AAV vectors for delivery of nucleic acids encoding FGF21 and methods of treating lung diseases using the same

Abstract

According to various aspects of this disclosure, the present disclosure relates to a method for treating or reducing pulmonary inflammation and / or pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a ubiquitous promoter.

Description

AAV VECTORS FOR DELIVERY OF NUCLEIC ACIDS ENCODING FGF21 AND METHODS OF TREATING LUNG DISEASES USING THE SAMECROSS REFERENCE TO RELATED APPLICATIONS[00011 The present application claims the priority benefit of U.S. Provisional Application No. 63 / 734,583, filed December 16, 2024; which is hereby incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 5344_012PC01_SequenceListing_ST26.xml; Size: 102,845 bytes; and Date of Creation: December 15, 2025), filed with the application, is incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to the medical field, including nucleic acid therapies for treating and preventing lung diseases.BACKGROUND OF THE DISCLOSURE

[0004] The pathological increase in adiposity that occurs in obesity has been shown to cause detrimental effects in the lung, leading to inflammation and subsequent fibrosis of this tissue (Cheng, X., et al, Lipids Health Dis. 22(l):201 (2023)). Interestingly, FGF receptors and co-receptor beta Klotho (KLB) are expressed in pulmonary tissue (Joannes, A., et al., American Journal of Physiology. Lung Cellular and Molecular Physiology 310:L615-L629 (2016)). A recent investigation suggested the anti-fibrotic potential of FGF21 in pulmonary fibrosis, demonstrating that a combination of systemic FGF21 overexpression and treatment with peghelfermin (a PEGylated FGF21 analogue) effectively reduced pulmonary fibrosis in mice induced after a bleomycin challenge. Conversely, the deletion of FGF21 in mice was associated with an augmented fibroticresponse in a model of pulmonary fibrosis (Ghanem, M., et al., Pharmacol Ther. 259: 108669 (2024)).|0005] Furthermore, an increased in lung inflammation associated with aging has been described (Faniyi, A., et al., British Journal of Pharmacology 179: 1790-1807 (2022); Torrelles, J., et al., Frontiers in Aging 3:818700 (2022); Wang, Y., et al., Frontiers in Immunology 15: 1383503 (2024); Joshi, P.R., Geriatrics 9:34 (2024); and Li, X., et al., Signal Transduction and Targeted Therapy 8:239 (2023)).BRIEF SUMMARY

[0006] Certain aspects of the present disclosure provide a method for treating or reducing pulmonary inflammation and / or pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a fibroblast growth factor 21 (FGF21) protein or functional fragment thereof operably linked to a promoter (e.g., a ubiquitous promoter).

[0007] Certain aspects of the present disclosure provide a method for treating or reducing an inflammatory lung disease in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) protein or functional fragment thereof operably linked to a promoter (e.g., a ubiquitous promoter).

[0008] Certain aspects of the present disclosure provide a method for treating, preventing or reducing the likelihood of pulmonary fibrosis, e.g. idiopathic pulmonary fibrosis, in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growthfactor 21 (FGF21) protein or a functional fragment thereof operably linked to a promoter (e.g., a ubiquitous promoter).(0009] In some aspects, the pulmonary inflammation is caused by or associated with an infection, an inflammatory condition, a genetic condition, a medication, aging and / or a chemical exposure.100101 In some aspects, the pulmonary inflammation is a pneumonitis.

[0011] In some aspects, the pneumonitis is a hypersensitivity pneumonitis, a drug- induced pneumonitis, or a radiation-induced pneumonitis.

[0012] In some aspects, the pneumonitis is a hypersensitivity pneumonitis.|0013] In some aspects, the hypersensitivity pneumonitis is caused by exposure to a mold, a bacteria, a bird (e.g., exposure to bird feathers or bird droppings), dust, chemicals, or animal fur (e.g., exposure to skin cells, fur, or droppings from animals).(0014] In some aspects, the pulmonary inflammation is a chronic inflammation.

[0015] In some aspects, the pulmonary inflammation is caused by chronic obstructive pulmonary disease (COPD).

[0016] In some aspects, the inflammatory lung disease, lung disorder, or lung condition is COPD.

[0017] In some aspects, the pulmonary inflammation is a pulmonary sarcoidosis.10018] In some aspects, the inflammatory lung disease, lung disorder, or lung condition is asthma.

[0019] In some aspects, the inflammatory lung disease, lung disorder, or lung condition is bronchitis.

[0020] In some aspects, the subject suffers from asbestosis. In some aspects, the inflammatory lung disease, lung disorder, or lung condition is caused by asbestosis.

[0021] In some aspects, the subject suffers from obesity and / or insulin resistance.

[0022] In some aspects, the subject does not suffer from obesity and / or insulin resistance.

[0023] In some aspects, the ubiquitous promoter comprises a small chicken beta-actin promoter / cytomegalovirus enhancer (smCBA) promoter, a eukaryotic translation elongation factor 1 a (EFla) promoter, a simian virus 40 (SV40) promoter, a cytomegalovirus (CMV) promoter or a CAG promoter.

[0024] In some aspects, the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter.

[0025] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence encoding an amino acid sequence having at least 85%, 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 amino acids 29-209 of SEQ ID NO: 1 and a leader sequence.10026] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence encoding an amino acid sequence having at least 85%, 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.

[0027] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 nucleic acids 85-627 of any of SEQ ID NO: 4, 5, 6, or 7.

[0028] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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, 5, 6, or 7.

[0029] In some aspects, the leader sequence comprises a signal peptide selected from a human FGF21 signal peptide, a human IL- 10 (hIL-10) signal peptide, a human IL-6 (hlL- 6) signal peptide, a human FSH signal peptide, a human Albumin signal peptide, a human CSF signal peptide, a mouse immunoglobulin kappa light chain signal peptide (KASP), a Gaussia Luciferase signal peptide (GLSP), a human Igk signal peptide, a human Apolipoprotein signal peptide, a human PEDF signal peptide, a human SPARC signal peptide, a human tPA signal / propeptide (amino acids 1-32), a human tPA signal / propeptide (amino acids 1-35), a human IgVH signal peptide, a human GCG signal peptide, a human GIP signal peptide + GIP propeptide, or a human Prothrombin signal peptide.

[0030] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof further comprises a nucleotide sequence encoding a leader sequence comprising a signal peptide selected from a human IL-10 (hIL-10) signalpeptide, a human IL-6 (hIL-6) signal peptide, a human FGF21 signal peptide, a human FSH signal peptide, a human Albumin signal peptide, a human CSF signal peptide, a mouse immunoglobulin kappa light chain signal peptide (KASP), a Gaussia Luciferase signal peptide (GLSP), a human Igk signal peptide, a human Apolipoprotein signal peptide, a human PEDF signal peptide, a human SPARC signal peptide, a human tPA signal / propeptide (amino acids 1-32), a human tPA signal / propeptide (amino acids 1-35), a human IgVH signal peptide, a human GCG signal peptide, a human GIP signal peptide + GIP propeptide, or a human Prothrombin signal peptide.

[0031] In some aspects, the rAAV comprises a polynucleotide having at least 85%, 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: 14.

[0032] In some aspects, the expression construct further comprises one or more of a polyadenylation signal, at least one target sequence of a microRNA, and / or an enhancer sequence.

[0033] In some aspects, the rAAV vector is administered as a single dose.

[0034] In some aspects, the rAAV vector is administered over multiple doses.

[0035] In some aspects, the subject has at least a 0.1-6.0 fold reduction in a relative expression of an inflammatory gene marker selected from the group consisting of Cluster of Differentiation 68 (CD68), F4 / 80, Tumor necrosis factor-alpha (TNFa), Monocyte Chemoattractant Protein- 1 (MCP1), Interleukin-6 (IL-6), or any combination thereof, optionally wherein the relative expression is measured by qRT-PCR.

[0036] In some aspects, the subject has at least a 0.1-6.0 fold reduction in a relative expression of a fibrosis gene marker selected from the group consisting of collagen type I alpha 1 chain (Collal), collagen type III alpha 1 chain (Col3al), transforming growth factor beta 1 (Tgf-fB), fibronectin 1 (Fnl) and alpha smooth muscle actin (a-Sma), or any combination thereof, optionally, wherein the relative expression is measured by qRT- PCR.

[0037] In some aspects, the methods of the disclosure treat or prevent one or more symptoms associated with the lung disorder, the lung condition, the pulmonary inflammation and / or the pulmonary fibrosis. In some aspects, the symptoms associated with the inflammatory lung disease, the lung disorder, the lung condition, the pulmonaryinflammation and / or the pulmonary fibrosis comprise shortness of breath, a persistent cough (e.g., dry cough), fatigue, or any combination thereof.DESCRIPTION OF FIGURES

[0038] FIGs. 1A-1B show histological assessment of pulmonary inflammation.Representative images of lung sections from mice fed control chow (left), high-fat diet (HFD) (middle), or HFD and treated with AAV-FGF21 (right) and immunostained against the macrophage-specific marker MAC2 (bright field in FIG. 1A, fluorescence in FIG. IB). Arrows indicate MAC2+ cells. Scale bars: 200 pm (FIG. 1A, upper panel) and 400 pm. (FIG. 1A, lower panel and FIG. IB).(0039] FIGs. 2A-2E shows AAV-FGF21 treatment counteracted pulmonary inflammation induced by HFD. (FIGs. 2A-2E) Expression levels of the inflammatory markers Tnfa (FIG. 2A), Mcpl (FIG. 2B), CD68 (FIG. 2C), F4 / 80 (FIG. 2D) and IL-6 (FIG. 2E) are shown in the lung. Results are shown as mean ± SEM. n= 5-7 animals / group. *P<0.05, **P<0.01, and ***P<0.001.

[0040] FIGs. 3A-3E shows that long-term HFD-feeding did not induce pulmonary fibrosis. (FIGs. 3A-3E) Expression levels of the fibrotic markers Col la (FIG. 3A), Col3a (FIG. 3B), Tgfp (FIG. 3C), Fnl (FIG. 3D) and aSma (FIG. 3E) are shown in the lung. Results are shown as mean ± SEM. n= 5-7 animals / group.(0041] FIG. 4 shows histological analysis of fibrosis in lung. Representative images of Picrosirius Red staining of lung sections showing collagen fibers (in red). Middle and lower panels are insets of images shown in the upper panel. Scale bars: 100 pm (upper panel), 200 pm (middle panel) and 400 pm (lower panel).(0042] FIGs. 5A-5B show histological assessment of pulmonary inflammation.Representative images of lung sections from db / db mouse control (left) or treated with AAV-FGF21 (right) and immunostained against the macrophage-specific marker MAC2 (bright field in FIG. 5A, fluorescence in FIG. 5B). Arrows indicate MAC2+ cells. Scale bars: 200 pm (FIG. 5A, upper panel) and 400 pm (FIG. 5A, lower panel and FIG. 5B).

[0043] FIGs. 6A-6E show that treatment with AAV-FGF21 vectors reduced pulmonary inflammation in db / db mice. Expression levels of the inflammatory markers Tnfa (FIG. 6A), F4 / 0 (FIG. 6B), CD68 (FIG. 6C), Mcpl (FIG. 6D) and IL-6 (FIG. 6E) are shown in lung. Results are shown as mean ± SEM. n= 6-8 animals / group. *P<0.05, **P<0.01.100441 FIGs. 7A-7E show decreased expression of fibrotic markers in lungs of db / db mice treated IM with AAV-vectors. Expression levels of the fibrotic markers Col la (FIG. 7A), Col3a (FIG. 7B), Tgf (FIG. 7C), Fnl (FIG. 7D) and aSma (FIG. 7E) are shown in the lung. Results are shown as mean ± SEM. n= 6-8 animals / group. **P<0.01.

[0045] FIG. 8 shows histological analyses of lung sections by picrosirius red staining. Representative images of Picrosirius Red staining of lung sections showing collagen fibers are displayed. Middle and lower panels are insets of the upper panel. Scale bars: 100 pm (upper panel), 200 pm (middle panel) 400 pm (lower panel).

[0046] FIGs. 9A-9C show expression level of lung inflammatory markers: Tumor necrotic factor alpha (Tnfla) (FIG. 9A), F4 / 80 (FIG. 9B), and Transforming growth factor beta-1 (Tgfb) (FIG. 9C). Data are presented as mean ± SEM. *P<0.05, **P<0.01 by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test. FC, Fold change. N = 5-8 animals / group.

[0047] FIG. 10 shows Mac2 immunostaining of lung sections of healthy control mice (7- month-old), aged control mice (26-month-old), and aged mice (26-month-old) treated intramuscularly with AAV1-FGF21 at 13 months of age.

[0048] FIG. 11 shows histological analysis of fibrosis in lung in aged mice at 26 months for both control and AAV-FGF21 treated mice. Representative images of Picrosirius Red staining of lung sections showing collagen fibers. Scale bars: 400 pm.

