WO2025240607A2

WO2025240607A2 - Bicistronic gene expression systems and methods of use

Info

Publication number: WO2025240607A2
Application number: PCT/US2025/029350
Authority: WO
Inventors: Noah DAVIDSOHN; Christopher DE SOLIS
Original assignee: Rejuvenate Bio Inc
Current assignee: Rejuvenate Bio Inc
Priority date: 2024-05-14
Filing date: 2025-05-14
Publication date: 2025-11-20
Anticipated expiration: 2026-11-14

Abstract

Provided herein arc polynucleotides comprising at least two coding sequences for expression of coding sequences of interest in a cell. Also provided are vectors, recombinant viral genomes, and recombinant virus compositions comprising said polynucleotides, as well as associated methods.

Description

BICISTRONIC GENE EXPRESSION SYSTEMS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application number 63/647,134 filed on May 14, 2024, which is incorporated herein by reference it its entirety.

BACKGROUND

[0002] In certain gene transfer and therapy applications, expression of multiple coding sequences within the same target cells is required. In certain gene transfer and therapy applications, expression of multiple coding sequences within the same target cells is required. In such cases, coordinating coding sequence expression is important for when the resultant activity depends on multiple subunits encoded by different genes, or the synergism of separate molecules.

[0003] However, achieving coordinated, high-level expression of multiple coding sequences in the majority of target cells remains a significant challenge for gene transfer technology. Expression of two different coding sequences can result from the use of two separate vectors, but only a fraction of target cells is transduced by both vectors. Even successful transduction results in a heterogeneous population of cells that express either one or two genes in different ratios. This inconsistent transduction and expression profile can prevent reliable studies and/or efficacious applications.

[0004] Accordingly, there is a need for improved gene expression systems for the efficient and sustained expression of multiple coding sequences.

SUMMARY

[0005] Provided herein are polynucleotides including dual promoter expression cassettes, useful for expressing at least two coding sequences of interest in a cell. Various embodiments of polynucleotides comprise bidirectional dual promoter expression cassettes. Also provided are vectors, recombinant adeno-associated virus (rAAV) genomes including said polynucleotides, and rAAV particles, as well as associated methods for making and using polynucleotides and rAAV particles.

[0006] Accordingly, the present disclosure provides an isolated polynucleotide. In certain embodiments, an isolated polynucleotide includes a first nucleic acid. In certain embodiments, a first nucleic acid includes a first coding sequence. In certain embodiments, a first nucleic acid includes a first transcriptional regulatory element (TRE). In certain embodiments, a first TRE is operatively linked to a first coding sequence. In certain embodiments, a first TRE is or includes: a promoter, one or more enhancer elements, or any combination thereof.

[0007] In certain embodiments, an isolated polynucleotide includes a second nucleic acid. In certain embodiments, a second nucleic acid includes a second coding sequence. In certain embodiments, a second nucleic acid includes a second transcriptional regulatory element (TRE). In certain embodiments, a second TRE is operatively linked to a second coding sequence. In certain embodiments, a second TRE is or includes: a promoter, one or more enhancer elements, or any combination thereof.

[0008] In certain embodiments, a promoter is or includes a human albumin (hAlb) promoter. In certain embodiments, a promoter includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

[0009] In certain embodiments, a promoter is or includes a human alpha 1-antitrypsin (hAAT) promoter. In certain embodiments, a promoter includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0010] In certain embodiments, one or more enhancer elements comprise an apolipoprotein E (ApoE) enhancer element. In certain embodiments, one or more enhancer elements comprise an alpha-fetoprotein (AFP) enhancer element. In certain embodiments, one or more enhancer elements comprise at least two ApoE enhancer elements. In certain embodiments, one or more enhancer elements comprise at least three ApoE enhancer elements. In certain embodiments, one or more enhancer elements each comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. In certain embodiments, one or more enhancer elements each comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. [0011] In certain embodiments, an isolated polynucleotide includes one or more intron elements. In certain embodiments, one or more intron elements each include a P-globin intron element. In certain embodiments, one or more intron elements each include a MVM intron element. In certain embodiments, a P-globin intron element includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3. In certain embodiments, a P-globin intron element is 3’ of a first TRE. In certain embodiments, a P-globin intron element is 3’ of a second TRE. In certain embodiments, a P-globin intron element is 3’ of a first TRE and a second TRE.

[0012] In certain embodiments, a MVM intron element includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. In certain embodiments, a MVM intron element is 3’ of a first TRE. In certain embodiments, a MVM intron element is 3’ of a second TRE. In certain embodiments, a MVM intron element is 3’ of a first TRE and a second TRE.

[0013] In certain embodiments, a first TRE includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 27-29.

[0014] In certain embodiments, a second TRE includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or 11.

[0015] In certain embodiments, an isolated polynucleotide includes a first nucleic acid including a first post-transcriptional regulatory element (PRE). In certain embodiments, an isolated polynucleotide includes a second nucleic acid including a second post-transcriptional regulatory element (PRE).

[0016] In certain embodiments, a first PRE includes a polyadenylation sequence. In certain embodiments, a first PRE includes a WPRE sequence. In certain embodiments, a first PRE includes a polyadenylation sequence and a WPRE sequence. [0017] In certain embodiments, a second PRE includes a polyadenylation sequence. In certain embodiments, a second PRE includes a WPRE sequence. In certain embodiments, a second PRE includes a poly adenylation sequence and a WPRE sequence.

[0018] In certain embodiments, (a) a first PRE include any combination(s) of (1) a polyadenylation sequence, (2) a WPRE sequence, and (3) a polyadenylation sequence and a WPRE sequence; and (b) a second PRE include any combination(s) of (1) a poly adenylation sequence, (2) a WPRE sequence, and (3) a polyadenylation sequence and a WPRE sequence.

[0019] In certain embodiments, a poly adenylation sequence is or includes a bovine growth hormone polyadenylation (BGHpA) sequence. In certain embodiments, a polyadenylation sequence is or includes a simian virus 40 polyadenylation (SV40pA) sequence. In certain embodiments, a polyadenylation sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 or 23. In certain embodiments, a polyadenylation sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 9.

[0020] In certain embodiments, a WPRE sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0021] In certain embodiments, an isolated polynucleotide includes a Kozak consensus sequence 5’ to a first coding sequence. In certain embodiments, an isolated polynucleotide includes a Kozak consensus sequence 5’ to a second coding sequence. In certain embodiments, an isolated polynucleotide includes a Kozak consensus sequence 5’ to a first coding sequence and a Kozak consensus sequence 5’ to a second coding sequence.

[0022] In certain embodiments, a first nucleic acid and a second nucleic acid are in a same orientation in an isolated polynucleotide. In certain embodiments, a first nucleic acid and a second nucleic acid are separated by a polycistronic element. In certain embodiments, a polycistronic element is an IRES or 2A sequence. [0023] In certain embodiments, an isolated polynucleotide includes from 5’ to 3’: (a) a first TRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27; (b) a first coding sequence; (c) a first PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31; (d) a second TRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; (e) a second coding sequence; and (f) a second PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22.

