of an rAAV, wherein the packaging system comprises: (a) a first nucleotide sequence encoding one or more AAV Rep proteins; (b) a second nucleotide sequence encoding an AAV capsid protein; and (c) a third nucleotide sequence comprising the rAAV genome sequence of any one of embodiments 44-47. Embodiment 54. The packaging system of embodiment 53, wherein the packaging system comprises a first vector comprising the first nucleotide sequence and the second nucleotide sequence, and a second vector comprising the third nucleotide sequence. Embodiment 55. The packaging system of embodiment 54, further comprising a fourth nucleotide sequence comprising one or more helper virus genes, optionally wherein the fourth nucleotide sequence is comprised within a third vector.Embodiment 56. The packaging system of embodiment 55, wherein the fourth nucleotide sequence comprises one or more genes from a virus selected from the group consisting of adenovirus, herpesvirus, vaccinia virus, and cytomegalovirus (CMV). Embodiment 57. The packaging system of embodiment 55 or 56, wherein the first vector, second vector, and / or the third vector is a plasmid. Embodiment 58. A method for recombinant preparation of an rAAV, the method comprising introducing the packaging system of any one of embodiments 53-57 into a cell under conditions whereby the rAAV is produced. Embodiment 59. A method comprising introducing into a cell the polynucleotide of any one of embodiments 1-38, the vector of any one of embodiments 39-43, the rAAV genome of any one of embodiments 44-47, the rAAV of any one of embodiments 48-50, or the pharmaceutical composition of embodiment 51. Embodiment 60. A method of expressing an FGF21 coding sequence in a cell, comprising introducing into the cell the polynucleotide of any one of embodiments 1-38, the vector of any one of embodiments 39-43, the rAAV genome of any one of embodiments 44-47, the rAAV of any one of embodiments 48-50, or the pharmaceutical composition of embodiment 51. Embodiment 61. A method of expressing an FGF21 coding sequence in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of embodiments 1-38, the vector of any one of embodiments 39-43, the rAAV genome of any one of embodiments 44-47, the rAAV of any one of embodiments 48-50, or the pharmaceutical composition of embodiment 51. Embodiment 62. A method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of embodiments 1-38, the vector of any one of embodiments 39-43, the rAAV genomeof any one of embodiments 44-47, the rAAV of any one of embodiments 48-50, or the pharmaceutical composition of embodiment 51. Embodiment 63. The method of embodiment 62, wherein the disease or disorder is selected from the group consisting of an arrhythmogenic cardiomyopathy (ACM), a mitral valve disease (MVD), and a familial partial lipodystrophy (FPL). Embodiment 64. The method of embodiment 63, wherein the ACM is selected from the group consisting of arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), and biventricular arrhythmogenic cardiomyopathy. Embodiment 65. The method of embodiment 63, wherein the MVD is selected from the group consisting of myxomatous mitral valve disease, mitral valve stenosis, mitral valve prolapse, and mitral valve regurgitation. Embodiment 66. The method of embodiment 63, wherein the FPL is selected from the group consisting of type 1 FPL, type 2 FPL, type 3 FPL, type 4 FPL, type 5 FPL, and type 6 FPL. Embodiment 67. The method of any one of embodiments 61-66, wherein the subject is selected from the group consisting of a human, a non-human primate, a canine, a feline, an equine, a bovine, a swine, an avian, and a rodent. Embodiment 68. The method of any one of embodiments 61-67, wherein the subject is human.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide comprising a coding sequence, wherein the coding sequence encodes a polypeptide comprising a fibroblast growth factor 21 (FGF21) pathway activating agent.

2. The polynucleotide of claim 1, wherein the FGF21 pathway activating agent comprises: (i) an FGF21 polypeptide or variant thereof, (ii) an FGF21 fusion polypeptide or variant thereof, or (iii) an antibody or antigen binding fragment.

3. The polynucleotide of claim 1 or 2, wherein the FGF21 pathway activating agent comprises an FGF21 polypeptide or variant thereof.