[0049] FIG. 12 shows the percent area stained with picrosirius red in 26 month old control and AAV-FGF21 treated mice. Results are shown as mean ± SEM. n= 5 animals / group by unpaired Student's t-test. **P<0.01.DETAILED DESCRIPTION

[0050] Certain aspects of the present disclosure provide a method for treating or reducing pulmonary inflammation and / or pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a promoter (e.g., a ubiquitous promoter).|0051| Certain aspects of the present disclosure provide a method for treating or reducing an inflammatory lung disease, lung disorder, or lung condition in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a promoter (e.g., a ubiquitous promoter).

[0052] Certain aspects of the present disclosure provide a method for treating, preventing or reducing the likelihood of pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or a functional fragment thereof operably linked to a promoter (e.g., a ubiquitous promoter).I. Definitions

[0053] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0054] Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the detailed description and from the claims.

[0055] In order to further define this disclosure, the following terms and definitions are provided.|0056| The singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. In certain aspects, the term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "multiple."

[0057] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0058] The term "at least" prior to a number or series of numbers is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21 -nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).

[0059] Throughout this disclosure, various aspects are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Numeric ranges recited are inclusive of the numbers defining the range and include each integer within the defined range.

[0060] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integervalue, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0061] Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0062] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0063] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of are also provided.

[0064] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, formulations, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.|0065| The term "excipient" refers to any substance, not itself a therapeutic agent, which may be used in a composition for delivery of an active therapeutic agent to a subject or combined with an active therapeutic agent (e.g., to create a pharmaceutical composition) to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition. Excipients include, but are not limited to, solvents, penetration enhancers, wetting agents, antioxidants, lubricants, emollients, substances added to improve appearance or texture of the composition and substances used to form hydrogels. Any such excipients can be used in any dosage forms according to the present disclosure. The foregoing classes of excipients are not meant to be exhaustive but merely illustrative as a person of ordinary skill in the art would recognize that additional types and combinations of excipients could be used to achieve the desired goals for delivery of a drug. The excipient can be an inert substance, an inactive substance, and / or a not medicinally active substance. The excipient can serve various purposes. A person skilled in the art can select one or more excipients with respect to the particular desired properties by routine experimentation and without any undue burden. The amount of each excipient used can vary within ranges conventional in the art. Techniques and excipients which can be used to formulate dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009); and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

[0066] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0067] A "coding sequence" or a sequence "encoding" a particular molecule (e.g., a therapeutic molecule) is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of mRNA) into polypeptide, in vitro or in vivo, when operablylinked to an appropriate regulatory sequence, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence.

[0068] As used herein, the term "synthetic" means designed, produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present disclosure can be chemical or enzymatic.10069] "Nucleic acid," "polynucleotide," and "oligonucleotide," are used interchangeably in the present application. These terms refer to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms "nucleic acid," "polynucleotide," and "oligonucleotide," as used herein, are defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides can also be referred to as nucleic acid molecules or oligomers.Polynucleotides can be made, e.g., recombinantly, enzymatically, or synthetically, e.g., by solid-phase chemical synthesis followed by purification. When referring to a sequence of the polynucleotide or nucleic acid, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The disclosure of a polynucleotide or nucleic acid sequence can be understood to disclose the complementary sequences as well.10070] A kilobase (abbreviated: Kb) is a unit of length of DNA that is equal to 1,000 nucleotide bases (also referred to herein as nucleotides or bases).

[0071] The term "mRNA," as used herein, refers to a single stranded messenger RNA that encodes the amino acid sequence of one or more polypeptide chains.

[0072] "Introns" are noncoding sections of an RNA transcript, or the DNA encoding it, that are spliced out before the RNA molecule is translated into a protein.

[0073] The term "promoter" as used herein refers to a DNA sequence recognized by the machinery of the cell, or introduced synthetic machinery, required to initiate the transcription of a gene or coding sequence. The term "promoter" is also meant to encompass those nucleic acid elements sufficient for promoter-dependent gene or coding sequence expression controllable for cell-type specific, tissue-specific or inducible byexternal signals or agents; such elements can be located in the 5' or 3' regions of the gene. In some aspects, the promoter is a constitutively active promoter, a cell-type specific promoter, or an inducible promoter.

[0074] As used herein, "CAG promoter" refers to a synthetic promoter for driving gene expression which is constructed from a CMV early enhancer element; a promoter, first exon, and first intron of chicken beta-actin gene; and a splice acceptor of the rabbit betaglobin gene.

[0075] The terms "operatively linked," "operatively inserted," "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene or coding sequence. In some aspects, the term "operably linked" means that a DNA sequence and a regulatory sequence(s) are connected in such a way as to permit gene or coding sequence expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). In some aspects, the term "operably inserted" means that the DNA of interest introduced into the cell is positioned adjacent a DNA sequence which directs transcription and translation of the introduced DNA (i.e., facilitates the production of, e.g., a FGF21 protein encoded by a DNA of interest).

[0076] The terms "expression vector", "expression construct" or "expression cassette" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. Typically, an expression cassette comprises a promoter operably linked to a nucleic acid (e.g., a gene of interest).

[0077] The term "adeno-associated virus vector" or "AAV vector" as used herein refers to any vector that comprises or derives from components of an adeno-associated virus. The term AAV vector can be used to designate a vector comprising an AAV genome sequence (e.g., an AAV ITR) and a payload. In some aspects, the AAV vector is suitable to infect mammalian cells, preferably human cells. The term AAV vector can be used to designate an AAV-type viral particle or virion comprising a payload. The AAV vector can be derived from various serotypes, including combinations of serotypes (i.e., "pseudotyped" AAV) or from various genomes (e.g., single stranded or self-complementary). In addition, the AAV vector can be replication defective and / or targeted. As used herein, the term "adeno-associated virus" (AAV), includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAVtype 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh8, AAVrhlO, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), and any other AAV now known or later discovered. See, e.g., FIELDS et al.VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some aspects, an "AAV vector" includes a derivative of a known AAV vector. In some aspects, an "AAV vector" includes a modified or an artificial AAV vector. The terms "AAV capsid" and "AAV particle" can be used interchangeably. In some aspects, the AAV vector is modified or mutated relative to the wild-type AAV serotype sequence. In some aspects, the AAV particle that can comprise an AAV vector comprising at least one payload region (e.g., a polynucleotide encoding a gene of interest) and at least one AAV inverted terminal repeat (ITR) region, which is suitable to infect mammalian cells, preferably human cells.

[0078] As used herein, recombinant AAV vectors or rAAV vectors refer to AAV particles lacking viral genes and packaged with payload DNA encoding heterologous genes of interest. In some aspects, the rAAV vectors of the disclosure comprise a polynucleotide encoding a gene of interest flanked by ITRs, which is encapsulated in an AAV particle.

[0079] "scAAV" or "self-complementary adeno-associated virus" refers to an adeno- associated viral vector that comprises duplex nucleic acid sequence that are complementary to one another. The two complementary sequences will associate to form one double stranded DNA unit for replication and transcription.

[0080] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide or nucleoside sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C, or 70 °C, for 12-16 hours followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions,such as physiologically relevant conditions as can be encountered inside an organism, can be used. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides or nucleosides.

[0081] Complementary sequences can include, or be formed entirely from, non-Watson- Crick base pairs and / or base pairs formed from non-natural and alternative nucleotides or nucleosides, as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing. Complementary sequences within a dsRNA, or between an oligonucleotide and a target sequence as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide or nucleoside sequence to an oligonucleotide or polynucleotide comprising a second nucleotide or nucleoside sequence over the entire length of one or both nucleotide or nucleoside sequences. Such sequences can be referred to as "fully complementary" or "100% complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., reduction of expression via a RISC pathway. "Substantially complementary" can refer to a polynucleotide that is substantially complementary to a contiguous portion of the nucleic acid of interest (e.g., a DNA or an mRNA encoding a FGF21 protein). For example, a polynucleotide is complementary to at least a part of a nucleic acid molecule if the sequence is substantially complementary to a non-interrupted portion of a nucleic acid molecule encoding a therapeutic molecule. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a shRNA comprising one oligonucleotide of 21 linked nucleosides in length and another oligonucleotide of 23 nucleosides in length, wherein the longer oligonucleotide comprises a sequence of 21 linked nucleosides that is fully complementary to the shorter oligonucleotide, can be referred to as "fully complementary" for the purposes described herein.100821 "Inverted terminal repeat" (abbreviated: ITR) refers to a single stranded sequence of nucleotides, typically, followed downstream by its reverse complement sequence or a sequence similar to the reverse complement. Naturally occurring ITR sequences are approximately 145 bases each (AAV-1 has ITRs of 143 nucleotides), and AAV plasmids typically include two ITR sequences. The DNA sequence between the ITRs is packaged into the AAV molecule. The intervening sequence of nucleotides between the initial sequence and the reverse complement (or a sequence similar to the reverse complement) can be any length. ITRs can adopt orientations at the ends of the AAV genome that are described, e.g., as FLIP / FLOP, FLOP / FLIP, FLIP / FLIP, or FLOP / FLOP.

[0083] "Flanking" or "flanked by" as used herein in the context of the relationship or location of sequences relative to one another, refers to one or more sequences (e.g., ITRs) being on each or on one side of another sequence (e.g., an expression cassette). In some aspects, a sequence can flank another sequence that is immediately beside the sequence. In some aspects, a sequence can flank another sequence where there is an intervening sequence between the sequences.

[0084] The phrase "contacting a cell" (e.g., contacting a cell with the AAV vectors, the AAV capsids, or the pharmaceutical compositions of the disclosure) as used herein, includes contacting a cell directly or indirectly. In some aspects, contacting a cell with the expression vector, the rAAV vector, or the pharmaceutical composition includes contacting a cell in vitro with the expression vector, the rAAV vector, or the pharmaceutical composition, or contacting a cell in vivo with the expression vector, the rAAV vector, or the pharmaceutical composition. Thus, for example, the expression vector, the rAAV vector, or the pharmaceutical composition can be put into physical contact with the cell by the individual performing the method, or alternatively, the expression vector, the rAAV vector, or the pharmaceutical composition can be put into a situation that will permit or cause it to subsequently come into contact with the cell.

[0085] In some aspects, contacting a cell in vitro can be done, for example, by incubating the cell with the expression vector, the rAAV vector, or the pharmaceutical composition. In some aspects, contacting a cell in vivo can be done, for example, by injecting the expression vector, the rAAV vector, or the pharmaceutical composition of the disclosure into or near the tissue where the cell is located (e.g., the skeletal muscle), or by injecting the expression vector, the rAAV vector, or the pharmaceutical composition into another area, such that the agent will subsequently reach the tissue where the cell to be contactedis located. For example, the expression vector can be encapsulated by an AAV capsid that directs the AAV vector to a site of interest. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell can be contacted in vitro with the expression vector, the rAAV vector, or the pharmaceutical composition and subsequently transplanted into a subject.100861 In some aspects, contacting a cell with the expression vector, the rAAV vector, or the pharmaceutical composition of the present disclosure includes "introducing" or "delivering" (directly or indirectly) the expression vector, the rAAV vector, or the pharmaceutical composition into the cell by facilitating or effecting uptake or absorption into the cell. Introducing the expression vector, the rAAV vector, or the pharmaceutical composition into a cell can be in vitro and / or in vivo. For example, for in vivo introduction the expression vector, the rAAV vector, or the pharmaceutical composition can be injected into a specific tissue site (e.g., the locus where a therapeutic effect is desired) or administered systemically (e.g., administering an AAV vector targeted to a locus where a therapeutic effect is desired). In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.

[0087] As used herein, the terms "effective amount," "therapeutically effective amount," and a "sufficient amount" of, e.g., expression vector, the rAAV vector, or the pharmaceutical composition disclosed herein refer to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount" or synonym thereto depends on the context in which it is being applied. In some aspects, a therapeutically effective amount of an agent (e.g. expression vector, the rAAV vector, or the pharmaceutical composition disclosed herein) is an amount that results in a beneficial or desired result in a subject as compared to a control.

[0088] The amount of a given agent (e.g., the expression vector, the rAAV vector, or the pharmaceutical composition disclosed herein) will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, and / or weight) or host being treated, and the like.