[0024] In certain embodiments, a first nucleic acid and a second nucleic acid are in a opposite orientation in an isolated polynucleotide. In certain embodiments, an isolated polynucleotide includes from 5’ to 3’: (a) a second PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; (b) a second coding sequence; (c) a second TRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11; (d) a first TRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28; (e) a first coding sequence; and (f) a first PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31. In certain embodiments, an isolated polynucleotide includes from 5’ to 3”: (a) a second PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; (b) a second coding sequence; (c) a second TRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11; (d) a first TRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29; (e) a first coding sequence; and (f) a first PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31. In certain embodiments, an isolated polynucleotide includes from 5’ to 3’: (a) a second PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23; (b) a second coding sequence; (c) a second TRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11; (d) a first TRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity SEQ ID NO: 27; (e) a first coding sequence; and (f) a first PRE including a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31 or 32.

[0025] In certain embodiments, a first coding sequence encodes a miRNA, a shRNA, a siRNA, an antisense RNA, a gRNA, an antagomir, a miRNA sponge, an RNA aptazyme, an RNA aptamer, a IncRNA, a ribozyme or a mRNA. In certain embodiments, a second coding sequence encodes a miRNA, a shRNA, a siRNA, an antisense RNA, a gRNA, an antagomir, a miRNA sponge, an RNA aptazyme, an RNA aptamer, a IncRNA, a ribozyme or a mRNA. In certain embodiments, a first coding sequence and a second coding sequence each encode a miRNA, a shRNA, a siRNA, an antisense RNA, a gRNA, an antagomir, a miRNA sponge, an RNA aptazyme, an RNA aptamer, a IncRNA, a ribozyme or a mRNA.

[0026] In certain embodiments, a first coding sequence encodes one or more polypeptides. In certain embodiments, a second coding sequence encodes one or more polypeptides. In certain embodiments, a first coding sequence and a second coding sequence each encode one or more polypeptides.

[0027] In certain embodiments, a first coding sequence encodes a growth factor or receptor thereof. In certain embodiments, a second coding sequence encodes a growth factor or receptor thereof. In certain embodiments, a first coding sequence and a second coding sequence each encode a growth factor or receptor thereof.

[0028] In certain embodiments, an isolated polynucleotide includes a first nucleic acid including a first coding sequence encoding a first growth factor or receptor thereof and a first transcriptional regulatory element (TRE). In certain embodiments, an isolated polynucleotide includes a second nucleic acid including a second TRE and a second coding sequence encoding a second growth factor or receptor thereof.

[0029] In certain embodiments, a first coding sequence encodes fibroblast growth factor 21 (FGF21). In certain embodiments, a first coding sequence encodes a canine FGF21. In certain embodiments, a first coding sequence encodes a mouse FGF21. In certain embodiments, a first coding sequence encodes a human FGF21. In certain embodiments, a first coding sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 4-6.

[0030] In certain embodiments, a second coding sequence encodes a growth factor receptor. In certain embodiments, a second coding sequence encodes a transforming growth factor beta receptor 2 (TGPFR2). In certain embodiments, a second coding sequence encodes a canine TGPFR2. In certain embodiments, a second coding sequence encodes a mouse TGPFR2. In certain embodiments, a second coding sequence encodes a human TGPFR2. [0031] In certain embodiments, a second coding sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-18.

[0032] In certain embodiments, a second coding sequence encodes a soluble growth factor receptor. In certain embodiments, a second coding sequence encodes a soluble transforming growth factor beta receptor 2 (sTG0FR2). In certain embodiments, a growth factor receptor is linked to an Fc region. In certain embodiments, a Fc region is or includes a canine Fc region. In certain embodiments, a Fc region is or includes an IgG2 Fc region. In certain embodiments, a Fc region is or includes an IgGl Fc region.

[0033] In certain embodiments, a second coding sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33-35.

[0034] In certain embodiments, a growth factor or receptor thereof is linked to a signal peptide. In certain embodiments, a signal peptide is or includes a human TGF R2 signal peptide. In certain embodiments, is or includes a signal peptide is a canine AAT signal peptide.

[0035] In certain embodiments, a growth factor receptor is soluble transforming growth factor beta receptor 2 (sTGpFR2).

[0036] In certain embodiments, a second coding sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 36-39.

[0037] In certain embodiments, a growth factor receptor includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 40-43.

[0038] In certain embodiments, a first coding sequence does not encode a functional antibody. In certain embodiments, a second coding sequence does not encode a functional antibody. In certain embodiments, a first coding sequence and a second coding sequence do not encode a functional antibody. [0039] In certain embodiments, an isolated polynucleotide comprising (a) a first nucleic acid comprising: (1) a first coding sequence encoding a first growth factor or receptor thereof, and (2) a first transcriptional regulatory element (TRE); and (b) a second nucleic acid comprising: (1) a second coding sequence encoding a second TRE, and (2) a second coding sequence encoding a second growth factor or receptor thereof. In certain embodiments, an isolated polynucleotide includes a first post-transcriptional regulatory element (PRE). In certain embodiments, a first PRE includes a polyadenylation sequence. In certain embodiments, a first PRE includes a WPRE sequence. In ceriain embodiments, a first PRE includes a polyadenylation sequence and a WPRE sequence. In certain embodiments, a first PRE is 5’ of a first coding sequence. In certain embodiments, an isolated polynucleotide includes a second PRE. In certain embodiments, a second PRE includes a polyadenylation sequence. In certain embodiments, a second PRE includes a WPRE sequence. In certain embodiments, a second PRE includes a polyadenylation sequence and a WPRE sequence. In certain embodiments, a second PRE is 3’ of a second coding sequence. In certain embodiments, a first coding sequence encodes a growth factor receptor. In certain embodiments, a second coding sequence encodes a growth factor receptor. In certain embodiments, a growth factor receptor is a soluble growth factor receptor. In certain embodiments, a first coding sequence encodes a soluble transforming growth factor beta receptor 2 (sTGpFR2). In certain embodiments, a second coding sequence encodes a soluble transforming growth factor beta receptor 2 (sTGpFR2). In certain embodiments, a sTGpFR2 is linked to an Fc region. In certain embodiments, a first coding sequence encodes a growth factor, optionally wherein a growth factor is fibroblast growth factor 21 (FGF21). In certain embodiments, a second coding sequence encodes a growth factor. In certain embodiments, a growth factor is or includes fibroblast growth factor 21 (FGF21).

[0040] In certain embodiments, a growth factor is linked to a signal peptide.

[0041] In certain embodiments, a first TRE and a second TRE are the same. In certain embodiments, a first TRE and a second TRE are different.

[0042] In certain embodiments, a first TRE is liver- specific. In certain embodiments, a second TRE is liver- specific. In certain embodiments, a first TRE and a second TRE are liverspecific. In certain embodiments, a liver- specific TRE includes one or more elements comprising an ApoA-I promoter, an ApoA-II promoter, an ApoA-IV promoter, an ApoB promoter, an ApoC- I promoter, an ApoC-II promoter, an ApoC-III promoter, an ApoE promoter, an albumin promoter, an a- fetoprotein promoter, a phosphoenolpyruvate carboxykinase 1 (PCK1 ) promoter, a phosphocnolpyruvatc carboxykinasc 2 (PCK2) promoter, a transthyretin (TTR) promoter, an a- antitrypsin (AAT or SERPINA1) promoter, a hemopexin promoter, an alcohol dehydrogenase 6 promoter, a cholesterol 7alpha-hydroxylase promoter, a factor IX promoter, or an a- microglobulin promoter.