4. The polynucleotide of claim 2 or 3, wherein the FGF21 polypeptide or variant thereof comprises one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

5. The polynucleotide of any one of claims 2-4, wherein the FGF21 polypeptide or variant thereof comprises one or more amino acid deletions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

6. The polynucleotide of any one of claims 2-5, wherein the FGF21 polypeptide or variant thereof comprises one or more amino acid insertions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

7. The polynucleotide of claim 6, wherein the one or more amino acid insertions comprise a tag (e.g., for increased efficacy, e.g., for activation of one or more pathways, e.g., for simultaneous activation of one or more pathways, e.g., for activation of an FGF21 pathway, e.g., for activation of a GLP1 pathway, e.g., for simultaneous activation of an FGF21 pathway and GLP1 pathway).

8. The polynucleotide of any one of claims 2-7, wherein the FGF21 polypeptide or variant thereof comprises one or more structural modifications.

9. The polynucleotide of claim 8, wherein the one or more structural modifications comprise methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, or any combination thereof, of one or more amino acid residues.

10. The polynucleotide of claim 8 or 9, wherein the one or more substitutions, the one or more amino acid deletions, and / or the one or more structural modifications modulates a function of the FGF21 polypeptide or variant thereof.

11. The polynucleotide of claim 10, wherein the modulation of a function comprises increasing half-life, improving physicochemical properties, reducing aggregation, and / or reducing proteolysis of the FGF21 polypeptide or variant thereof.

12. The polynucleotide of claim 10 or 11, wherein the modulation of a function comprises increasing binding of the FGF21 polypeptide or variant thereof to a β-Klotho and / or FGF receptor as compared to a reference.

13. The polynucleotide of any one of claims 2-10, wherein the FGF21 polypeptide or variant thereof comprises an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 27, 43, 45, or 47.

14. The polynucleotide of claim 1 or 2, wherein the FGF21 pathway activating agent comprises an FGF21 fusion polypeptide or variant thereof.

15. The polynucleotide of claim 14, wherein the FGF21 fusion polypeptide or variant thereof comprises the FGF21 polypeptide or variant thereof of any one of claims 2-13.

16. The polynucleotide of claim 15, wherein the FGF21 fusion polypeptide or variant thereof further comprises an agent associated with the FGF21 polypeptide or variant thereof of any one of claims 2-13.

17. The polynucleotide of claim 16, wherein the agent extends a half-life of the FGF21 polypeptide or variant thereof to a longer length of time relative to a half-life of a reference polypeptide that does not include the agent.

18. The polynucleotide of claim 17, wherein the half-life is a serum half-life.

19. The polynucleotide of any one of claims 16-18, wherein the agent comprises an antibody or a fragment thereof.

20. The polynucleotide of claim 19, wherein the antibody or a fragment thereof comprises an IgG constant domain.

21. The polynucleotide of claim 20, wherein the IgG constant domain comprises IgG1.

22. The polynucleotide of any one of claims 16-18, wherein the agent comprises albumin or a fragment thereof.

23. The polynucleotide of claim 1 or 2, wherein the FGF21 pathway activating agent comprises an antibody or antigen binding fragment.

24. The polynucleotide of claim 2 or 23, wherein the antibody or antigen binding fragment activates an FGF receptor.

25. The polynucleotide of claim 2 or 23, wherein the antibody or antigen binding fragment activates β-Klotho.

26. The polynucleotide of any one of claims 2 and 23-25, wherein the antibody or antigen binding fragment activates a β-Klotho and FGF receptor complex.

27. The polynucleotide of any one of the preceding claims, wherein the polynucleotide further comprises one or more transcriptional regulatory elements (TREs).

28. The polynucleotide of any one of claims 1-27, wherein the polynucleotide comprises one or more TREs operably linked to the coding sequence.

29. The polynucleotide of claim 27 or 28, wherein the polynucleotide comprises one or more constitutively active TREs, one or more inducible TREs, one or more ubiquitous TREs, one or more tissue-specific TREs, or any combination thereof.

30. The polynucleotide of claim 29, wherein the polynucleotide comprises one or more tissue- specific TREs, and wherein the one or more tissue-specific TREs comprise one or more liver- specific TREs.