[0089] The term "prophylactically effective amount," as used herein, includes the amount of an agent, (e.g., the expression vector, the rAAV vector, or the pharmaceutical composition disclosed herein) that, when administered to a subject having or predisposedto have a disease or disorder (e.g., a lung disease or disorder), is sufficient to prevent, reduce the risk of, reduce the symptoms of, or ameliorate the disease or disorder or one or more symptoms of the disease or disorder. Ameliorating the disease or disorder includes slowing the course of the disease or disorder or reducing the severity of later-developing disease or disorder. The "prophylactically effective amount" can vary depending on the characteristics of the agent, e.g., the expression vector, the rAAV vector, or the pharmaceutical composition, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0090] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).10091] As used herein, the term "in vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0092] As used herein, the term "transfection" refers to methods to introduce exogenous nucleic acids into a cell. Methods of transfection include, but are not limited to, chemical methods, physical treatments and cationic lipids or mixtures. The list of agents that can be transfected into a cell is large and includes, e.g., mRNA, siRNA, shRNA, sense and / or anti-sense sequences, DNA encoding one or more genes or coding sequences including those organized into an expression plasmid, e.g., a vector.|0093] "Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST.|0094| The terms "administer," "administering," "administration," and the like, as used herein, refer to methods that may be used to enable delivery of a drug, e.g., the expression vector, the rAAV vector, or the pharmaceutical composition disclosed herein, to the desired site of biological action. Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0095] The terms "treat," "treatment," or "treating," as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition. The term also includes prophylaxis or prevention of a disease or condition or its symptoms thereof, unless indicated otherwise.

[0096] The terms "subject," "patient," "individual," and "host," and variants thereof are used interchangeably herein and refer to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications. As used herein, the phrase "subject in need thereof includes subjects, such as mammalian subjects, that would benefit from administration of a therapeutic agent, e.g., the expression vector, the rAAV vector, or the pharmaceutical composition disclosed herein.

[0097] As used herein, the terms "derived from" or "derivative" refer to a component that is isolated from or made using a specified molecule, or information (e.g., a nucleic acid sequence) from the specified molecule. For example, a polynucleotide sequence that is derived from another polynucleotide sequence can include a polynucleotide sequence that is identical or substantially similar to the polynucleotide sequence it derives from. In the case of polynucleotides, the derived species can be obtained by, for example, naturally occurring mutagenesis, artificial directed mutagenesis, or artificial random mutagenesis. The mutagenesis used to derive polynucleotides can be intentionally directed or intentionally random, or a mixture of each. The mutagenesis of a polynucleotide to createa different polynucleotide derived from the first polynucleotide can be a random event (e.g., caused by polymerase infidelity) and the identification of the derived polynucleotide can be made by appropriate screening methods known in the art. In some aspects, a polynucleotide sequence that is derived from a first polynucleotide sequence has a sequence identity of at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identity to the first polynucleotide sequence, respectively, wherein the derived polynucleotide sequence retains the biological activity of the original polynucleotide.[0098| As used herein, the term "functional fragment thereof' refers to a fragment or portion of a protein that is still capable of one or more functions associated with the full protein (e.g., stimulating, modulating, regulating, or modifying). In some aspects, a functional fragment can comprise a truncated protein.[ 00991 As used herein, "truncated" refers to a molecule or sequence (e.g., protein, polypeptide, nucleic acid, etc.) that is shortened by removing one or more amino acids or portions therein. The truncation(s) can be in any portion(s) throughout the molecule, e.g., not limited to the N or C terminal portions.[01001 As used herein, the term "delivery vector" or "vector" refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. A vector can be a replicon to which another nucleic acid segment can be attached so as to bring about the replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term "delivery vector" or "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini. Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / orselection of host cells that incorporate and express other coding regions contained on the vector. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), P-galactosidase (LacZ), P-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters. In some aspects, the delivery vector is selected from the group consisting of a viral vector (e.g., a recombinant AAV vector), a plasmid, a lipid, a protein particle, a bacterial vector, and a lysosome.

[0101] Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. In some aspects, treatment includes eliciting a clinically significant response without excessive levels of side effects. In some aspects, treatment includes prolonging survival as compared to expected survival if not receiving treatment. As used herein, the term "amelioration" or "ameliorating" refers to a lessening of severity of at least one indicator of a condition or disease. In some aspects, "preventing" or "prevention" includes delaying or forestalling the onset, development or progression of a condition or disease for a period of time, including weeks, months, or years.

[0102] "Disorder" is used interchangeably herein with "disease", "condition", and similar words to refer to any impairment of health or state of abnormal functioning of an organism, e.g., any state in which medical and / or surgical management is indicated or for which a subject appropriately seeks medical and / or surgical attention. It should also be understood that the listing of a particular disorder within a particular category is for convenience and is not intended to limit the invention. It will be understood that certain disorders could appropriately be listed in multiple categories.

[0103] As used herein, "skeletal muscle" refers to the tissue composed of muscle fibers.A muscle fiber, also known as a myofiber, is a single multinucleated or syncytial cell that results from the fusion of many hundreds of myoblasts, some of which remain in the mature muscle as undifferentiated cells known as satellite cells. Individual muscle fibers are surrounded by a connective tissue called endomysium. Around 10 to 100 musclefibers form fascicles, or bundles, which are themselves surrounded by another connective tissue layer called the perimysium. Finally, the skeletal muscle is formed by groups of fascicles that are surrounded also by another connective tissue layer called the epimysium. In addition to muscle fibers, skeletal muscle is also composed of numerous blood vessels and nerves. The ends of muscles converge in dense connective tissue structures, the tendons and aponeuroses that mediate attachment of muscles to the periosteum of bones or to the connective tissue of other muscles.

[0104] As used herein, the term "lung inflammation" or "pulmonary inflammation" refers to a condition in which the cells in lung tissue become inflamed and may have a reduced ability to transfer oxygen into the bloodstream. In some aspects, the pulmonary inflammation can be caused by infection, inflammatory conditions, genetic conditions, medications, aging, or exposure to certain chemicals. In some aspects, without treatment and / or control, pulmonary inflammation can lead to lung damage or pulmonary fibrosis. In some aspects, gene markers associated with pulmonary inflammation can include one or more of the following: F4 / 80, Monocyte chemotactic protein 1 (Mcpl), CD68, Tumor necrotic factor alpha (TNFla), and Interleukin-6 (IL-6).

[0105] As used herein, "pulmonary fibrosis" or "pulmonary fibrosis" refers to the overgrowth, hardening, and / or scarring of lung tissues related to excess deposition of extracellular matrix components. In some aspects, gene markers associated with pulmonary fibrosis can include one or more of the following: collagen type I alpha 1 chain (Col lai), collagen type III alpha 1 chain (Col3al), transforming growth factor beta 1 (Tgf-P), fibronectin 1 (Fnl) and alpha smooth muscle actin (a-Sma).II. Fibroblast growth factor 21 (FGF21)

[0106] In some aspects, the Fibroblast growth factor 21 (FGF21) amino acid or nucleic acid sequence provided herein is a human FGF21 sequence or a functional fragment thereof. In some aspects, the Fibroblast growth factor 21 (FGF21) amino acid or nucleic acid sequence is a human FGF21 sequence. In some aspects, the FGF21 amino acid or nucleic acid sequence is a full length human FGF21 sequence. In some aspects, the FGF21 amino acid or nucleic acid sequence is a functional fragment of a full length human FGF21 sequence.

[0107] In some aspects, the FGF21 protein or a functional fragment thereof comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 1.

[0108] In some aspects, the FGF21 protein or a functional fragment thereof comprises an amino acid sequence having at least 85%, 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 amino acids 29-209 of SEQ ID NO: 1 and a leader sequence.

[0109] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 4, 5, 6, or 7.

[0110] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 nucleic acids 85-627 of any of SEQ ID NO: 4, 5, 6, or 7.

[0111] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 4.10112] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 5.

[0113] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 6.| 0114 | In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 7.|0115| In some aspects, the leader sequence comprises a signal peptide selected from a human FGF21 signal peptide, a human IL- 10 (hIL-10) signal peptide, a human IL-6 (hIL-6) signal peptide, a human FSH signal peptide, a human Albumin signal peptide, a human CSF signal peptide, a mouse immunoglobulin kappa light chain signal peptide (KASP), a Gaussia Luciferase signal peptide (GLSP), a human Igk signal peptide, a human Apolipoprotein signal peptide, a human PEDF signal peptide, a human SPARC signal peptide, a human tPA signal / propeptide (amino acids 1-32), a human tPA signal / propeptide (amino acids 1-35), a human IgVH signal peptide, a human GCG signal peptide, a human GIP signal peptide + GIP propeptide, or a human Prothrombin signal peptide.

[0116] In some aspects, the leader sequence comprises an amino acid sequence having at least 80%, at least 85%, 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: 34-51.

[0117] In some aspects, the leader sequence is a human FGF21 signal peptide.

[0118] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof further comprises a nucleotide sequence encoding a leader sequence comprising a signal peptide selected from a human IL-10 (hIL-10) signal peptide, a human IL-6 (hIL-6) signal peptide, a human FGF21 signal peptide, a human FSH signal peptide, a human Albumin signal peptide, a human CSF signal peptide, a mouse immunoglobulin kappa light chain signal peptide (KASP), a Gaussia Luciferase signal peptide (GLSP), a human Igk signal peptide, a human Apolipoprotein signal peptide, a human PEDF signal peptide, a human SPARC signal peptide, a human tPA signal / propeptide (amino acids 1-32), a human tPA signal / propeptide (amino acids 1-35), a human IgVH signal peptide, a human GCG signal peptide, a human GIP signal peptide + GIP propeptide, or a human Prothrombin signal peptide.

[0119] In some aspects, the nucleotide sequence encoding a leader sequence comprises a nucleic acid sequence having at least 80%, at least 85%, 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: 52-69.III. Expression Constructs

[0120] In some aspects, the expression constructs provided herein comprise a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a promoter. In some aspects, the FGF21 nucleic acid sequence isoperably linked to a ubiquitous promoter. In some aspects, the ubiquitous promoter is a small chicken beta-actin promoter / cytomegalovirus enhancer (smCBA) promoter (e.g., SEQ ID NO: 25), a eukaryotic translation elongation factor 1 a (EFla) promoter (e.g., SEQ ID NO: 24 or 77), a simian virus 40 (SV40) promoter (e.g., SEQ ID NO: 26), a cytomegalovirus (CMV) promoter (e.g., SEQ ID NOs: 17, 70, or 71) or a CAG promoter (e.g., SEQ ID NO: 16).

[0121] In some aspects, the ubiquitous promoter comprises a nucleotide sequence having at least 85%, 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: 17.

[0122] In some aspects, the ubiquitous promoter comprises a nucleotide sequence having at least 85%, 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: 70.

[0123] In some aspects, the ubiquitous promoter comprises a nucleotide sequence having at least 85%, 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: 71.

[0124] In some aspects, the ubiquitous promoter comprises a nucleotide sequence having at least 85%, 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: 16.

[0125] In some aspects, the ubiquitous promoter comprises a nucleotide sequence having at least 85%, 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: 24 or 77.

[0126] In some aspects, the ubiquitous promoter comprises a nucleotide sequence having at least 85%, 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: 25.

[0127] In some aspects, the ubiquitous promoter comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 26.

[0128] In some aspects, the expression construct comprises the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof having at least 85%, 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 SEQ ID NO: 1.

[0129] In some aspects, the expression construct comprises the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 4, 5, 6, or 7.

[0130] In some aspects, the expression construct comprises the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 4.

[0011] In some aspects, the expression construct comprises the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 5.

[0132] In some aspects, the expression construct comprises the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 6.10133] In some aspects, the expression construct comprises the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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 SEQ ID NO 7.

[0014] In some aspects, the expression construct comprises a FGF21 nucleic acid sequence which is operably linked to a small chicken beta-actinpromoter / cytomegalovirus enhancer (smCBA) promoter (e.g., SEQ ID NO: 25), a eukaryotic translation elongation factor 1 a (EFla) promoter (e.g., SEQ ID NO: 24 or 77), a simian virus 40 (SV40) promoter (e.g., SEQ ID NO: 26), a cytomegalovirus (CMV) promoter (e.g., SEQ ID NO: 17, 70, or 71) or a CAG promoter (e.g., SEQ ID NO: 16).

[0135] In some aspects, the expression construct comprises a FGF21 nucleic acid sequence which is operably linked to a cytomegalovirus (CMV) promoter having at least 85%, 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: 17, 70, or 71.

[0136] In some aspects, the expression construct comprises a FGF21 nucleic acid sequence which is operably linked to a CAG promoter having at least 85%, 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: 16.[01.37] In some aspects, the expression construct comprises a FGF21 nucleic acid sequence which is operably linked to a small chicken beta-actin promoter / cytomegalovirus enhancer (smCBA) promoter having at least 85%, 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: 25.

[0138] In some aspects, the expression construct comprises a FGF21 nucleic acid sequence which is operably linked to a eukaryotic translation elongation factor 1 a (EFla) promoter having at least 85%, 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 NO: 24 or 77.

[0139] In some aspects, the expression construct comprises a FGF21 nucleic acid sequence which is operably linked to a simian virus 40 (SV40) promoter having at least 85%, 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: 26.

[0140] In some aspects, the expression construct is a viral expression construct.

[0141] In some aspects, the viral expression construct further comprises one or more of a polyadenylation signal, at least one target sequence of a microRNA, and / or an enhancer sequence.101421 In some aspects, the polyadenylation signal is a SV40 polyA or a rabbit betaglobin polyA.