[0043] In certain embodiments, a first TRE is muscle- specific. In certain embodiments, a second TRE is muscle- specific. In certain embodiments, a first TRE and a second TRE are musclespecific. In certain embodiments, a muscle-specific TRE includes one or more elements comprising a human skeletal muscle a-actin (HSA) promoter, a muscle creatine kinase (MCK) promoter, a MHCK7 promoter, a dMCK promoter, a tMCK promoter, a CK6 promoter, a CK8 promoter, a CK8e promoter, a human desmin (DES) promoter or variant thereof, a cardiac troponin T (cTnT) promoter, a myosin light-chain (MLC2v) promoter, a human a-myosin heavy chain gene (aMHC) promoter, a MLC promoter, a human troponin I (TNNI1) promoter, a AUSEx3 promoter, a SPcA5-12 promoter, a SP-301 promoter, a MH promoter, or a Sk-CRM4/DES promoter.

[0044] In certain embodiments, a first TRE includes an intron element. In certain embodiments, a second TRE include an intron element. In certain embodiments, a first TRE and second TRE each include an intron element.

[0045] In certain embodiments, a first coding sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-18 or 33-39. In certain embodiments, a second coding sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-18 or 33-39.

[0046] In certain embodiments, a first coding sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 4-6. In certain embodiments, a second coding sequence includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 4-6.

[0047] In certain embodiments, an isolated polynucleotide includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 44-61.

[0048] The present disclosure also provides an isolated polynucleotide that is a complement of any of the isolated polynucleotides described herein.

[0049] In certain embodiments, an isolated polynucleotide that is a reverse complement of any of the isolated polynucleotides described herein.

[0050] Among other things, the present disclosure provides a vector including any of the polynucleotides described herein.

[0051] In certain embodiments, a vector is or includes a plasmid. In certain embodiments, a vector is or includes a viral vector. In certain embodiments, a vector is or includes a DNA minimal vector. In certain embodiments, a vector is or includes an expression vector. In certain embodiments, a viral vector is or includes an adenoviral vector. In certain embodiments, a viral vector is or includes an adeno-associated virus (AAV) vector. In certain embodiments, a viral vector is or includes a lentiviral vector. In certain embodiments, a vector is or includes an AAV vector.

[0052] Among other things, the present disclosure provides a recombinant adeno- associated virus (rAAV) genome including any of the isolated polynucleotides described herein.

[0053] In certain embodiments, a rAAV genome includes a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence. In certain embodiments, a rAAV genome includes a 3’ inverted terminal repeat (3’ ITR) nucleotide sequence. In certain embodiments, a rAAV genome includes a ‘5 ITR nucleotide sequence and 3TTR nucleotide sequence. In certain embodiments, a 5’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24. In certain embodiments, a 3’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25 or 26. [0054] In certain embodiments, a rAAV genome includes a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 62-68.

[0055] Among other things, the present disclosure provides a recombinant AAV particle including an AAV capsid and any of the rAAV genomes described herein.

[0056] In certain embodiments, an AAV capsid protein is derived from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof. In certain embodiments, an AAV capsid protein includes a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof. In certain embodiments, an AAV capsid protein is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof. In certain embodiments, an AAV capsid protein includes an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69, 70, and/or 71.

[0057] Among other things, the present disclosure provides a pharmaceutical composition including any of the polynucleotides, vectors, rAAV genomes, or rAAV particles described herein. [0058] Among other things, the present disclosure provides any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein for use in various technologies. In certain embodiments, the present disclosure provides any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein for use in medicine. In certain embodiments, the present disclosure provides any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein for use as therapy. In certain embodiments, the present disclosure provides any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein for use as a medicament. [0059] Among other things, the present disclosure provides a packaging system for preparation of an rAAV particle. In certain embodiments, a packaging system includes: a first nucleotide sequence encoding one or more AAV Rep proteins. In certain embodiments, a second nucleotide sequence encoding an AAV capsid protein. In certain embodiments, a third nucleotide sequence includes any of the rAAV genomes described herein.

[0060] In certain embodiments, a packaging system includes a first vector. In certain embodiments, a first vector includes a first nucleotide sequence and a second nucleotide sequence. In certain embodiments, a packaging system includes a second vector. In certain embodiments, a second vector includes a third nucleotide sequence.

[0061] In certain embodiments, a packaging system includes a fourth nucleotide sequence. In certain embodiments a fourth nucleotide sequence includes one or more helper virus genes. In certain embodiments, a fourth nucleotide sequence includes one or more genes from a virus. In certain embodiments, one or genes are selected from an adenovirus. In certain embodiments, one or genes are selected from a herpesvirus. In certain embodiments, one or genes are selected from a vaccinia virus. In certain embodiments, one or genes are selected from a cytomegalovirus (CMV). In certain embodiment a packaging system includes a third vector. In certain embodiments, a third vector includes a fourth nucleotide sequence.

[0062] In certain embodiments, a first vector is a plasmid. In certain embodiments, a second vector is a plasmid. In certain embodiments, a third vector is a plasmid. In certain embodiments, a first vector, second vector, and a third vector is a plasmid.

[0063] Among other things, the present disclosure provides methods including any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein.

[0064] In certain embodiments, a method includes recombinant preparation of an rAAV particle. In certain embodiments, a method includes introducing any of the packaging systems described herein into a cell. In certain embodiments, a method includes introducing any of the packaging systems described herein into a cell under conditions whereby a rAAV particle is produced.

[0065] In certain embodiments, a method include introducing into a cell any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein. [0066] In certain embodiments, a method includes expressing a first coding sequence in a cell. In certain embodiments, a method includes expressing a second coding sequence in a cell. In certain embodiments, a method includes expressing a first coding sequence and a second coding sequence in a cell. In certain embodiments, said methods includes introducing into a cell any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein.

[0067] In certain embodiments, a method includes expressing a first coding sequence in a subject. In certain embodiments, a method includes expressing a second coding sequence in a subject. In certain embodiments, a method includes expressing a first coding sequence and a second coding sequence in a subject. In certain embodiments, a method includes administering to a subject an effective amount of any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein.

[0068] In certain embodiments, a method includes a method of treating an arrhythmogenic cardiomyopathy (ACM) in a subject. In certain embodiments, said method includes administering to a subject an effective amount of any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein. In certain embodiments, ACM comprises arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), or biventricular’ arrhythmogenic cardiomyopathy. In certain embodiments, ACM is ARVC.