31. The polynucleotide of any one of claims 27-30, wherein the one or more TREs comprise one or more enhancer elements.

32. The polynucleotide of claim 31, wherein the one or more enhancer elements comprise one or more ApoE enhancer elements.

33. The polynucleotide of claim 31 or 32, wherein the one or more enhancer elements comprise two or more ApoE enhancer elements.

34. The polynucleotide of any one of claims 31-33, wherein the one or more enhancer elements comprise three ApoE enhancer elements.

35. The polynucleotide of claim 33, wherein the two or more ApoE enhancer elements each comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 28, 40, or 41.

36. The polynucleotide of claim 34, wherein the three or more ApoE enhancer elements each comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 28, 40, or 41.

37. The polynucleotide of claim 35, wherein the two or more ApoE enhancer elements are not the same.

38. The polynucleotide of claim 35, wherein the two or more ApoE enhancer elements are the same.

39. The polynucleotide of claim 36, wherein the three or more ApoE enhancer elements are not the same.

40. The polynucleotide of claim 36, wherein the three or more ApoE enhancer elements are the same.

41. The polynucleotide of any one of claims 27-40, wherein the one or more TREs comprises one or more promoters.

42. The polynucleotide of 41, wherein the one or more promoters comprise a human alpha 1- antitrypsin (hAAT) promoter.

43. The polynucleotide of claim 42, wherein the hAAT promoter comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

4.

44. The polynucleotide of any one of claims 27-43, wherein the one or more TREs comprise an intron element.

45. The polynucleotide of claim 44, wherein the intron element comprises a β-globin intron.

46. The polynucleotide of claim 44 or 45, wherein the intron element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

5.

47. The polynucleotide of any one of claims 27-46, wherein the one or more TREs comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 29, 30, or 31.

48. The polynucleotide of any one of claims 27-47, wherein the one or more TREs comprise: (i) two or more enhancer elements, optionally three enhancer elements; (ii) a promoter; (iii) an intron element; or (iv) any combination thereof.

49. The polynucleotide of claim 48, wherein: (i) the two or more enhancer elements comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 3 , 28 , 40 , or 41, optionally wherein the enhancer elements are not the same;(ii) the promoter comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4; or (iii) the intron element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

5.

50. The polynucleotide of any one of the preceding claims, wherein the polynucleotide comprises one or more post-transcriptional regulatory elements (PREs).

51. The polynucleotide of claim 50, wherein the one or more PREs are operably linked to the coding sequence.

52. The polynucleotide of claim 50 or 51, wherein the one or more PREs comprise a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE).

53. The polynucleotide of claim 52, wherein the WPRE comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

7.

54. The polynucleotide of any one of claims 50-53, wherein the one or more PREs comprise a polyadenylation sequence.

55. The polynucleotide of claim 54, wherein the polyadenylation sequence comprises a simian virus 40 polyadenylation (SV40pA) sequence or variant thereof.

56. The polynucleotide of claim 54 or 55, wherein the polyadenylation sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 8, 32, or 33.

57. The polynucleotide of any one of claims 47-56, wherein the PRE comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 34 or 35.

58. A polynucleotide comprising, from 5’ to 3’: (i) one or more TREs comprising from 5’ to 3’: (a) one or more ApoE enhancer elements, (b) a human alpha 1-antitrypsin (hAAT) promoter, and (c) an intron element; (ii) a coding sequence encoding a polypeptide comprising an FGF21 pathway activating agent; and (iii) one or more PREs comprising: (a) a WPRE, and (b) a polyadenylation sequence.

59. The polynucleotide of claim 58, wherein the FGF21 pathway activating agent comprises: (i) an FGF21 polypeptide or variant thereof, (ii) an FGF21 fusion polypeptide or variant thereof, or (iii) an antibody or antigen binding fragment.

60. The polynucleotide of claim 58 or 59, wherein the FGF21 pathway activating agent comprises an FGF21 polypeptide or variant thereof.