[0143] In some aspects, the polyadenylation signal is a SV40 polyA. In some aspects, the polyadenylation signal comprises a sequence having at least 85%, 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: 21.

[0144] In some aspects, the polyadenylation signal is a rabbit beta-globin polyA. In some aspects, the polyadenylation signal comprises a sequence having at least 85%, 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: 22.

[0145] In some aspects, the polyadenylation sequence is a human growth hormone poly (A) sequence.(0146] In some aspects, the human growth hormone poly(A) sequence comprises a nucleic acid sequence corresponding to SEQ ID NO: 74.|0147| In some aspects, the polyadenylation sequence is a synthetic poly(A) sequence.

[0148] In some aspects, the synthetic poly(A) sequence comprises a nucleic acid sequence corresponding to SEQ ID NO: 75.

[0149] In some aspects, the polyadenylation sequence is a bovine growth hormone poly (A) sequence.

[0150] In some aspects, the bovine growth hormone poly(A) sequence comprises a nucleic acid sequence corresponding to SEQ ID NO: 76.

[0151] In some aspects, the viral expression construct comprises a chimeric intron, a CMV enhancer, and / or a CMV promoter / enhancer sequence.

[0152] In some aspects, the chimeric intron comprises a sequence having at least 85%, 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: 15 or 72.10153] In some aspects, the CMV enhancer comprises a sequence having at least 85%, 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: 18.10154] In some aspects, the CMV promoter / enhancer comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23.IV. Adeno-Associated Virus (AAV) Vectors and Capsids|0155] Adeno-Associated Virus (AAV), a parvovirus belonging to the genus Dependovirus, has several attractive features not found in other viruses. For example, AAV can infect a wide range of host cells, including non-dividing cells. Furthermore, AAV can infect cells from different species. Importantly, AAV has not been associated with any human or animal disease, and does not appear to alter the physiological properties of the host cell upon integration. Finally, AAV is stable at a wide range of physical and chemical conditions, which lends itself to production, storage, and transportation requirements.

[0156] The AAV genome, a linear, single-stranded DNA molecule containing approximately 4700 nucleotides (the AAV2 genome consists of 4681 nucleotides), generally comprises an internal non-repeating segment flanked on each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 nucleotides in length (AAV1 has ITRs of 143 nucleotides) and have multiple functions, including serving as origins of replication, and as packaging signals for the viral genome.10157] The internal non-repeated portion of the genome includes two large open reading frames (ORFs), known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode replication and capsid gene products, respectively: replication and capsid gene products (i.e., proteins) allow for the replication, assembly, and packaging of a complete AAV virion. More specifically, a family of at least four viral proteins are expressed from the AAV rep region: Rep 78, Rep 68, Rep 52, and Rep 40, all of which are named for their apparent molecular weights. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.

[0158] AAV is a helper-dependent virus, requiring co-infection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) in order to form functionally complete AAV virions. In the absence of co-infection with a helper virus, AAV establishes a latent state in which the viral genome inserts into a host cell chromosome or exists in an episomal form, but infectious virions are not produced. Subsequent infection by a helper virus "rescues" the integrated genome, allowing it to be replicated and packaged into viral capsids, thereby reconstituting the infectious virion. While AAV can infect cells fromdifferent species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells that have been co-infected with a canine adenovirus.

[0159] In some aspects, AAV vectors of the present disclosure can comprise or be derived from any natural or recombinant AAV serotype. According to the present disclosure, the AAV serotype can be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, and AAV12, and variants thereof.

[0160] In some aspects, the AAV is a self-complementary adeno-associated virus (sc AAV).[01611 In some aspects, the viral expression construct, vector genome, or the AAV vector disclosed herein comprises a promoter. In some aspects, the promoter is a constitutively active promoter, a cell-type specific promoter, or an inducible promoter.10162] Promoters can be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters and mammalian promoters. In some aspects, the promoters can be human promoters. In some aspects, the promoter can be truncated. In some aspects, the promoter comprises a small chicken beta-actin promoter / cytomegalovirus enhancer (smCBA) promoter, a simian virus 40 (SV40) promoter, a eukaryotic translation elongation factor 1 a (EFla, also referred to as EFl alpha or EFla) promoter, cytomegalovirus (CMV) immediate-early enhancer and / or promoter, chicken P-actin (CBA) and its derivative CAG, P glucuronidase (GUSB), or ubiquitin C (UBC). In some aspects, the promoter is a CBA promoter, a CMV promoter, an EF-la (Elongation Factor la) promoter, aRSV (Rous Sarcoma Virus) promoter, an Ubiquitin (UbC) promoter, or any combination thereof. In some aspects, tissue-specific expression elements can be used to restrict expression to certain cell types such as, but not limited to, muscle specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, liver promoters, or nervous system promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0163] In some aspects, the promoter comprises a CBA promoter, a CMV promoter, an EF-la (Elongation Factor la) promoter, a RSV (Rous Sarcoma Virus) promoter, anUbiquitin (UbC) promoter, a CAG promoter, a smCBA promoter or any combination thereof.

[0164] In some aspects, the promoter is a skeletal muscle-specific promoter selected from the group consisting of a muscle creatine kinase (MCK) promoter, a double MCK (dMCK) promoter, and a triple MCK (tMCK) promoter, a MHCK7 promoter, a CK8 promoter, a desmin promoter, a SPc5-12 promoter, a SP-301 promoter, a MH promoter, a Sk-CRM4 / DES promoter, or a myogenin promoter.

[0015] In some aspects, the promoter is a liver specific promoter. In some aspects the liver specific promoter comprises a hAAT promoter, a human thyroxine binding globulin (TBG) promoter, an apolipoprotein E / human alpha-antitrypsin (ApoE / hAAT) promoter, an apolipoprotein E promoter, a hepatic locus control region- 1 (HCR) promoter, or DC 172 promoter.

[0166] In some aspects, the promoter is a hAAT promoter. In some aspects, the promoter is a TBG promoter. In some aspects, the promoter comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31. In some aspects, the promoter can be less than 1 kb. In some aspects, the promoter can have a length between about 15-20, about 10-50, about 20-30, about 30-40, about 40-50, about 50-60, about 50-100, about 60-70, about 70-80, about 80-90, about 90-100, about 100-110, about 100-150, about 110-120, about 120-130, about 130-140, about 140-150, about 150-160, about 150-200, about 160-170, about 170-180, about 180- 190, about 190-200, about 200-210, about 200-250, about 210-220, about 220-230, about 230-240, about 240-250, about 250-260, about 250-300, about 260-270, about 270-280, about 280-290, about 290-300, about 200-300, about 200-400, about 200-500, about 200- 600, about 200-700, about 200-800, about 300-400, about 300-500, about 300-600, about 300-700, about 300-800, about 400-500, about 400-600, about 400-700, about 400-800, about 500-600, about 500-700, about 500-800, about 600-700, about 600-800 or about 700-800 nucleotides.

[0167] In some aspects, the promoter is a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include, e.g., CMV, CBA (including derivatives CAG, CBh, etc.), smCBA, EFla, SV40, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1- CBX3).101681 In some aspects, the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter or a C AG promoter.

[0169] In some aspects, the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter.] 0.170] In some aspects, the ubiquitous promoter comprises a CAG promoter.

[0171] In some aspects, the promoter is not cell specific. In some aspects, the promoter is an ubiquitin c (UBC) promoter. The UBC promoter can have a size of 300-350 nucleotides. In some aspects, the UBC promoter is 332 nucleotides. In some aspects, the promoter is a P- glucuronidase (GUSB) promoter. The GUSB promoter can have a size of 350-400 nucleotides. In some aspects, the GUSB promoter is 378 nucleotides. In some aspects, the promoter is a neurofilament light (NFL) promoter. The NFL promoter can have a size of 600-700 nucleotides. In some aspects, the NFL promoter is 650 nucleotides. In some aspects, the construct can be AAV-promoter-CMV / globin intron- modulatory polynucleotide-RBG, where the AAV can be self-complementary and the AAV can be the DJ serotype.

[0172] In some aspects, the viral expression construct, vector genome, or the AAV vector further comprises a Kozak sequence. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5' UTRs. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another G.

[0173] In some aspects, the viral expression construct, vector genome, or the AAV vector further comprises one or more of a polyadenylation signal, at least one target sequence of a microRNA, and / or an enhancer sequence.

[0174] In some aspects, the viral expression construct, vector genome, or the AAV vector further comprises an enhancer sequence.

[0175] In some aspects, the enhancer is a CMV enhancer sequence (SEQ ID NO: 18).

[0176] In some aspects, the viral expression construct, vector genome, or the AAV vector further comprises an intron sequence.]0177] In some aspects, the viral expression construct, vector genome, or the AAV vector further comprises an intron sequence, e.g., a SV40 intron sequence (e.g., SEQ ID NO: 73) or a CAG intron sequence.101781 In some aspects, the viral expression construct, vector genome, or the AAV vector further comprises an Internal Ribosome Entry Site (IRES).

[0179] In some aspects, the viral expression construct, vector genome, or the AAV vector comprises further comprises a poly(A) sequence. In some aspects, the polyadenylation sequence or "polyA sequence" can range from 50 to about 500 nucleotides in length.

[0180] In some aspects, the polyadenylation sequence is about 50-100, about 50-150, about 50-160, about 50-200, about 60-100, about 60-150, about 60-160, about 60-200, about 70-100, about 70-150, about 70-160, about 70-200, about 80-100, about 80-150, about 80-160, about 80-200, about 90-100, about 90-150, about 90-160, or about 90-200 nucleotides in length.

[0181] In some aspects, the polyadenylation sequence is a rabbit globin polyadenylation (poly A) signal sequence. In some aspects, the polyadenylation sequence is a human growth hormone polyadenylation (poly A) signal sequence. In some aspects, the polyadenylation sequence is a bovine growth hormone polyadenylation (poly A) signal sequence. In some aspects, the polyadenylation sequence is a synthetic polyadenylation (poly A) signal sequence. In some aspects, the polyadenylation sequence is a SV40 polyA signal sequence.

[0182] In some aspects, the poly(A) sequence comprises a SV40 polyadenylation signal or a Rabbit beta-globin polyadenylation signal.

[0183] In some aspects, the poly(A) sequence is a SV40 poly(A) signal sequence.

[0184] In some aspects, the poly(A) sequence is a SV40 poly(A) signal sequence comprises a nucleic acid sequence corresponding to SEQ ID NO: 21.

[0185] In some aspects, the poly(A) sequence is a Rabbit beta-globin polyadenylation signal sequence.

[0186] In some aspects, the Rabbit beta-globin polyadenylation signal sequence comprises a nucleic acid sequence corresponding to SEQ ID NO: 22.

[0187] In some aspects, the polyadenylation sequence is a human growth hormone poly (A) sequence.

[0188] In some aspects, the human growth hormone poly (A) sequence comprises a nucleic acid sequence corresponding to SEQ ID NO: 74.

[0189] In some aspects, the polyadenylation sequence is a synthetic poly(A) sequence.

[0010] In some aspects, the synthetic poly(A) sequence comprises a nucleic acid sequence corresponding to SEQ ID NO: 75.[01 11 In some aspects, the polyadenylation sequence is a bovine growth hormone poly (A) sequence.

[0012] In some aspects, the bovine growth hormone poly(A) sequence comprises a nucleic acid sequence corresponding to SEQ ID NO: 76.101931 In some aspects, the viral expression construct, vector genome, or the AAV vector further comprises two inverted terminal repeat (ITR) sequences.

[0194] In some aspects, the pair of ITR sequences are derived from an AAV serotype selected from AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variants thereof. In some aspects, the pair of ITR sequences are derived from an AAV serotype AAV2.

[0195] In some aspects, the viral expression construct, vector genome, or the AAV vector comprises at least one ITR region and a payload region, e.g., a polynucleotide or expression cassette for expression of the FGF21 protein. In some aspects, the AAV vector comprises two ITRs. In some aspects, these two ITRs flank the payload region at the 5' and 3' ends. The ITRs can function as origins of replication comprising recognition sites for replication. In some aspects, the ITRs comprise sequence regions that can be complementary and symmetrically arranged. In some aspects, the ITRs incorporated into the AAV vector of the present disclosure can be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0196] The ITRs can be derived from the same serotype as the desired capsid, selected from any of the serotypes disclosed herein, or a derivative thereof. The ITR can be of a different serotype from the desired capsid. In some aspects, the ITRs are of the same serotype as one another. In some aspects, the ITRs are of different serotypes. Nonlimiting examples include zero, one or both of the ITRs having the same serotype as the desired capsid.(0197] Independently, each ITR can be about 75 to about 175 nucleotides in length. In some aspects, the ITR can be about 100-150 nucleotides in length, about 140-150 nucleotides in length or about 140-155 nucleotides in length. Non-limiting examples of ITR length are about 140, about 141, about 142, about 145 nucleotides in length, and those having at least 95% identity thereto.