[0069] In certain embodiments, a subject is a mammal. In certain embodiments, a subject is a human. In certain embodiments, a subject is a non-human primate. In certain embodiments, a subject is a canine. In certain embodiments, a subject is a feline. In certain embodiments, a subject is an equine. In certain embodiments, a subject is a bovine. In certain embodiments, a subject is a swine. In certain embodiments, a subject is an avian. In certain embodiments, a subject is a rodent. [0070] Among other things, the present disclosure provides a kit including any of the polynucleotides, vectors, rAAV genomes, rAAV particles, or pharmaceutical compositions described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIGS. 1A and IB are bar graphs showing plasma expression levels of canine FGF21 (cFGF21) and canine TGFPR2 (cTGFpR2), respectively, in mice injected with different constructs, as detected by ELISA at 6 weeks post-injection. In these figures, error bars represent range and within-box line represents average.

[0072] FIGS. 2A-2D are graphs showing plasma expression levels of canine cFGF21 and cTGFpR2 in beagles injected with different constructs, as detected by ELISA, at 2 weeks, 4 weeks, 6 weeks, and 8 weeks post-injection. FIG. 2A shows mean values of plasma cFGF21 levels measured at each time point, across all injected subjects. FIG. 2B shows values of plasma cFGF21 levels measured at each time point, for each individual injected subject. FIG. 2C shows the mean values of plasma cTGFpR2 levels measured at each time point, across all injected subjects. FIG. 2D shows the values of plasma cTGFPR2 levels measured at each time point, for each individual injected subject. In these figures, error bars represent S.E.M.

[0073] FIGS. 3A and 3B are graphs showing plasma expression levels of mouse FGF21 (mFGF21) and mouse TGFPR2 (mTGFpR2), respectively, in mice injected with different constructs, as detected by ELISA at 2 weeks, 4 weeks, 6 weeks, and 8 weeks post-injection. In these figures, error bars represent S.E.M.

[0074] FIG. 4A and 4B are graphs showing levels of FGF21 (FIG. 4A) and levels of sTGF R2 (FIG. 4B) expression in PKP2cKO mice administered AAV8-p608, AAV8-mFGF21, and AAV8-msTGFpR2-Fc at a dose of 1.5E13 vg/kg each. Wild type mice administered vehicle (WT) were used as control. Means with standard error of the mean are presented.

[0075] FIGS. 5A-5C are graphs showing cardiac structure and function of PKP2cKO mice administered AAV8-p608, as measured by left ventricle ejection fraction (LVEF; FIG. 5A), fractional shortening (FS; FIG. 5B) and right ventricle area (RV area; FIG. 5C). Wild type mice administered vehicle (WT) were used as control. Means with standard deviation are presented. * Indicates a p value of <0.05 and ** indicates a p value of <0.01 by one-way ANOVA and Dunn’s multiple comparisons test.

[0076] FIG. 6 is a graph showing arrhythmia burden of PKP2cKO mice administered AAV8-p6O8, measured as the percent of premature ventricular contractions (PVCs) observed in 30 minutes following an isoproterenol challenge. The numbers refer to the number of PVCs observed in 30 minutes following an isoproterenol challenge.

[0077] FIGS. 7A and 7B are graphs showing the percentage of collagen in cardiac tissues of PKP2cKO mice administered AAV8-p6O8, indicative of left ventricle (LV) fibrosis (FIG. 7A), and right ventricle (RV) fibrosis (FIG. 7B). Means with standard deviation are presented. DETAILED DESCRIPTION

[0078] Provided herein are polynucleotides comprising dual promoter expression cassettes, useful for expressing at least two coding sequences of interest in a cell. Various embodiments of polynucleotides comprise bidirectional dual promoter expression cassettes. Also provided are vectors, recombinant adeno-associated virus (rAAV) genomes comprising said polynucleotides, and rAAV particles, as well as associated methods for making and using polynucleotides and rAAV particles.

I. Definitions

[0079] AAV: As used herein, the term "AAV" is a standard abbreviation for adeno- associated virus.

[0080] Administration: As used herein, the term “administration” typically refers to administration of a composition to a subject or system to achieve delivery of an agent to a subject or system. In certain embodiments, an agent is, or is included in, a composition; In certain embodiments, an agent is generated through metabolism of a composition or one or more components thereof. A variety of routes are available for administration of compositions; for example, some compositions may be administered by one or more routes such as ocular, oral, parenteral, topical, etc. For example, in certain embodiments, administration may be systematic or local. In certain embodiments, a systematic administration can be intravenous. In certain embodiments, administration can be local. In certain embodiments, administration may involve only a single dose. In certain embodiments, administration may involve application of a fixed number of doses. In certain embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In certain embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0081] Agent: As used herein, the term “agent,” may refer to a physical entity. In certain embodiments, an agent may be characterized by a particular feature and/or effect. For example, as used herein, the term “therapeutic agent” refers to a physical entity has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In certain embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or any combination or complex thereof. [0082] Amelioration: As used herein, the term “amelioration” refers to prevention, reduction or palliation of a state, or improvement of a state of a subject. Amelioration may include, but does not require, complete recovery or complete prevention of a disease, disorder or condition. [0083] Associated: As used herein, the term “associated” describes two events or entities as “associated” with one another, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In certain embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In certain embodiments, two or more entities that are physically associated with one another are covalently linked to one another; In certain embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0084] Characteristic portion'. As used herein, the term “characteristic portion,” in the broadest sense, refers to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance. In certain embodiments, a characteristic portion of a substance is a portion that is found in a given substance and in related substances that share a particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in certain embodiments, a “characteristic portion” of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. In certain embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a substance (e.g., of a protein, antibody, etc.) is one that, in addition to a sequence and/or structural identity specified above, shares at least one functional characteristic with the relevant intact substance. In certain embodiments, a characteristic portion may be biologically active. [0085] Characteristic sequence'. As used herein, the term “characteristic sequence” is a sequence that is found in all members of a family of polypeptides or nucleic acids, and can be used to define members of the family.

[0086] Characteristic sequence element'. As used herein, the phrase “characteristic sequence element” refers to a sequence element found in a polymer (e.g., in a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In certain embodiments, presence of a characteristic sequence element correlates with presence or level of a particular activity or property of a polymer. In certain embodiments, presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not a member) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In certain embodiments, a characteristic sequence element includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., contiguously linked monomers). In certain embodiments, a characteristic sequence element includes at least first and second stretches of contiguous monomers spaced apart by one or more spacer regions whose length may or may not vary across polymers that share a sequence element.

[0087] Coding sequence: As used herein, the term “coding sequence” refers to a non-AAV nucleic acid sequence that encodes a polypeptide e.g., a therapeutic protein) or non-coding RNA (e.g., a miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, or RNA aptamer).

[0088] Effective Amount: As used herein, the term “effective amount” in the context of the administration of a polynucleotide, vector, composition, or rAAV particle to a subject refers to the amount of the polynucleotide, vector, composition, or rAAV particle that achieves a desired prophylactic or therapeutic effect.

[0089] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In certain embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In certain embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a noncoding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.

[0090] Expression Vector: As used herein, an “expression vector” refers to a vector comprising transcriptional regulatory elements operably linked to a gene of interest (e.g., a polynucleotide described herein) that facilitate the expression of the gene of interest in a cell and/or a cell free expression system.