61. The polynucleotide of claim 59 or 60, wherein the FGF21 polypeptide or variant thereof comprises one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

62. The polynucleotide of any one of claims 59-61, wherein the FGF21 polypeptide or variant thereof comprises one or more amino acid deletions relative to the amino acid sequence of SEQ ID NO: 27, 43, 45, or 47.

63. The polynucleotide of any one of claims 59-62, wherein the FGF21 polypeptide or variant thereof comprises one or more structural modifications.

64. The polynucleotide of claim 63, wherein the one or more structural modifications comprise methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, or any combination thereof, of one or more amino acid residues.

65. The polynucleotide of any one of claims 63-64, wherein the one or more substitutions, the one or more amino acid deletions, and / or the one or more structural modifications modulates a function of the FGF21 polypeptide or variant thereof.

66. The polynucleotide of claim 65, wherein the modulation of a function comprises increasing half-life, improving physicochemical properties, reducing aggregation, and / or reducing proteolysis of the FGF21 polypeptide or variant thereof.

67. The polynucleotide of claim 65 or 66, wherein the modulation of a function comprises increasing binding of the FGF21 polypeptide or variant thereof to a β-Klotho and / or FGF receptor as compared to a reference.

68. The polynucleotide of any one of claims 59-67, wherein the FGF21 polypeptide or variant thereof comprises an amino acid sequence at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 27, 43, 45, or 47.

69. The polynucleotide of claim 58 or 59, wherein the FGF21 pathway activating agent comprises an FGF21 fusion polypeptide or variant thereof.

70. The polynucleotide of claim 69, wherein the FGF21 fusion polypeptide or variant thereof comprises the FGF21 polypeptide or variant thereof of any one of claims 59-68.

71. The polynucleotide of any one of claims 59-70, wherein the FGF21 fusion polypeptide or variant thereof further comprises an agent associated with the FGF21 polypeptide or variant thereof of any one of claims 59-68.

72. The polynucleotide of claim 71, wherein the agent extends a half-life of the FGF21 polypeptide or variant thereof to a longer length of time relative to a half-life of a reference polypeptide that does not include the agent.

73. The polynucleotide of claim 72, wherein the half-life is a serum half-life.

74. The polynucleotide of any one of claims 71-73, wherein the agent comprises an antibody or a fragment thereof.

75. The polynucleotide of claim 74, wherein the antibody or a fragment thereof comprises an IgG constant domain.

76. The polynucleotide of claim 75, wherein the IgG constant domain comprises IgG1.

77. The polynucleotide of any one of claims 71-73, wherein the agent comprises albumin or a fragment thereof.

78. The polynucleotide of claim 58 or 59, wherein the FGF21 pathway activating agent comprises an antibody or antigen binding fragment.

79. The polynucleotide of claim 59 or 78, wherein the antibody or antigen binding fragment activates an FGF receptor.

80. The polynucleotide of claim 59 or 78, wherein the antibody or antigen binding fragment activates β-Klotho.

81. The polynucleotide of any one of claims 59 and 79-80, wherein the antibody or antigen binding fragment activates a β-Klotho and FGF receptor complex.

82. The polynucleotide of any one of claims 58-81, wherein the coding sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 6, 42, 44, 46, or 48.

83. The polynucleotide of any one of claims 58-82, wherein the coding sequence encodes a polypeptide that comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 27, 43, 45, or 47.

84. The polynucleotide of any one of claims 58-83, wherein the one or more TRE sequences comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 3, 4, 5, 28, 29, 30, 31, 40, or 41.

85. The polynucleotide of any one of claims 58-84, wherein the one or more PRE sequences comprise a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 7, 8, 32, 33, 34, or 35.

86. The polynucleotide of any one of claims 58-85, further comprising a Kozak consensus sequence 5’ to the coding sequence.

87. The polynucleotide of any one of claims 58-86, wherein the polynucleotide comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

36.

88. The polynucleotide of any one of claims 58-87, further comprising a 5’ stuffer sequence positioned 5’ to the one or more TREs.

89. The polynucleotide of any one of claims 58-87, further comprising a 3’ stuffer sequence positioned 3’ to the one or more PREs.