[0018] In some aspects, the viral expression construct, vector genome, or the AAV vector comprises at least one inverted terminal repeat having a length of about 75-80, about 75- 85, about 75-100, about 80- 85, about 80-90, about 80-105, about 85-90, about 85-95,about 85-110, about 90-95, about 90-100, about 90-115, about 95-100, about 95-105, about 95-120, about 100-105, about 100-110, about 100-125, about 105-110, about 105- 115, about 105-130, about 110-115, about 110-120, about 110-135, about 115-120, about 115-125, about 115-140, about 120-125, about 120-130, about 120-145, about 125-130, about 125-135, about 125-150, about 130-135, about 130-140, about 130-155, about 135- 140, about 135-145, about 135-160, about 140-145, about 140-150, about 140-165, about 145-150, about 145-155, about 145-170, about 150-155, about 150-160, about 150-175, about 155-160, about 155-165, about 160-165, about 160-170, about 165-170, about 165- 175, or about 170-175 nucleotides.1019 1 In some aspects, the length of a first and / or a second ITR region for AAV vector can be about 75-80, about 75-85, about 75-100, about 80-85, about 80-90, about 80-105, about 85-90, about 85-95, about 85-110, about 90-95, about 90-100, about 90-115, about 95-100, about 95-105, about 95-120, about 100-105, about 100-110, about 100-125, about 105-110, about 105-115, about 105-130, about 110-115, about 110-120, about 110-135, about 115-120, about 115-125, about 115-140, about 120-125, about 120-130, about 120- 145, about 125-130, about 125-135, about 125-150, about 130-135, about 130-140, about 130-155, about 135-140, about 135-145, about 135-160, about 140-145, about 140-150, about 140-165, about 145-150, about 145-155, about 145-170, about 150-155, about 150- 160, about 150-175, about 155-160, about 155-165, about 160-165, about 160-170, about 165-170, about 165-175, or about 170-175 nucleotides.

[0200] In some aspects, the nucleic acid of interest is located near the 5' end of the flip ITR in the vector. In some aspects, the nucleic acid of interest is located near the 3' end of the flip ITR in the vector. In some aspects, the nucleic acid of interest is located near the 5' end of the flop ITR in the vector. In some aspects, the nucleic acid of interest is located near the 3' end of the flop ITR in the vector. In some aspects, the nucleic acid of interest is located between the 5' end of the flip ITR and the 3' end of the flop ITR in the vector. In some aspects, the nucleic acid of interest is located between (e.g., half-way between the 5' end of the flip ITR and 3' end of the flop ITR or the 3' end of the flop ITR and the 5' end of the flip ITR), the 3' end of the flip ITR and the 5' end of the flip ITR in the vector.

[0201] In some aspects, the nucleic acid of interest is located within about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30or more than about 30 nucleotides downstream or upstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in the vector.

[0202] As another non-limiting example, the nucleic acid of interest is located within about 1-5, about 1- 10, about 1-15, about 1-20, about 1-25, about 1-30, about 5-10, about 5-15, about 5-20, about 5-25, about 5-30, about 10-15, about 10-20, about 10-25, about 10-30, about 15-20, about 15-25, about 15-30, about 20-25, about 20-30 or about 25-30 nucleotides downstream or upstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in the vector.

[0203] In some aspects, the nucleic acid of interest is located within the first about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25% or more than about 25% of the nucleotides upstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in the vector.

[0204] As another non-limiting example, the nucleic acid of interest is located with the first about 1-5%, about 1-10%, about 1-15%, about 1-20%, about 1-25%, about 5-10%, about 5-15%, about 5-20%, about 5-25%, about 10-15%, about 10-20%, about 10-25%, about 15-20%, about 15-25%, or about 20-25% downstream from the 5' or 3' end of an ITR (e.g., Flip or Flop ITR) in the vector.

[0205] In some aspects, the ITRs comprise a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 19, 20, or combinations thereof.

[0206] In some aspects, the ITRs comprise a 5' ITR comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19, and a 3' ITR comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 20.

[0207] In some aspects, the AAV comprises an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, and AAV12.

[0208] In some aspects, the viral expression construct, vector genome, or the AAV vector comprises ITRs having an AAV2 serotype.

[0209] In some aspects, the nucleic acid sequence has been modified to reduce the number of CpG sites.

[0210] Certain aspects of the disclosure are directed to an adeno-associated virus (AAV) vector comprising a polynucleotide (e.g., expression construct disclosed herein) comprising a promoter (e.g., ubiquitous promoter) operably linked to a nucleic acid sequence encoding a FGF21 protein or functional fragment thereof flanked by AAV inverted terminal repeats (ITRs). In some aspects, the AAV vector is encapsidated by an AAV capsid (e.g., AAV1 serotype).

[0211] In some aspects, provided herein is an adeno-associated virus (AAV) vector comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding a FGF21 protein or functional fragment thereof where the expression cassette is flanked by ITRs, wherein the nucleic acid sequence encoding a FGF21 protein or functional fragment thereof comprises a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any of SEQ ID NOs: 4, 5, 6, or 7.

[0212] In some aspects, the AAV vector comprises an expression construct comprising a nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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.

[0021] In some aspects, the AAV vector comprises an expression construct comprising a nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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.

[0214] In some aspects, the AAV vector comprises an expression construct comprising a nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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: 6.

[0215] In some aspects, the AAV vector comprises an expression construct comprising a nucleotide sequence encoding the FGF21 protein or a functional fragment thereof which comprises a nucleotide sequence having at least 85%, 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.

[0216] In some aspects, the AAV vector comprises an expression construct comprising a FGF21 nucleic acid sequence which is operably linked to a cytomegalovirus (CMV) promoter (e.g., SEQ ID NO: 17) or a CAG promoter (e.g., SEQ ID NO: 16).

[0217] In some aspects, the AAV vector comprises an expression construct comprising a FGF21 nucleic acid sequence which is operably linked to a cytomegalovirus (CMV) promoter having at least 85%, 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: 17.

[0218] In some aspects, the AAV vector comprises an expression construct comprising a FGF21 nucleic acid sequence which is operably linked to a CAG promoter having at least 85%, 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: 16.

[0219] In some aspects, the AAV vector comprises an expression construct comprising a FGF21 nucleic acid sequence which is operably linked to a small chicken beta-actin promoter / cytomegalovirus enhancer (smCBA) promoter having at least 85%, 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: 25.

[0220] In some aspects, the AAV vector comprises an expression construct comprising a FGF21 nucleic acid sequence which is operably linked to a eukaryotic translation elongation factor 1 a (EFla) promoter having at least 85%, 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 NO: 24 or 77.

[0221] In some aspects, the AAV vector comprises an expression construct comprising a FGF21 nucleic acid sequence which is operably linked to a simian virus 40 (SV40) promoter having at least 85%, 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: 26.

[0222] In some aspects, the AAV vector comprises an expression construct further comprising one or more of a polyadenylation signal, at least one target sequence of a microRNA, and / or an enhancer sequence.

[0223] In some aspects, the polyadenylation signal is a SV40 polyA or a rabbit betaglobin polyA.

[0224] In some aspects, the polyadenylation signal is a SV40 polyA. In some aspects, the polyadenylation signal comprises a sequence having at least 85%, 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: 21.10225] In some aspects, the polyadenylation signal is a rabbit beta-globin polyA. In some aspects, the polyadenylation signal comprises a sequence having at least 85%, 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: 22.

[0226] In some aspects, the polyadenylation signal is a human growth hormone poly(A) sequence.

[0227] In some aspects, the polyadenylation signal comprises a sequence having at least 85%, 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: 74.

[0228] In some aspects, the polyadenylation signal is a synthetic poly(A) sequence.

[0229] In some aspects, the polyadenylation signal comprises a sequence having at least85%, 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: 75.

[0230] In some aspects, the polyadenylation signal is a bovine growth hormone poly(A) sequence.

[0231] In some aspects, the polyadenylation signal comprises a sequence having at least 85%, 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: 76.

[0232] In some aspects, the AAV vector comprises an expression construct comprising a chimeric intron, a CMV enhancer, and / or a CMV promoter / enhancer sequence.

[0233] In some aspects, the chimeric intron comprises a sequence having at least 85%, 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: 15.

[0234] In some aspects, the CMV enhancer comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18.(0235] In some aspects, the CMV promoter / enhancer comprises a sequence having at least 85%, 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: 23.

[0236] In some aspects, the AAV vector comprises an expression construct flanked by AAV ITRs, wherein the AAV ITRs are AAV2 serotype.

[0237] In some aspects, the AAV ITRs comprise a 5' ITR and a 3' ITR. In some aspects, the 5' ITR comprises a sequence having at least 85%, 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: 19. In some aspects, the 3' ITR comprises a sequence having at least 85%, 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: 20.

[0238] In some aspects, the 5' ITR comprises a sequence having at least 85%, 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: 32. In some aspects, the 3' ITR comprises a sequence having at least 85%, 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: 33.

[0239] In some aspects, the AAV vector comprises a sequence having at least 85%, 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: 14.

[0240] In some aspects, the AAV vector comprises a nucleic acid sequence comprising any of the SEQ ID NOs disclosed in Table 1, or any combinations thereof.Table 1 - Exemplary AAV construct sequences

[0241] In some aspects, the viral expression construct, vector genome, or the AAV vector disclosed herein is encapsidated in an adeno-associated (AAV) capsid. In some aspects, provided herein is a recombinant adeno-associated (rAAV) vector comprising a capsid and any of the viral expression construct, vector genome, or the AAV vector disclosed herein.

[0242] In some aspects, the AAV capsid serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, and AAV12. In some aspects, the AAV capsid serotype is AAV1.

[0243] In some aspects, the disclosure is directed to an isolated rAAV particle comprising the vector genome of any of the AAV vectors described herein. In some aspects, the rAAV particle is produced by any of the methods of packaging described herein or known in the art.

[0244] In some aspects, the disclosure is directed to a plurality of rAAV particles. In some aspects, the rAAV particles are isolated or purified.|0245| The present disclosure also provides for cells containing the viral expression construct, vector genome, or the AAV vector disclosed herein. In some aspects, the cell comprises mammalian cells. In some aspects, the cell provides helper functions that package the viral expression construct, vector genome, or the AAV vector into a viral particle. In some aspects, the cell provides AAV helper functions. In some aspects, the cell provides AAV Rep and / or Cap proteins. In some aspects, the cell is stably or transiently transfected with polynucleotide(s) encoding Rep and / or Cap protein sequence(s). In some aspects, the cell provides Rep78 or / and Rep68 proteins. In some aspects, the cell is stably or transiently transfected with Rep78 and Rep68 proteins polynucleotide encoding sequence(s).

[0246] In some aspects, the present disclosure provides for a method for packaging a nucleic acid of interest in an AAV capsid. In some aspects, the method comprises transfecting a cell in vitro with any viral expression construct, vector genome, or the AAV vector described herein and one or more plasmids comprising Rep / Cap genes and adenovirus genes. In some aspects, the nucleic acid of interest is packaged into the AAV capsid.

[0247] In some aspects, a rAAV particle is produced by a method comprising: (1) co-transfecting competent bacterial cells with a bacmid vector and either a viral construct vector and / or AAV payload construct vector, (2) isolating the resultant viral construct expression vector and AAV payload construct expression vector and separately transfecting viral replication cells, (3) isolating and purifying resultant payload and viral construct particles comprising viral construct expression vector or AAV payload construct expression vector, (4) co-infecting a viral replication cell with both the AAV payload and viral construct particles comprising viral construct expression vector or AAV payload construct expression vector, and (5) harvesting and purifying the viral particle comprising a parvoviral genome.

[0248] In some aspects, the present disclosure provides a method for producing an rAAV particle comprising the steps of (1) simultaneously co-transfecting mammalian cells, such as, but not limited to HEK293 cells, with: (i) AAV vector of the disclosure comprising a payload region (e.g., polynucleotide encoding a FGF21 protein), (ii) a construct expressing rep and cap genes and (iii) a helper construct, and (2) harvesting and purifying the rAAV particle comprising a viral genome.102491 In some aspects, the rAAV particles can be produced in a viral replication cell that comprises an insect cell. Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well known in the art, see, e.g., U.S. Patent No. 6,204,059.

[0250] The viral replication cell can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells. Viral replication cells can comprise mammalian cells such as A549, WEH1, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals. Viral replication cells comprise cells derived from mammalian species including, but not limited to, human, monkey, mouse, rat, rabbit, and hamster or cell type, including but not limited to fibroblast, hepatocyte, tumor cell, cell line transformed cell, etc.|025l] Viral production disclosed herein describes processes and methods for producing rAAV particles that contact a target cell to deliver a payload, e.g. a recombinant viral construct, which comprises a polynucleotide or expression construct disclosed herein.

[0252] In some aspects, the rAAV particles can be produced in a viral replication cell that comprises a mammalian cell. Viral replication cells commonly used for production of recombinant AAV particles include, but are not limited to 293 cells, COS cells, HeLa cells, and KB cells.