[0091] Identity: As used herein, the term “identity” refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In certain embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules are considered to be “substantially identical” to one another if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. [0092] Increased, Induced, or Reduced’. As used herein, these terms or grammatically comparable comparative terms, indicate values that arc relative to a comparable reference measurement. For example, in certain embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be “increased” relative to that obtained with a comparable reference composition. Alternatively or additionally, In certain embodiments, an assessed value achieved in a subject may be “increased” relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In certain embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). In a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance can be determined. In certain embodiments, the term “reduced”, or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In certain embodiments, the term “reduced”, or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In certain embodiments, the term “increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.

[0093] Isolated polynucleotide: As used herein, an “isolated polynucleotide” refers to a polynucleotide that has been separated from one or more nucleic acid molecules present in the natural source of the polynucleotide.

[0094] Linker. As used herein, the term “linker” refers to a portion of a polypeptide that connects different regions, portions, or antigens to one another.

[0095] Nucleic acid or polynucleotide '. As used herein, in its broadest sense, nucleic acid or polynucleotide refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In certain embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in certain embodiments, "nucleic acid" or "polynucleotide" refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in certain embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In certain embodiments, a "nucleic acid" is or comprises RNA; in certain embodiments, a "nucleic acid" or "polynucleotide" is or comprises DNA. In certain embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In certain embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In certain embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in certain embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively, or additionally, in certain embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N- phosphoramidite linkages rather than phosphodiester bonds. In certain embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine). In certain embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyiTolo-pyrimidine, 3 - methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 - propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In certain embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In certain embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In certain embodiments, a nucleic acid includes one or more introns. In certain embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In certain embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In certain embodiments, a nucleic acid is partly or wholly single stranded; in certain embodiments, a nucleic acid is partly or wholly double stranded. In certain embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In certain embodiments, a nucleic acid has enzymatic activity.

[0096] Nucleotide sequence encoding an AAV capsid protein: As used herein, the term “nucleotide sequence encoding an AAV capsid protein” refers to a nucleic acid sequence that encodes a capsid protein. For AAV, the capsid protein may be VP1, VP2, or VP3. VP1, VP2, and/or VP3 capsid proteins assemble into a capsid that surrounds the rAAV genome.

[0097] Nucleotide sequence encoding one or more AAV Rep proteins: As used herein, the term “nucleotide sequence encoding one or more AAV Rep proteins” refers to one or more nucleic acid sequences that encode the non-structural proteins (e.g., rep78, rep68, rep52, and rep40) required for the replication and production of an AAV.

[0098] Operably linked: As used herein, the term “operably linked” is used to describe the connection between a TRE and/or a PRE and a polynucleotide sequence (e.g., a coding sequence described herein) to be transcribed. Typically, gene expression is placed under the control of a TRE comprising one or more promoter and/or enhancer elements and/or a PRE comprising, e.g., a transcription termination sequence. A coding sequence is “operably linked” to the TRE if the transcription of the coding sequence is controlled or influenced by the TRE. A coding sequence is “operably linked” to the PRE if the RNA molecule encoded by the coding sequence is controlled or influenced by the PRE. The elements of the TRE and/or PRE may be in any orientation and/or at any distance from the coding sequence, as long as the desired transcriptional and/or post- transcriptional activity is obtained. In an embodiment, the TRE is upstream from the coding sequence. In an embodiment, the PRE is downstream from the coding sequence.

[0099] Percent identity: As used herein, the “percentage identity” between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between the pair of aligned sequences by 100, and dividing by the length of the aligned region, including internal gaps. Identity scoring only counts perfect matches, and does not consider the degree of similarity of amino acids to one another. Note that only internal gaps are included in the length, not gaps at the sequence ends. [0100] Polypeptide: As used herein, the term “polypeptide” refers to a polymeric chain of amino acids. In certain embodiments, a polypeptide has an amino acid sequence that occurs in nature. In certain embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In certain embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In certain embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In certain embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In certain embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In certain embodiments, a polypeptide may comprise only D-amino acids. In certain embodiments, a polypeptide may comprise only L- amino acids. In certain embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In certain embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In certain embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In certain embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In certain embodiments, a polypeptide is linear. In certain embodiments, a polypeptide may be or comprise a stapled polypeptide. In certain embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. In certain embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In certain embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (In certain embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; In certain embodiments with all polypeptides within the class). For example, In certain embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may In certain embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; In certain embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In certain embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

[0101] Post-transcriptional regulatory element: As used herein, the term “post- transcriptional regulatory element” or “PRE” refers to a cis-acting nucleotide sequence, for example, a DNA sequence, that regulates (e.g., controls, increases, or reduces) the production, stability, processing, or levels of an RNA molecule (e.g., an RNA molecule that is being transcribed or has been transcribed from a coding sequence). A post-transcriptional regulatory element may be, e.g., any sequence that effectively terminates transcription. Post-transcriptional regulatory element sequences can be isolated from any genomic loci that are associated with expression of genes in a cell in which transcription of a coding sequence is desired.

[0102] rAAV: As used herein interchangeably, the terms “recombinant adeno-associated virus” or “rAAV” refer to an AAV comprising a genome lacking functional rep and cap genes.

[0103] rAAV genome: As used herein, the term “rAAV genome” refers to a nucleic acid molecule (e.g., DNA and/or RNA) comprising the genome sequence of an rAAV. It is an insight of the present disclosure that where an rAAV genome comprises a coding sequence (e.g., a polypeptide encoding a therapeutic protein operably linked to a transcriptional regulatory element (i.e., pay load)), the rAAV genome can be in the sense or antisense orientation relative to the direction of transcription of the coding sequence.

[0104] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in certain embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In certain embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In certain embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. A reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. [0105] Transcriptional Regulatory Element: As used herein, the term “transcriptional regulatory clement” or “TRE” refers to a cis-acting nucleotide sequence, for example, a DNA sequence, which regulates (e.g., controls, increases, or reduces) transcription of an operably linked nucleotide sequence by an RNA polymerase to form an RNA molecule. A TRE may comprise one or more promoter elements and/or enhancer elements. Promoter and enhancer elements in a gene may be close in location, and the term “promoter” may refer to a sequence comprising a promoter element and an enhancer element. Thus, the term “promoter” does not exclude an enhancer element in the sequence. The promoter and enhancer elements do not need to be derived from the same gene or species, and the sequence of each promoter or enhancer element may be either identical or substantially identical to the corresponding endogenous sequence in the genome.

[0106] Treat, Treating, or Treatment: As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration of a polynucleotide to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0107] Variant: As used herein, the term “variant” refers to a molecule that shows significant structural (e.g., primary or secondary) identity with a reference molecule but differs structurally from the reference molecule. For example, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In certain embodiments, a variant polypeptide comprises a characteristic portion of a reference polypeptide. [0108] Vector: As used herein, a “vector” refers to a nucleic acid molecule that is a vehicle for introducing a nucleic acid molecule (e.g., a polynucleotide described herein) into a cell.