90. The polynucleotide of any one of claims 58-89, further comprising a 5’ stuffer sequence positioned 5’ to the one or more TREs and a 3’ stuffer sequence positioned 3’ to the one or more PREs.

91. The polynucleotide of any one of claims 88-90, wherein the 5’ stuffer sequence and / or the 3’ stuffer sequence comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID Nos: 2, 10, 11, 12, 13, and 14.

92. The polynucleotide of any one of claims 59-91, wherein the polynucleotide comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 15, 17, 19, 21, 23, and 25.

93. A vector comprising the polynucleotide of any one of the preceding claims.

94. The vector of claim 93, wherein the vector is or comprises a plasmid, a viral vector, or a DNA minimal vector.

95. The vector of claim 93 or 94, wherein the vector is or comprises an expression vector.

96. The vector of claim 94, wherein the viral vector is or comprises an adenoviral vector, an adeno-associated virus (AAV) vector, or a lentiviral vector.

97. The vector of any one of claims 93-96, wherein the vector is an AAV vector.

98. A recombinant adeno-associated virus (rAAV) genome comprising the polynucleotide of any one of claims 1-92.

99. The rAAV genome of claim 98, wherein the polynucleotide further comprises a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence and a 3’ inverted terminal repeat (3’ ITR) nucleotide sequence.

100. The rAAV genome of claim 99, wherein the 5’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and / or the 3’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

9.

101. The rAAV genome of any one of claims 98-100, wherein the rAAV genome comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16, 18, 20, 22, 24, or 26.

102. An rAAV particle comprising an AAV capsid and the rAAV genome of any one of claims 98-101.

103. The rAAV particle of claim 102, wherein the AAV capsid polypeptide is derived from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3,AAV4, AAV8 AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid polypeptide, or an engineered variant thereof.

104. The rAAV particle of claim 102 or 103, wherein the AAV capsid polypeptide comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 37, 38, or 39.

105. A composition comprising a fibroblast growth factor 21 (FGF21) pathway activating agent, wherein the FGF21 pathway activating agent comprises: (i) an FGF21 polypeptide or variant thereof, (ii) a peptide, (iii) an FGF21 fusion polypeptide or variant thereof, (iv) an antibody or antigen binding fragment, or (v) a combination thereof.

106. The composition of claim 105, wherein the pathway activating agent activates a FGF receptor.

107. The composition of claim 105, wherein the pathway activating agent activates β-Klotho.

108. The composition of claim 105, wherein the pathway activating agent activates a β-Klotho and FGF receptor complex.

109. A pharmaceutical composition comprising the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108.

110. The polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, orthe composition of any one of claims 105-108, or the pharmaceutical composition of claim 10 for use in medicine, for use as therapy, optionally gene therapy, or for use as a medicament.

111. A packaging system for preparation of an rAAV, wherein the packaging system comprises: (i) a first nucleotide sequence encoding one or more AAV Rep polypeptides; (ii) a second nucleotide sequence encoding an AAV capsid polypeptide; and (iii) a third nucleotide sequence comprising the rAAV genome sequence of any one of claims 98-101.

112. The packaging system of claim 111, wherein the packaging system comprises a first vector comprising the first nucleotide sequence and the second nucleotide sequence, and a second vector comprising the third nucleotide sequence.

113. The packaging system of claim 112, further comprising a fourth nucleotide sequence comprising one or more helper virus genes, optionally wherein the fourth nucleotide sequence is comprised within a third vector.

114. The packaging system of claim 113, wherein the fourth nucleotide sequence comprises one or more genes from a virus selected from the group consisting of adenovirus, herpesvirus, vaccinia virus, and cytomegalovirus (CMV).

115. The packaging system of claim 112 or 113, wherein the first vector, second vector, and / or the third vector is a plasmid.

116. A method for recombinant preparation of an rAAV, the method comprising introducing the packaging system of any one of claims 111-115 into a cell under conditions whereby the rAAV is produced.

117. A method comprising introducing into a cell the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAVparticle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109.

118. A method of expressing an FGF21 coding sequence in a cell, comprising introducing into the cell the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109.

119. A method of expressing an FGF21 coding sequence in a subject, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109.