[0253] In some aspects, rAAV particles are produced in mammalian cells wherein all three VP proteins are expressed at a stoichiometry approaching 1 : 1 : 10 (VP1 :VP2:VP3). In some aspects, the VP proteins are expressed at a stoichiometry such that VP3 is higher than VP1 and VP2. In some aspects, the VP proteins are expressed at a stoichiometry such that VP1 and VP2 are at approximately the same level. The regulatory mechanisms that allow this controlled level of expression include the production of two mRNAs, one for VP1, and the other for VP2 and VP3, produced by differential splicing.

[0254] In some aspects, rAAV particles are produced in mammalian cells using a triple transfection method wherein a payload construct (e.g., an AAV vector disclosed herein), parvoviral Rep and parvoviral Cap and a helper construct are comprised within three different constructs. The triple transfection method of the three components of AAV particle production can be utilized to produce small lots of virus for assays including transduction efficiency, target tissue (tropism) evaluation, and stability.102551 In some aspects, the viral expression construct, vector genome, or the AAV vector can be each incorporated by a transposon donor / acceptor system into a bacmid, also known as a baculovirus plasmid, by standard molecular biology techniques known and performed by a person skilled in the art. Transfection of separate viral replication cell populations produces two baculoviruses, one that comprises the viral construct expression vector, and another that comprises the AAV payload construct expression vector. The two baculoviruses can be used to infect a single viral replication cell population for production of AAV particles.

[0256] Baculovirus expression vectors for producing viral particles in insect cells, including but not limited to Spodoptera frugiperda (Sf9) cells, provide high titers of viral particle product. Recombinant baculovirus encoding the viral construct expression vector and AAV vector initiates a productive infection of viral replicating cells. Infectious baculovirus particles released from the primary infection secondarily infect additional cells in the culture, exponentially infecting the entire cell culture population in a number of infection cycles that is a function of the initial multiplicity of infection, see, e.g., Urabe, M. et al., J Virol. 2006 Feb; 80 (4): 1874-85, the contents of which are herein incorporated by reference in their entirety.

[0257] Production of rAAV particles with baculovirus in an insect cell system can address known baculovirus genetic and physical instability. Baculovirus-infected viral producing cells are harvested into aliquots that can be cryopreserved in liquid nitrogen; the aliquots retain viability and infectivity for infection of large-scale viral producing cell culture (Wasilko DJ et al., Protein Expr Purif. 2009 Jun; 65(2): 122-32).

[0258] In some aspects, stable viral replication cells permissive for baculovirus infection are engineered with at least one stable integrated copy of any of the elements necessary for AAV replication and viral particle production including, but not limited to, the entire AAV genome, Rep and Cap genes, Rep genes, Cap genes, each Rep protein as a separate transcription cassette, each VP protein as a separate transcription cassette, the AAP (assembly activation protein), or at least one of the baculovirus helper genes with native or non-native promoters.

[0259] In some aspects, rAAV particle production can be modified to increase the scale of production. Transfection of replication cells in large-scale culture formats can be carried out according to any methods known in the art.102601 In some aspects, cell culture bioreactors can be used for large scale viral production. In some cases, bioreactors comprise stirred tank reactors.(0261 ] Cells of the disclosure, including, but not limited to viral production cells, can be subjected to cell lysis according to any methods known in the art. In some aspects, the cells of the disclosure are not subjected to cell lysis. Cell lysis can be carried out to obtain one or more agents (e.g. viral particles) present within any cells of the disclosure.

[0262] In some aspects, a method for harvesting rAAV particles without lysis can be used for efficient and scalable AAV particle production. In a non-limiting example, AAV particles can be produced by culturing an AAV particle lacking a heparin binding site, thereby allowing the AAV particle to pass into the supernatant, in a cell culture, collecting supernatant from the culture; and isolating the AAV particle from the supernatant, as described in U.S. Patent Application 2009 / 0275107.

[0263] Cell lysates comprising viral particles can be subjected to clarification. Clarification refers to initial steps taken in purification of viral particles from cell lysates. Clarification serves to prepare lysates for further purification by removing larger, insoluble debris. Clarification steps can include, but are not limited to centrifugation and filtration.

[0264] In some aspects, rAAV particles can be purified from clarified cell lysates by one or more methods of chromatography.V. Pharmaceutical Compositions

[0265] In some aspects, provided herein is a pharmaceutical composition comprising any of the expression constructs, the viral expression constructs, the vector genomes, the AAV vectors, or rAAV vectors disclosed herein.

[0266] In some aspects, the pharmaceutical composition is in a form suitable for administration to a subject in need thereof. In some aspects, the pharmaceutical composition is in a form suitable for intramuscular administration to a subject in need thereof.

[0267] In some aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0268] In some aspects, pharmaceutically acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition.|0269| Also provided herein are pharmaceutical compositions comprising rAAV vectors disclosed herein having the desired degree of purity, and a pharmaceutically acceptable carrier or excipient, in a form suitable for administration to a subject. Pharmaceutically acceptable excipients or carriers can be determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions comprising a plurality of vectors, e.g., AAV vectors described herein. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 21st ed. (2005)). The pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0270] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients (e.g., animals or humans) at the dosages and concentrations employed.

[0271] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Except insofar as any conventional media or compound is incompatible with the rAAV vectors disclosed herein, use thereof in the compositions is contemplated. In some aspects, a pharmaceutical composition is formulated to be compatible with its intended route of administration.

[0272] The rAAV vectors disclosed herein can be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution.VI. Kits

[0273] The present disclosure also provides kits, or products of manufacture, comprising (i) any of the AAV vectors, the rAAV vectors, or the pharmaceutical compositions disclosed herein, and (ii) optionally instructions for use (e.g., a package insert with instructions to perform any of the methods described herein).

[0274] In some aspects, the kit or product of manufacture comprises (i) viral expression construct, vector genome, or the AAV vector disclosed herein disclosed herein (ii) optionally, an additional agent (e.g., rep / cap plasmid and / or helper plasmid), and (iii) optionally, instructions for use (e.g., a package insert with instructions to perform any of the methods described herein are also contemplated).102751 One skilled in the art will readily recognize that the AAV vectors, the rAAV vectors, or the pharmaceutical compositions of the present disclosure, can be readily incorporated into one of the established kit formats which are well known in the art.VII. Administration

[0276] The AAV vectors, rAAV vectors, or pharmaceutical compositions disclosed herein can be administered by any route which results in a therapeutically effective outcome. In some aspects, the methods disclosed herein can comprise intramuscularly administering an AAV vector, rAAV vector, or pharmaceutical composition of the present disclosure to a subject disclosed herein.

[0277] In some aspects, the methods disclosed herein comprise administering any of the vectors, rAAV vectors, or pharmaceutical compositions disclosed herein to a subject, e.g., a subject suffering from a lung disease.

[0278] The AAV vector, rAAV vector, or pharmaceutical composition disclosed herein can be formulated to be suitable for administration to a cell, tissue and / or an organ in vivo of individuals affected by or at risk of developing a pulmonary inflammation or pulmonary fibrosis, and may be administered in vivo, ex vivo or in vitro.

[0279] In some aspects, the AAV vector, rAAV vector, or pharmaceutical composition disclosed herein is administered to a human skeletal muscle.

[0280] In some aspects, the AAV vector, rAAV vector, or pharmaceutical composition disclosed herein is injected into a muscle selected from the group consisting of quadriceps, gastrocnemius, tibialis, and any combination thereof.

[0281] In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a tricep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and / or a latissimus dorsi (lat). In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and / or a hamstring. In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a hamstring, or any combination thereof.

[0282] In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a tricep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat).102831 In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring.(0284] The AAV vector, rAAV vector, or pharmaceutical composition disclosed herein can be administered in any suitable form, either as a liquid solution or suspension, as a solid form suitable for liquid solution or suspension in a liquid solution.

[0285] The optimal effective amount of the AAV vector, rAAV vector, or pharmaceutical composition disclosed herein can be determined empirically and will depend on the type and severity of the disease, route of administration, disease progression and health, mass and body area of the individual.10286] The AAV vector, rAAV vector, or pharmaceutical composition disclosed herein may be administered in a single dose, or in multiple doses. The AAV vector, rAAV vector, or pharmaceutical composition disclosed herein may be administered by injection in various locations in the skeletal muscle, such as quadriceps, gastrocnemius, tibialis, and any combination thereof.

[0287] In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a tricep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and / or a latissimus dorsi (lat). In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and / or a hamstring. In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a hamstring, or any combination thereof.J0288] In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a tricep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat).

[0289] In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring.

[0290] In some aspects, the rAAV vector is administered as a single dose.

[0291] In some aspects, the rAAV vector is administered over multiple doses.

[0292] In some aspects, the rAAV vector is administered to the same skeletal muscle(e.g., the left quadriceps, left gastrocnemius, the left tibialis, the right quadriceps, right gastrocnemius, or the right tibialis). In some aspects, the rAAV vector is administered to different skeletal muscles (e.g., one dose is given to the left quadriceps and the left gastrocnemius, one dose is given to the left quadriceps and the right quadriceps, etc.).VIII. Methods of Treatment for Pulmonary inflammation and / or Pulmonary fibrosis

[0293] Certain aspects of the present disclosure provide a method for treating or reducing pulmonary inflammation and / or pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a promoter, e.g., a ubiquitous promoter.

[0294] In some aspects, the pulmonary inflammation is caused by or associated with an infection, an inflammatory condition, a genetic condition, a medication, aging and / or a chemical exposure. In some aspects, the pulmonary inflammation is caused by or associated with an infection. In some aspects, the pulmonary inflammation is caused by or associated with an inflammatory condition. In some aspects, the pulmonary inflammation is caused by or associated with a genetic condition. In some aspects, the pulmonary inflammation is caused by or associated with a medication. In some aspects, the pulmonary inflammation is caused by or associated with aging. In some aspects, the pulmonary inflammation is caused by or associated with a chemical exposure.

[0295] Certain aspects of the present disclosure provide a method for treating or reducing an inflammatory lung disease, lung disorder, or lung condition in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a promoter, e.g., a ubiquitous promoter.

[0296] Certain aspects of the present disclosure provide a method for treating, preventing or reducing the likelihood of pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising anucleotide sequence encoding a fibroblast growth factor 21 (FGF21) or a functional fragment thereof operably linked to a promoter, e.g., a ubiquitous promoter.

[0297] In some aspects, the subject suffers from idiopathic pulmonary fibrosis. In some aspects, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

[0298] In some aspects, the subject suffers from a pneumonitis. In some aspects, the pulmonary inflammation is a pneumonitis.

[0299] In some aspects, the pneumonitis is a hypersensitivity pneumonitis, a drug- induced pneumonitis, or a radiation-induced pneumonitis.

[0300] In some aspects, the subject suffers from a hypersensitivity pneumonitis. In some aspects, the pneumonitis is a hypersensitivity pneumonitis.

[0301] In some aspects, the hypersensitivity pneumonitis is caused by exposure to a mold, a bacteria, a bird (e.g., exposure to bird feathers or bird droppings), dust, chemicals, or animal fur (e.g., exposure to skin cells, fur, or droppings from animals).

[0302] In some aspects, the pulmonary inflammation is a chronic inflammation.

[0303] In some aspects, the subject suffers from chronic obstructive pulmonary disease(COPD). In some aspects, the pulmonary inflammation is caused by chronic obstructive pulmonary disease (COPD).

[0304] In some aspects, the inflammatory lung disease, lung disorder, or lung condition is COPD.

[0305] In some aspects, the subject suffers from pulmonary sarcoidosis. In some aspects, the pulmonary inflammation is a pulmonary sarcoidosis.

[0306] In some aspects, the subject suffers from asthma. In some aspects, the inflammatory lung disease, lung disorder, or lung condition is asthma.

[0307] In some aspects, the subject suffers from bronchitis. In some aspects, the inflammatory lung disease, lung disorder, or lung condition is bronchitis.

[0308] Asbestosis is a lung disease that can occur when someone inhales asbestos fibers. In some aspects, the subject suffers from asbestosis. In some aspects, the inflammatory lung disease, lung disorder, or lung condition is caused by asbestosis.

[0309] In some aspects, the subject suffers from obesity and / or insulin resistance.

[0310] In some aspects, the subject does not suffer from obesity and / or insulin resistance.

[0311] In some aspects, the subject has at least 0.1-6 fold reduction in a relative expression of an inflammatory gene marker selected from the group consisting of Cluster of Differentiation 68 (CD68), F4 / 80, Tumor necrosis factor-alpha (TNFa), MonocyteChemoattractant Protein- 1 (MCP1), Interleukin-6 (IL-6), or any combination thereof, optionally wherein the relative expression is measured by qRT-PCR.