II. Polynucleotides, Vectors, and Compositions

[0109] Among other things, the present disclosure provides polynucleotides or nucleic acid sequences (e.g., isolated polynucleotides, vectors, etc.) comprising dual promoter expression cassettes, useful for expressing at least two coding sequences of interest in a cell. As described herein and demonstrated in the Examples, various embodiments of nucleic acid sequences comprise bidirectional dual promoter expression cassettes useful for promoting expression of one or more coding sequences in a cell (e. ., according to the methods provided herein). The embodiments of nucleic acid sequences described herein are non-limiting configurations of the coding sequences and additional elements e.g., linear arrangement of the coding sequences and additional elements within the provided nucleic acid sequences, sense/antisense orientation of the coding sequences and/or additional elements relative to each other, etc.).

Coding Sequence

[0110] In certain embodiments, a nucleic acid sequence provided herein comprises a coding sequence. In certain embodiments, nucleic acid sequence comprises one or more coding sequences. In certain embodiments, nucleic acid sequence comprises two coding sequences. In certain embodiments, the coding sequences are the same. In certain embodiments, the coding sequences are different.

[0111] In certain embodiments, a coding sequence encodes an RNA molecule. In certain embodiments, an RNA molecule is non-coding RNA. In certain embodiments, a coding sequence encodes a miRNA, a shRNA, a siRNA, an antisense RNA, a gRNA, an antagomir, a miRNA sponge, an RNA aptazyme, an RNA aptamer, a IncRNA, a ribozyme, or a mRNA. In certain embodiments, a coding sequence encodes a synthetic RNA.

[0112] In certain embodiments, a coding sequence encodes one or more polypeptides, or a fragment or fragments thereof. Such coding sequences can comprise a complete coding sequence of a polypeptide, or only a fragment of a coding sequence of a polypeptide. In certain embodiments, a coding sequence encodes a polypeptide that is useful to treat a disease or disorder in a subject. Suitable polypeptides include, without limitation, P-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; colony stimulating factors (CSF); interleukins, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, etc.; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factor (FGF, such as basic FGF, acidic FGF, and FGF21), hepatocyte growth factor (HGF), insulin-like growth factors (IGFs), bone morphogenetic protein (BMP), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-0) and soluble fragments thereof, and the like; soluble receptors, such as soluble TGFp receptors (e.g., soluble TGFPR2 receptors (sTGFpR2), or soluble TGFPR2 receptors fused to an Fc (sTGFpR2- Fc)), soluble TNF-a receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), soluble y/A T cell receptors, ligand-binding fragments of a soluble receptor, and the like; enzymes, such as a-glucosidase, imiglucerase, P-glucocerebrosidase, and alglucerase; enzyme activators, such as tissue plasminogen activator; chemokines, such as IP- 10, monokine induced by interferon-gamma (Mig), Groa/IL-8, RANTES, MIP-la, MIP-ip, MCP-1, PF-4, and the like; angiogenic agents, such as vascular' endothelial growth factors (VEGFs, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), glioma-derived growth factor, angiogenin, angiogenin-2; and the like; anti- angiogenic agents, such as a soluble VEGF receptor; protein vaccine; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastrin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagons, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, a sleep peptide, and the like; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein; follicle stimulating hormone (FSH); human alpha- 1 antitrypsin; leukemia inhibitory factor (LIF); tissue factors; macrophage activating factors; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); tissue inhibitors of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotrofin; fibrin; hirudin; IL-1 receptor antagonists; ciliary neurotrophic factor (CNTF); brain-derived neurotrophic factor (BDNF); neurotrophins 3 and 4/5 (NT-3 and -4/5); glial cell derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); Factor VIII, Factor IX, Factor X; dystrophin or minidystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disease- related enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporter, aldolase A, -enolase, glycogen synthase; lysosomal enzymes, such as iduronate-2-sulfatase (128), and arylsulfatase A; and mitochondrial proteins, such as frataxin. [0113] In certain embodiments, a coding sequence encodes an immune cell receptor. Suitable immune cell receptors include T cell receptors (TCRs) and chimeric antigen receptors (CARs).

[0114] In certain embodiments, a coding sequence encodes a protein that may be defective in one or more lysosomal storage diseases. Suitable proteins include, without limitation, a- sialidase, cathepsin A, a-mannosidase, P-mannosidase, glycosylasparaginase, a-fucosidase, a-N- acetylglucosaminidase, -galactosidase, -hexosaminidase a-subunit, P-hexosaminidase P- subunit, GM2 activator protein, glucocerebrosidase, Saposin C, Arylsulfatase A, Saposin B, formyl-glycine generating enzyme, P-galactosylceramidase, a-galactosidase A, iduronate sulfatase, a-iduronidase, heparan N-sulfatase, acetyl-CoA transferase, N-acetyl glucosaminidase, P-glucuronidase, N-acetyl glucosamine 6-sulfatase, N-acetylgalactosamine 4-sulfatase, galactose 6-sulfatase, hyaluronidase, a-glucosidase, acid sphingomyelinase, acid ceramidase, acid lipase, capthepsin K, tripeptidyl peptidase, palmitoyl-protein thioesterase, cystinosin, sialin, UDP-N- acetylglucosamine, phosphotransferase y-subunit, mucolipin-1, LAMP-2, NPC1, CLN3, CLN 6, CLN 8, LYST, MYOV, RAB27A, melanophilin, and AP3 P-subunit.

[0115] In certain embodiments, a coding sequence encodes an antibody or a fragment thereof (e.g., a Fab, scFv, or full-length antibody). Suitable antibodies include, without limitation, muromonab-cd3, efalizumab, tositumomab, daclizumab, nebacumab, catumaxomab, edrecolomab, abeiximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, adalimumab, ibritumomab tiuxetan, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, canakinumab, golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab vedotin, pertuzumab, raxibacumab, obinutuzumab, alemtuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumab ozogamicin, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab ozogamicin, durvalumab, burosumab, erenumab, galcanezumab, lanadelumab, mogamulizumab, tildrakizumab, cemiplimab, fremanezumab, ravulizumab, emapalumab, ibalizumab, moxetumomab, caplacizumab, romosozumab, risankizumab, polatuzumab, eptinezumab, leronlimab, sacituzumab, brolucizumab, isatuximab, and tcprotumumab.

[0116] In certain embodiments, a coding sequence encodes an scFv, nanobody, or VHH. In certain embodiments, a coding sequence does not encode a functional antibody.

[0117] In certain embodiments, a coding sequence encodes one or more transcription factors.

[0118] In certain embodiments, a coding sequence encodes a nuclease. Suitable nucleases include, without limitation, zinc fingers nucleases (ZFN) (see, e.g., Porieus, and Baltimore (2003) Science 300: 763; Miller et al. (2007) Nat. Biotechnol. 25:778-785; Sander et al. (2011) Nature Methods 8:67-69; and Wood et al. (2011) Science 333:307, each of which is hereby incorporated by reference in its entirety); transcription activator-like effectors nucleases (TALEN) (see, e.g., Wood et al. (2011) Science 333:307; Boch et al. (2009) Science 326:1509-1512; Moscou and Bogdanove (2009) Science 326;1501; Christian et al. (2010) Genetics 186:757-761; Miller et al. (2011) Nat. Biotechnol. 29:143-148; Zhang et al. (2011) Nat. Biotechnol. 29:149-153; and Reyon et al. (2012) Nat. Biotechnol. 30(5): 460-465, each of which is hereby incorporated by reference in its entirety); homing endonucleases; meganucleases (see, e.g., U.S. Patent Publication No. US 2014/0121115, which is hereby incorporated by reference in its entirety); and RNA-guided nucleases (see, e.g., Makarova et al. (2018) The CRISPR Journal 1(5): 325-336; and Adli (2018) Nat. Communications 9:1911, each of which is hereby incorporated by reference in its entirety).