120. A method of treating a disease, disorder, or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109.

121. The method of claim 120, wherein the disease, disorder, or condition is a cardiac disease, disorder, or condition associated with an arrhythmia.

122. The method of claim 120, wherein the disease, disorder, or condition is a neurological disease, disorder, or condition.

123. The method of claim 120, wherein the disease, disorder, or condition is associated with aberrant calcium signaling.

124. A method of treating atrial fibrillation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one ofclaims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98- 101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105- 108, or the pharmaceutical composition of claim 109.

125. A method of treating arrhythmogenic cardiomyopathy (ACM) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109.

126. The method of claim 125, wherein the ACM is associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), and / or biventricular arrhythmogenic cardiomyopathy.

127. A method of treating mitral valve disease (MVD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98- 101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105- 108, or the pharmaceutical composition of claim 109.

128. The method of claim 127, wherein the MVD is associated with myxomatous mitral valve disease, mitral valve stenosis, mitral valve prolapse, and / or mitral valve regurgitation.

129. A method of treating familial partial lipodystrophy (FPL) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109.

130. The method of claim 129, wherein the FPL is associated with type 1 FPL, type 2 FPL, type 3 FPL, type 4 FPL, type 5 FPL, and / or type 6 FPL.

131. The method of any one of claims 119-130, wherein the subject is a human, a non-human primate, a canine, a feline, an equine, a bovine, a swine, an avian, or a rodent.

132. The method of any one of claims 119-131, wherein the subject is human.

133. A method of measuring calcium signaling in a subject, the method comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98- 101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105- 108, or the pharmaceutical composition of claim 109.

134. The method of claim 133, wherein calcium signaling is measured from a sample.

135. The method of claim 134, wherein the sample is obtained from the subject.

136. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 in a method of treating of a disease or disorder in a subject in need thereof.

137. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 for the treatment of a disease or disorder in a subject in need thereof.

138. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 in the manufacture of a medicament for the treatment of a disease or disorder in a subject in need thereof.

139. The use of any one of claims 136-138, wherein the disease or disorder is a cardiac disease or disorder associated with arrhythmias.

140. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 in a method of treating ACM in a subject in need thereof.

141. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 for the treatment of ACM in a subject in need thereof.

142. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 in the manufacture of a medicament for the treatment of ACM in a subject in need thereof.

143. The use of any one of claims 140-142, wherein the ACM is associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), and / or biventricular arrhythmogenic cardiomyopathy.

144. Use of a polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109, or a pharmaceutical composition of claim 105 in a method of treating MVD in a subject in need thereof.

145. Use of a polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104,or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 for the treatment of MVD in a subject in need thereof.

146. Use of a polynucleotide of any one of claims 1-92, the vector of any one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109in the manufacture of a medicament for the treatment of MVD in a subject in need thereof.

147. The use of any one of claims 144-146, wherein the MVD is associated with myxomatous mitral valve disease, mitral valve stenosis, mitral valve prolapse, and / or mitral valve regurgitation.

148. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 in a method of treating FPL in a subject in need thereof.

149. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 for the treatment of FPL in a subject in need thereof.

150. Use of the polynucleotide of any one of claims 1-92, the vector of any one of claims 93- 97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102- 104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109 in the manufacture of a medicament for the treatment of FPL in a subject in need thereof.

151. The use of any one of claims 148-150, wherein the FPL is associated with type 1 FPL, type 2 FPL, type 3 FPL, type 4 FPL, type 5 FPL, and / or type 6 FPL.

152. A method of activating an FGF21 pathway, comprising administering to a subject a therapeutically effective amount of the polynucleotide of any one of claims 1-92, the vector ofany one of claims 93-97, the rAAV genome of any one of claims 98-101, the rAAV particle of any one of claims 102-104, or the composition of any one of claims 105-108, or the pharmaceutical composition of claim 109.

153. The method of claim 152, wherein the method comprises treating a disease, disorder or condition in a subject in need thereof.

154. The method of claim 153, wherein the disease, disorder or condition is one or more of ACM, MVD, and / or FPL.

Images