[0312] In some aspects, the reduction in the inflammatory gene marker is at least 0.1-6.0 fold, at least 0.5-6.0 fold, at least 1.0 to 6.0 fold, at least 1.5 to 6.0 fold, at least 2.0 to 6.0 fold, at least 2.5 to 6.0 fold, at least 3.0 to 6.0 fold, at least 3.5 to 6.0 fold, or at least 4.0 to 6.0 fold. In some aspects, the rejection is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, or at least 6 fold.

[0313] In some aspects, the reduction in the inflammatory gene marker is 0.1-6.0 fold, 0.5-6.0 fold, 1.0 to 6.0 fold, 1.5 to 6.0 fold, 2.0 to 6.0 fold, 2.5 to 6.0 fold, 3.0 to 6.0 fold, 3.5 to 6.0 fold, or 4.0 to 6.0 fold. In some aspects, the rejection is about 2 fold, about 3 fold, about 4 fold, about 5 fold, or about 6 fold.

[0314] In some aspects, the subject has at least 0.1-6 fold reduction in a relative expression of a fibrosis gene marker selected from the group consisting of collagen type I alpha 1 chain (Collal), collagen type III alpha 1 chain (Col3al), transforming growth factor beta 1 (Tgf-P), fibronectin 1 (Fnl) and alpha smooth muscle actin (a-Sma), or any combination thereof, optionally, wherein the relative expression is measured by qRT- PCR.

[0315] In some aspects, the reduction in the fibrosis gene marker is at least 0.1-6.0 fold, at least 0.5-6.0 fold, at least 1.0 to 6.0 fold, at least 1.5 to 6.0 fold, at least 2.0 to 6.0 fold, at least 2.5 to 6.0 fold, at least 3.0 to 6.0 fold, at least 3.5 to 6.0 fold, or at least 4.0 to 6.0 fold. In some aspects, the rejection is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, or at least 6 fold.10316 [ In some aspects, the reduction in the fibrosis gene marker is 0.1-6.0 fold, 0.5-6.0 fold, 1.0 to 6.0 fold, 1.5 to 6.0 fold, 2.0 to 6.0 fold, 2.5 to 6.0 fold, 3.0 to 6.0 fold, 3.5 to 6.0 fold, or 4.0 to 6.0 fold. In some aspects, the rejection is about 2 fold, about 3 fold, about 4 fold, about 5 fold, or about 6 fold.103.17] In some aspects, the methods of the disclosure treat or prevent one or more symptoms associated with a lung disease or disorder, a lung condition, pulmonary inflammation, and / or a pulmonary fibrosis. Common symptoms of a lung disease or disorder, pulmonary inflammation, and / or a pulmonary fibrosis can include shortness of breath, a persistent cough (e.g., dry cough), fatigue, or any combination thereof. In some aspects, the subject of the disclosure suffers from shortness of breath, a persistent cough (e.g., dry cough), fatigue, or any combination thereof.103181 In some aspects, the methods of the disclosure reduce one or more symptoms associated with the inflammatory lung disease, the lung disease or disorder, the lung condition, the pulmonary inflammation and / or pulmonary fibrosis comprising shortness of breath, a persistent cough (e.g., dry cough), fatigue, or any combination thereof.10319] In some aspects, the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter or a C AG promoter.

[0320] In some aspects, the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter.

[0321] In some aspects, the ubiquitous promoter comprises a CAG promoter.

[0322] In some aspects, the promoter is a liver specific promoter. In some aspects, the liver specific promoter comprises a hAAT promoter, a human thyroxine binding globulin (TBG) promoter, an apolipoprotein E / human alpha-antitrypsin (ApoE / hAAT) promoter, an apolipoprotein E promoter, a hepatic locus control region- 1 (HCR) promoter, or DC 172 promoter.

[0323] In some aspects, the viral expression construct further comprises one or more of a polyadenylation signal, at least one target sequence of a microRNA, and / or an enhancer sequence.

[0324] In some aspects, the polyadenylation signal is a SV40 polyA or a rabbit betaglobin polyA.

[0325] In some aspects, the AAV ITRs (e.g., a 5' ITR and a 3' ITR) are AAV2 serotype.]0326[ In some aspects, the administration is intramuscular.

[0327] In some aspects, the rAAV vector is injected into a muscle selected from the group consisting of quadriceps, gastrocnemius, tibialis, and any combination thereof.

[0328] In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a tricep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and / or a latissimus dorsi (lat). In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and / or a hamstring. In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a hamstring, or any combination thereof.

[0329] In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a tricep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, a hamstring, a deltoid, a trapezius, a pectoral muscle (e.g., pectoralis major), and a latissimus dorsi (lat).103301 In some aspects, the muscle is selected from the group consisting of a quadricep, a bicep, a gastrocnemius (e.g., a calf muscle), a gluteus maximus, and a hamstring.(0331 ] In some aspects, the rAAV vector is administered as a single dose.

[0332] In some aspects, the rAAV vector is administered over multiple doses.10333] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence encoding an amino acid sequence having at least 85%, 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.

[0334] In some aspects, the FGF21 protein or a functional fragment thereof comprises an amino acid sequence having at least 85%, 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 amino acids 29-209 of SEQ ID NO: 1 and a leader sequence.

[0335] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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, 5, 6, or 7.

[0336] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 nucleic acids 85-627 of any of SEQ ID NO: 4, 5, 6, or 7.

[0337] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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.

[0338] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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.

[0339] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, at least90%, 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: 6.

[0340] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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.[0341 J In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof encodes the amino acid sequence of SEQ ID NO: 1.

[0342] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises the sequence of SEQ ID NO: 4.

[0343] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises the sequence of SEQ ID NO: 5.

[0344] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises the sequence of SEQ ID NO: 6.

[0345] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises the sequence of SEQ ID NO: 7.

[0346] In some aspects, the rAAV comprises a polynucleotide having at least 85%, 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: 14.

[0347] In some aspects, the leader sequence comprises a signal peptide selected from a human FGF21 signal peptide, a human IL- 10 (hIL-10) signal peptide, a human IL-6 (hlL- 6) signal peptide, a human FSH signal peptide, a human Albumin signal peptide, a human CSF signal peptide, a mouse immunoglobulin kappa light chain signal peptide (KASP), a Gaussia Luciferase signal peptide (GLSP), a human Igk signal peptide, a human Apolipoprotein signal peptide, a human PEDF signal peptide, a human SPARC signal peptide, a human tPA signal / propeptide (amino acids 1-32), a human tPA signal / propeptide (amino acids 1-35), a human IgVH signal peptide, a human GCG signal peptide, a human GIP signal peptide + GIP propeptide, or a human Prothrombin signal peptide.

[0348] In some aspects, the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof further comprises a nucleotide sequence encoding a leader sequence comprising a signal peptide selected from a human IL-10 (hIL-10) signalpeptide, a human IL-6 (hIL-6) signal peptide, a human FGF21 signal peptide, a human FSH signal peptide, a human Albumin signal peptide, a human CSF signal peptide, a mouse immunoglobulin kappa light chain signal peptide (KASP), a Gaussia Luciferase signal peptide (GLSP), a human Igk signal peptide, a human Apolipoprotein signal peptide, a human PEDF signal peptide, a human SPARC signal peptide, a human tPA signal / propeptide (amino acids 1-32), a human tPA signal / propeptide (amino acids 1-35), a human IgVH signal peptide, a human GCG signal peptide, a human GIP signal peptide + GIP propeptide, or a human Prothrombin signal peptide.

[0349] In some aspects, the leader sequence comprises an amino acid sequence having at least 80%, at least 85%, 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: 34-51.

[0350] In some aspects, the nucleotide sequence encoding a leader sequence comprises a nucleic acid sequence having at least 80%, at least 85%, 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: 52-69.

[0351] The following examples are illustrative and are not intended to limit the full scope of the claimed aspects.EXAMPLESExample 1. AAV1-CMV-FGF21 construct

[0352] Recombinant AAV vectors.

[0353] AAV expression cassettes were obtained by cloning, between the ITRs of AAV2, the murine, canine or human optimized FGF21 coding sequences (e.g., SEQ ID NOs: 4- 11) under the control of the cytomegalovirus (CMV) promoter. Single-stranded AAV1 vectors were produced by triple transfection in HEK293 cells and purified using an optimized CsCl gradient-based purification protocol that renders vector preps of high purity with negligible amounts of empty capsids. Viral genome titers were determined by PicoGreen, using phage lambda DNA to generate the standard curve.Example 2. Effects of intramuscular AAV1-FGF21 on pulmonary inflammation and fibrosis.

[0354] The first objective of this study was to evaluate pulmonary inflammation and fibrosis in two mouse models of obesity (high fat diet fed mice and genetically obese db / db mice). Leptin receptor-deficient db / db mice are a widely studied mouse model of hyperglycemia and obesity but their cardiovascular and pulmonary pathology linked to metabolic syndrome make them also a useful mouse model to study pulmonary inflammatory and fibrotic development (Wang, Y., et al., Mol Med 29(1): 100 (2023); Aravani, D., et al., Thromb Haemost 121 (6):703-715 (2021)). The second objective of the study was to determine the potential therapeutic effect of the muscle-directed AAV1- FGF21 gene therapy to counteract pulmonary inflammation and fibrosis.Experimental design

[0355] For the evaluation of pulmonary inflammation and fibrosis in diet-induced obese mice, eight-week-old C57Bl / 6J01aHsd male mice were fed a high fat diet (HFD) for 20 weeks. Obese mice were then injected intramuscularly (IM) with 3xl0nvector genomes (vg) per animal of AAV vectors of serotype 1 (AAV1) coding for a murine optimized FGF21 coding sequence under the control of the cytomegalovirus (CMV) promoter AAVl-CMV-comFGF21. After AAV administration, AAV-treated mice were maintained on HFD until they were 70 weeks old, at which time they were euthanized and lung samples were obtained for both histological and transcriptomic analysis. As controls, age- matched untreated chow and HFD-fed C57Bl / 6J01aHsd mice were used.

[0356] For the evaluation of pulmonary inflammation and fibrosis in genetically obese mice, nine-week old male db / db mice were injected IM with 3xl0uvg of AAV1-CMV- comFGF21 vectors. Untreated age-matched db / db male mice were used as controls. Lung samples were obtained at sacrifice at 29 weeks of age.Methods

[0357] Animals. Diet-Induced Obesity Model: Congenic eight-week-old C57Bl / 6J01aHsd male mice were used. Mice were fed ad libitum with standard chow (2018S Teklad global diets®, Envigo) or high fat (TD.88137 Teklad Diets, Envigo) diets. Genetic Obesity Model: Nine-week-old BKS.Cg-+ Leprdb / + Leprdb / OlaHsd (db / db) mice were used. Db / db mice were fed ad libitum with a standard chow diet (2018STeklad Global Diets, Envigo). Mice were kept in a specific pathogen-free facility (SER- CBATEG, UAB) and maintained under a light-dark cycle of 12 hours at 22°C.(0358] Sample collection. At the time of euthanasia, mice were anesthetized by means of inhalational anesthetics Isoflurano (IsoFlo®, Abbott Animal Health, Illinois, USA) and decapitated. Tissues of interest were excised and kept at -80°C or with formalin, until analysis.

[0359] Histology. Tissues were fixed for 12-24 hours in 10% formalin, embedded in paraffin and sectioned. Sections were incubated overnight at 4°C with rat anti-Mac2 (1 :50; CL8942AP; Cedarlane). Biotinylated rabbit anti-rat (1 :300; E0467; Dako) was used as secondary antibody. The ABC peroxidase kit (Pierce) was used for immunodetection, and sections were counterstained in Mayer's hematoxylin. PicroSirius Red staining was used to evaluate fibrosis. Images were obtained with a Nikon Eclipse 90i microscope (Nikon).

[0360] RNA analysis. Total RNA was obtained from lung using isolation reagent (Tripure, Roche) and RNeasy Minikit (Qiagen) and treated with DNAsel (Qiagen). One microgram of RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (Takara). Real-time quantitative PCR (qRT-PCR) was performed in a QuantStudio 5 Real-Time PCR instrument (ThermoFisher Scientific) using the TB Green Premix Ex Taq (Tli RNase H Plus) (Takara) and primers for a-Sma, Cd68, Collal, Col3al, F4 / 80, Tgf- P, Fnl, Tnfa, Mcpl, 11-6, II- 1 P, RplpO. Data was normalized to RplpO expression.