[0119] In certain embodiments, a coding sequence encodes an RNA-guided nuclease. Suitable RNA-guided nucleases include, without limitation, Class I and Class II clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleases. Class I is divided into types I, III, and IV, and includes, without limitation, type I (Cas3), type I-A (Cas8a, Cas5), type I-B (Cas8b), type I-C (Cas8c), type 1-D (CaslOd), type I-E (Csel, Cse2), type I-F (Csyl, Csy2, Csy3), type I-U (GSU0054), type III (CaslO), type III-A (Csm2), type III-B (Cmr5), type III-C (CsxlO or Csxl l), type III-D (CsxlO), and type IV (Csfl). Class II is divided into types II, V, and VI, and includes, without limitation, type II (Cas9), type II-A (Csn2), type II-B (Cas4), type V (Cpfl, C2cl, C2c3), and type VI (Casl3a, Casl3b, Casl3c). RNA-guided nucleases also include naturally-occurring Class II CRISPR nucleases such as Cas9 (Type II) or Casl2a/Cpfl (Type V), as well as other nucleases derived or obtained therefrom. Exemplary Cas9 nucleases that may be used in the present invention include, but are not limited to, .S'. pyogenes Cas9 (SpCas9), .S'. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GcoCas9).

[0120] In certain embodiments, a coding sequence comprises a suicide gene. Cells that express a suicide gene are conferred sensitivity to an agent, e.g.. a drug, such that when a cell expressing a suicide gene is contacted with or exposed to an agent, it causes a cell to die. In certain embodiments, suicide genes include, for example, without limitation, Herpes Simplex Virus Thymidine Kinase (HSV-TK), for which an agent is ganciclovir, and inducible Caspase 9 (iCasp9), for which an agent is a small molecule AP20187 can be used in accordance with embodiments of the present disclosure. Other examples of suicide genes include, without limitation, sequences comprising a minimal epitope based on an epitope of CD20 that is recognized by a therapeutic anti-CD20 antibody (e.g., rituximab), described in e.g., PCT Publication No. WO2013153391A1, which is herein incorporated by reference in its entirety; and a truncated epidermal growth factor receptor (EGFR) that is recognized by a therapeutic anti-EGFR antibody (e.g., cetuximab), described in e.g., PCT Publication No. WO2011056894A2, which is herein incorporated by reference in its entirety.

[0121] In certain embodiments, a coding sequence encodes reporter sequences, which upon expression produce a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding P-lactamase, P -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein (RFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, CD2, CD4, CD8, and an influenza hemagglutinin protein to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.

[0122] In certain embodiments, a coding sequence encodes a polypeptide linked to an Fc region. In certain embodiments, a Fc region is a canine Fc region, an IgG2 Fc region, or an IgGl Fc region.

[0123] In certain embodiments, a coding sequence encodes a polypeptide linked to a signal peptide. In certain embodiments, a signal peptide is a human TGFPR2 signal peptide or a canine A AT signal peptide. [0124] In certain embodiments, a coding sequence encodes a growth factor or receptor thereof. In certain embodiments, a coding sequence encodes fibroblast growth factor 21 (FGF21). In certain embodiments, a coding sequence encodes a canine FGF21, a murine FGF21, and/or a human FGF21. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 4-6. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 4. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 5. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 6.

[0125] In certain embodiments, a coding sequence encodes a canine FGF21 having an exemplary amino acid sequence set forth in NCBI Reference Sequence XP_022279904.1. In certain embodiments, a coding sequence comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 72. In certain embodiments, a coding sequence encodes a murine FGF21 having an exemplary amino acid sequence set forth in NCBI Reference Sequence NP_064397.1. In certain embodiments, a coding sequence comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:

73. In certain embodiments, a coding sequence encodes a human FGF21 having an exemplary amino acid sequence set forth in NCBI Reference Sequence NP_061986.1. In certain embodiments, a coding sequence comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:

74.

[0126] In certain embodiments, a coding sequence encodes a growth factor receptor. In certain embodiments, a coding sequence encodes a transforming growth factor beta receptor 2 (TGFPR2). In certain embodiments, a coding sequence encodes a soluble form of a transforming growth factor beta receptor 2 (sTGFpR2). In certain embodiments, sTGFpR2 comprises the extracellular region of TGFPR2. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 16-18. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 16. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 18. [0127] In certain embodiments, a coding sequence encodes the extracellular region of a caninc TGFPR2 having an exemplary amino acid sequence set forth in NCBI Reference Sequence XP_005634388.1. In certain embodiments, a coding sequence encodes the extracellular region of a canine TGFPR2 comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 75. In certain embodiments, a coding sequence encodes the extracellular region of a murine TGFPR2 having an exemplary amino acid sequence set forth in NCBI Reference Sequence NP_033397.3. In certain embodiments, a coding sequence encodes the extracellular region of a canine TGFPR2 comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 76. In certain embodiments, a coding sequence encodes the extracellular region of a human TGFPR2 having an exemplary amino acid sequence set forth in NCBI Reference Sequence NP_001020018.1. In certain embodiments, a coding sequence encodes the extracellular region of a canine TGFPR2 comprising an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 77.

[0128] In certain embodiments, a coding sequence encodes a STGFPR2 linked to an Fc region. In certain embodiments, the Fc region is a canine Fc region, an IgG2 Fc region, or an IgGl Fc region. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 33-35. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 33. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 34. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 35.

[0129] In certain embodiments, a coding sequence is linked to a signal peptide. In certain embodiments, the sTGFpR2 is linked to a signal peptide. The signal peptide can be derived from the same protein it is linked to, or can be heterologous to the protein it is linked to. In certain embodiments, the signal peptide is derived from human TGFPR2 or canine AAT.

[0130] In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 36-39. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 36. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 37. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 38. In certain embodiments, a coding sequence comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 39.

[0131] In certain embodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 40-43. In certain embodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 40. In certain embodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 41. In certain embodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 42. In certain embodiments, a coding sequence encodes an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 43.

[0132] In certain embodiments, a nucleic acid sequence provided herein is optimized, e.g., by codon/RNA optimization, replacement with heterologous signal sequences, and/or elimination of mRNA instability elements. Methods to generate optimized polynucleotides for recombinant expression by introducing codon changes and/or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291 ,664; 6,414,132; and 6,794,498, accordingly, all of which are herein incorporated by reference in their entireties. For example, potential splice sites and instability elements (e.g., A/T or A/U rich elements) within the RNA can be mutated without altering the amino acids encoded by nucleic acid sequences to increase stability of the RNA for recombinant expression. The alterations utilize the degeneracy of the genetic code, e.g., using an alternative codon for an identical amino acid. In certain embodiments, it can be desirable to alter one or more codons to encode a conservative mutation, e.g., a similar amino acid with similar chemical structure and properties and/or function as the original amino acid. Such methods can increase expression of the encoded capsid protein relative to the expression of the capsid encoded by polynucleotides that have not been optimized.