[0361] Statistical analysis. Sample size determination was based on previous experience with similar studies. Each experimental group was caged separately to avoid any caging effects. All tests were performed by investigators blinded to the treatment. Statistics were calculated using GraphPad Prism. Statistical significance was calculated using a one-way analysis of variance (ANOVA) with Dunnett' s multiple comparison test except for those parameters involving comparison of only two experimental groups, in which case an unpaired Student's t-test was used. Differences were considered significant when P < 0.05.Results

[0362] Intra-muscular AAV-FGF21 treatment counteracts pulmonary inflammation in HFD-fed mice. Immunostaining against the macrophage-specific marker MAC2 in lung sections showed widespread macrophage infiltration in pulmonary alveoli andbronchi, which was more evident in the lobe borders, of animals fed a HFD (Figs. 1A and IB). Mice treated intramuscularly (IM) with AAVl-CMV-comFGF21 vector showed markedly reduced presence of macrophages in all the examined areas, resembling to their healthy control littermates fed a chow diet (Figs. 1A and IB). HFD-fed mice treated with AAVl-CMV-comFGF21 showed normalized expression levels of the pro-inflammatory cytokines TNFa and Mcpl and the macrophage markers F4 / 80 and CD68 in lung (Figs. 2A-2D). No changes in the expression levels of interleukin 6 (IL-6) were observed in control HFD-fed mice or HFD-fed mice treated IM with AAVl-CMV-comFGF21 vector (Fig. 2E). Expression levels of interleukin- 1 -a (IL-la) in lung remained undetectable in all the experimental groups when evaluated by qPCR. These results support that intramuscular treatment with AAVl-CMV-comFGF21 vector counteracted pulmonary inflammation in HFD-fed mice.

[0363] Long-term HDF feeding did not induce pulmonary fibrosis. As chronic inflammatory reactions may result in fibrosis (Ghanem, M., et al., Pharmacol Ther. 259: 108669 (2024)), expression of the fibrosis markers collagen type I alpha 1 chain (Collal), collagen type III alpha 1 chain (Col3al), transforming growth factor beta 1 (Tgf-P), fibronectin 1 (Fnl) and alpha smooth muscle actin (a-Sma) was determined in lung (Figs. 3A-3E). Expression levels of Collal, Col3al, Tgf-P, Fnl and a-Sma remained unchanged in HFD-fed mice (Figs. 3A-3E). Although not statistically significant, mice treated with AAVl-CMV-comFGF21 vector showed decreased expression of Collal, Col3al, Tgf-P, Fnl and a-Sma relative to chow-fed control mice (Figs. 3A-3E). In agreement, analyses of fibrosis by picrosirius red staining of lung sections revealed no marked changes in the degree of collagen deposition among experimental groups (Fig. 4).[0364[ Intra-muscular administration of AAV-FGF21 vectors decreased pulmonary inflammation in db / db mice. Immunostaining for the macrophage-specific marker MAC2 in lung sections of db / db mice showed severe macrophage infiltration in pulmonary alveoli and bronchi, which was more evident in the lobe borders, while macrophages were not detected in the lungs of AAVl-CMV-comFGF21-treated db / db mice (Figs. 5A-5B). IM treatment with AAVl-CMV-comFGF21 vectors decreased expression of Tnfla, F4 / 80, Cd68, Mcpl and 11-6 (Figs. 6A-6E). Expression levels of interleukin- 1 -a (IL-la) in lung remained undetectable in all the experimental groups when evaluated by qPCR.

[0365] AAV-FGF21 IM injection reduced pulmonary fibrosis in db / db mice. In agreement with the reduction of pulmonary inflammation, decreased expression of the fibrosis markers Collal, Tgf-|3, Col3al and Fnl was observed in lungs of db / db mice treated IM with AAVl-CMV-comFGF21 vectors (Figs. 7A-7D). No changes in the expression levels of a-Sma in the lung were observed (Fig. 7E). Histological analysis of lung sections by picrosirius red staining further revealed decreased collagen accumulation in lungs of AAV-FGF21 -treated db / db mice (Fig. 8).

[0366] Conclusions. The data showed that intra-muscular AAV-FGF21 treatment normalized pulmonary inflammation in HFD-induced obese mice, and decreased inflammation and fibrosis in the lung of db / db mice. Altogether, these results suggest that the muscle-directed AAV-FGF21 gene therapy may counteract not only pulmonary inflammation and fibrosis induced by obesity and insulin resistance but also in other pulmonary inflammatory diseases.Example 3. Effects of intramuscular AAV1-FGF21 on pulmonary inflammation and fibrosis.

[0367] A dose of 3xl0nviral genomes (vg) / mouse of AAV vectors of serotype 1 including an expression cassette encoding a murine optimized Fgf21 coding sequence (moFgf21) under the control of the cytomegalovirus (CMV) promoter (AAV1-FGF21) was administered into quadriceps, gastrocnemius and tibialis muscles of both hindlimbs of thirteen-months-old C57BL / 6J01aHsd male mice, which were followed up to 26 months of age. A littermate control group was administered the same dose of null vectors (AAVl-null) intramuscularly at thirteen months of age and followed up to 26 months. At 26 months of age, mice were euthanized and lung samples obtained for transcriptomic analysis. Another cohort of seven-months-old C57BL / 6J male mice were used as young controls.

[0368] In lung samples from 7, or 26-months-old mice (n=5-8 / group), quantification by qRT-PCR of the expression of key genes was performed. FIGs. 9A-9C provides expression level of lung inflammatory markers: Tumor necrotic factor alpha (Tnfla) (FIG. 9A), F4 / 80 (FIG. 9B), and Transforming growth factor beta-1 (Tgfb) (FIG. 9C). Data are presented as mean ± SEM. *P<0.05, **P<0.01 by one-way analysis of variance (ANOVA) with Tukey's multiple comparison test. FC, Fold change. N = 5-8 animal s / group.

[0369] Additionally, FIG. 10 shows Mac2 immunostaining of lung sections of healthy control mice (7-month-old), aged control mice (26-month-old) treated intramuscularly. The staining shows that AAV-FGF21 treatment prevents age-related pulmonary inflammation.

[0370] The data shows that aged healthy mice treated with an intramuscular injection of AAV1-FGF21 presented a decrease in inflammatory markers and in Mac 2 (macrophages) immunostaining of lung sections relative to the control cohort.

[0371] Picrosirius red staining of sections of a whole lung lobule at 26 months of age for control and AAV-FGF21 -treated mice showed that AAV-FGF21 gene therapy prevented development of severe lung fibrosis, a key hallmark of lung aging, as evidenced by reduced picrosirius red staining and reduced percentage of area stained (FIGs. 11-12). Analysis of a whole lung lobule allowed detection of the widespread fibrotic lesion in the control lungs compared to the lung from AAV-FGF21 -treated mice, which had normal, non-aged appearance with lack of fibrotic alterations.* * *

[0372] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.

[0373] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.Table 2: Sequences

Claims

WHAT IS CLAIMED IS:

1. A method for treating or reducing pulmonary inflammation and / or pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises a vector genome and an AAV capsid, optionally having an AAV1 serotype, wherein the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a ubiquitous promoter.

2. A method for treating or reducing an inflammatory lung disease, lung disorder, or lung condition in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises a vector genome and an AAV capsid, optionally having an AAV1 serotype, wherein the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a ubiquitous promoter.

3. A method for treating, preventing or reducing the likelihood of pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises a vector genome and an AAV capsid, optionally having an AAV1 serotype, wherein the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or a functional fragment thereof operably linked to a ubiquitous promoter.

4. The method of any one of claims 1-3, wherein the ubiquitous promoter comprises a small chicken beta-actin promoter / cytomegalovirus enhancer (smCBA) promoter, a eukaryotic translation elongation factor 1 a (EFla) promoter, a simian virus 40 (SV40) promoter, a cytomegalovirus (CMV) promoter or a CAG promoter.

5. The method of any one of claims 1-4, wherein the ubiquitous promoter comprises a cytomegalovirus (CMV) promoter.

6. The method of any one of claims 1-5, wherein the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence encoding an amino acid sequence having at least 85%, 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.

7. The method of any one of claims 1-5, wherein the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence encoding an amino acid sequence having at least 85%, 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 amino acids 29-209 of SEQ ID NO: 1 and a leader sequence.

8. The method of any one of claims 1-7, wherein the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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, 5, 6, or 7.

9. The method of any one of claims 1-7, wherein the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof comprises a nucleotide sequence having at least 85%, 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 nucleic acids 85-627 of any of SEQ ID NO: 4, 5, 6, or 7.

10. The method of claim 7, wherein the leader sequence comprises a signal peptide selected from a human FGF21 signal peptide, a human IL- 10 (hIL-10) signal peptide, a human IL- 6 (hIL-6) signal peptide, a human FSH signal peptide, a human Albumin signal peptide, a human CSF signal peptide, a mouse immunoglobulin kappa light chain signal peptide(KASP), a Gaussia Luciferase signal peptide (GLSP), a human Igk signal peptide, a human Apolipoprotein signal peptide, a human PEDF signal peptide, a human SPARC signal peptide, a human tPA signal / propeptide (amino acids 1-32), a human tPA signal / propeptide (amino acids 1-35), a human IgVH signal peptide, a human GCG signal peptide, a human GIP signal peptide + GIP propeptide, or a human Prothrombin signal peptide.

11. The method of claim 10, wherein the leader sequence comprises a FGF21 signal peptide.

12. The method of claim 9, wherein the nucleotide sequence encoding the FGF21 protein or a functional fragment thereof further comprises a nucleotide sequence encoding a leader sequence comprising a signal peptide selected from a human IL-10 (hIL-10) signal peptide, a human IL-6 (hIL-6) signal peptide, a human FGF21 signal peptide, a human FSH signal peptide, a human Albumin signal peptide, a human CSF signal peptide, a mouse immunoglobulin kappa light chain signal peptide (KASP), a Gaussia Luciferase signal peptide (GLSP), a human Igk signal peptide, a human Apolipoprotein signal peptide, a human PEDF signal peptide, a human SPARC signal peptide, a human tPA signal / propeptide (amino acids 1-32), a human tPA signal / propeptide (amino acids 1-35), a human IgVH signal peptide, a human GCG signal peptide, a human GIP signal peptide + GIP propeptide, or a human Prothrombin signal peptide.

13. The method of claim 12, wherein the leader sequence comprises a FGF21 signal peptide.

14. The method of any one of claims 1-13, wherein the rAAV comprises a polynucleotide having at least 85%, 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: 14.

15. The method of any one of the claims 1-14, wherein the expression construct further comprises one or more of a polyadenylation signal, at least one target sequence of a microRNA, and / or an enhancer sequence.

16. The method of any one of the claims 1-15, wherein the rAAV vector is administered as a single dose.

17. The method of any one of the claims 1-16, wherein the rAAV vector is administered over multiple doses.

18. The method of any one of claims 1-17, wherein the subject suffers from obesity and / or insulin resistance.

19. The method of any one of claims 1-17, the subject does not suffer from obesity and / or insulin resistance.

20. The method of any one of claims 1 and 4-17, wherein the pulmonary inflammation is caused by or associated with an infection, an inflammatory condition, a genetic condition, a medication, aging and / or a chemical exposure.

21. The method of any one of claims 1, 4-17, and 20, wherein the pulmonary inflammation is a pneumonitis.

22. The method of claim 21, wherein the pneumonitis is a hypersensitivity pneumonitis, a drug-induced pneumonitis, or a radiation-induced pneumonitis.

23. The method of any one of claims 1, 4-17, and 20-22, wherein the pulmonary inflammation is a chronic inflammation.

24. The method of any one of claims 1, 4-17, and 20-23, wherein, the pulmonary inflammation is caused by chronic obstructive pulmonary disease (COPD).

25. The method of any one of claims 1 and 4-17, wherein the pulmonary inflammation is a pulmonary sarcoidosis.

26. The method of any one of claims 2 and 4-17, wherein the inflammatory lung disease, lung disorder, or lung condition is COPD.

27. The method of any one of claims 2 and 4-17, wherein the inflammatory lung disease, lung disorder, or lung condition is asthma.

28. The method of any one of claims 2 and 4-17, wherein the inflammatory lung disease, lung disorder, or lung condition is bronchitis.

29. The method of any one of claims 1 and 3-17, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.

30. The method of any one of claims 1-29, wherein the subject suffers from asbestosis.

31. The method of any one of claims 1-30, wherein the subject has at least a 0.1-6.0 fold reduction in a relative expression of an inflammatory gene marker selected from the group consisting of Cluster of Differentiation 68 (CD68), F4 / 80, Tumor necrosis factoralpha (TNFa), Monocyte Chemoattractant Protein- 1 (MCP1), Interleukin-6 (IL-6), or any combination thereof.

32. The method of any one of claims 1-31, the subject has at least a 0.1-6.0 fold reduction in a relative expression of a fibrosis gene marker selected from the group consisting of collagen type I alpha 1 chain (Collal), collagen type III alpha 1 chain (Col3al), transforming growth factor beta 1 (Tgf-P), fibronectin 1 (Fnl) and alpha smooth muscle actin (a-Sma), or any combination thereof.

33. The method of any one of claims 1-32, wherein the method reduces one or more symptoms associated with the inflammatory lung disease, the lung disorder, the lung condition, the pulmonary inflammation and / or pulmonary fibrosis.

34. The method of claim 33, wherein the one or more symptoms comprising shortness of breath, a persistent cough, fatigue, or any combination thereof.

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