Transcriptional Regulatory Element (TRE}

[0133] In certain embodiments, a nucleic acid sequence provided herein comprises a transcriptional regulatory element (TRE). In certain embodiments, a TRE is operably linked to one or more coding sequences, i.e., to control expression of an RNA or polypeptide encoded by a coding sequence. In certain embodiments, a TRE comprises a constitutive promoter. In certain embodiments, a TRE can be active in any mammalian cell (e.g., any human cell). In certain embodiments, a TRE is active in a broad range of human cells. Such TREs may comprise constitutive promoter and/or enhancer elements including any of those described herein. In certain embodiments, a TRE comprises an inducible promoter. In certain embodiments, a TRE may be a tissue-specific TRE, i.e., it is active in specific tissue(s) and/or organ(s). A tissue- specific TRE comprises one or more tissue-specific promoter and/or enhancer elements, and optionally one or more constitutive promoter and/or enhancer elements. It is an insight of the present disclosure that tissue-specific promoter and/or enhancer elements can be isolated from genes specifically expressed in a tissue by methods well known in the art.

Promoters

[0134] Suitable promoters include, e.g., cytomegalovirus promoter (CMV) (Stinski et al. (1985) Journal of Virology 55(2): 431-441); CMV early enhancer/chicken P-actin (CBA) promoter/rabbit P-globin intron (CAG) (Miyazaki et al. (1989) Gene 79(2): 269-277); CB^SB (Jacobson et al. (2006) Molecular Therapy 13(6): 1074-1084); human elongation factor la promoter (EFla) (Kim et al. (1990) Gene 91 (2): 217-223); human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al. (1984) Gene 32(3): 409-417); mitochondrial heavy-strand promoter (Lodeiro et al. (2012) PNAS 109(17): 6513-6518); and ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105). In certain embodiments, a TRE comprises a cytomegalovirus (CMV) promoter/enhancer, an SV40 promoter, a chicken beta actin (CBA) promoter, an smCBA promoter, a human elongation factor 1 alpha (EFla) promoter, a minute virus of mouse (MVM) intron which comprises transcription factor binding sites, a human phosphoglycerate kinase (PGK1) promoter, a human ubiquitin C (Ubc) promoter, a human beta actin promoter, a human neuron- specific enolase (ENO2) promoter, a human beta-glucuronidase (GUSB) promoter, a rabbit beta-globin element, a human calmodulin 1 (CALM1) promoter, a human ApoE/C-I hepatic control region (HCR1), an ApoE enhancer, a human l -antitrypsin (hAAT) promoter, an extended HCR1, a HS-CRM8 element of an hAAT promoter, a human transthyretin (TTR) promoter, a human AFP enhancer, and/or a human Methyl-CpG Binding Protein 2 (MeCP2) promoter.

[0135] In certain embodiments, a TRE is liver- specific. Exemplary liver- specific TREs may comprise one or more elements from, without limitation, an ApoA-I promoter, an ApoA-II promoter, an ApoA-IV promoter, an ApoB promoter, an ApoC-I promoter, an ApoC-II promoter, an ApoC-III promoter, an ApoE promoter, an albumin promoter (e.g., a human albumin promoter), an a- fetoprotein promoter, a phosphoenolpyruvate carboxykinase 1 (PCK1) promoter, a phosphoenolpyruvate carboxykinase 2 (PCK2) promoter, a transthyretin (TTR) promoter, an al- antitrypsin promoter (e.g., a human al-antitrypsin (AAT or SERPINA1) promoter), a TK (thymidine kinase) promoter, a hemopexin promoter, an alcohol dehydrogenase 6 promoter, a cholesterol 7alpha-hydroxylase promoter, a factor IX promoter, an a-microglobulin promoter, a SV40 promoter, a CMV promoter, a Rous Sarcoma Virus-LTR promoter and a HBV promoter.

[0136] In certain embodiments, a TRE is muscle-specific. Exemplary muscle-specific TREs may comprise one or more elements from, without limitation, a human skeletal muscle a- actin (HSA) promoter, a muscle creatine kinase (MCK) promoter, a MHCK7 promoter, a dMCK promoter, a tMCK promoter, a CK6 promoter, a CK8 promoter, a CK8e promoter, a human desmin (DES) promoter or variant thereof, a cardiac troponin T (cTnT) promoter, a myosin light-chain (MLC2v) promoter, a human a-myosin heavy chain gene (aMHC) promoter, a MLC promoter, a human troponin I (TNNI1) promoter, a AUSEx3 promoter, a SPcA5-12 promoter, a SP-301 promoter, a MH promoter, and a Sk-CRM4/DES promoter.

[0137] In certain embodiments, a native promoter for a coding sequence may be used. A native promoter may be preferred when it is desired that expression of a coding sequence should mimic a native expression. A native promoter may be used when expression of a coding sequence must be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic a native expression.

[0138] In certain embodiments, a nucleic acid sequence provided herein comprises a human alpha 1-antitrypsin (hAAT) promoter. In certain embodiments, a hAAT promoter comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 2.

[0139] In certain embodiments, a nucleic acid sequence provided herein comprises a human albumin (hAlb) promoter. In certain embodiments, a hAlb promoter comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11.

Enhancers

[0140] In certain embodiments, a nucleic acid sequence provided herein comprises one or more ApoE enhancer elements. In certain embodiments, a nucleic acid sequence comprises one ApoE enhancer element. In certain embodiments, a nucleic acid sequence comprises two ApoE enhancer elements. In certain embodiments, a nucleic acid sequence comprises three ApoE enhancer elements. In certain embodiments, a nucleic acid sequence comprises more than three ApoE enhancer elements. In certain embodiments, one or more ApoE enhancer elements each comprise a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 1. In certain embodiments, a nucleic acid sequence provided herein comprises two ApoE enhancer elements. In certain embodiments, two ApoE enhancer elements comprise a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 120 or 121. In certain embodiments, a nucleic acid sequence provided herein comprises three ApoE enhancer elements. In certain embodiments, three ApoE enhancer elements comprise a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 122.

[0141] In certain embodiments, a nucleic acid sequence provided herein comprises a human AFP enhancer element. In certain embodiments, a human AFP enhancer element comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10.

5’ Cap

[0142] In certain embodiments, a polynucleotide sequence provided herein comprise a 5’ cap. As described herein, a 5’ cap (also termed an RNA cap, an RNA 7 -methylguanosine cap or an RNA m.sup.7G cap) is a modified guanine nucleotide that has been added to a “front” or 5’ end of a eukaryotic messenger RNA shortly after a start of transcription. In certain embodiments, a 5’ cap consists of a terminal group which is linked to a first transcribed nucleotide. Its presence is critical for recognition by a ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after start of transcription, a 5’ end of an mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes a chemical reaction that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. A capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.

Introns

[0143] In certain embodiments, a nucleic acid sequence provided herein comprises one or more intron elements. In certain embodiments, a nucleic acid sequence comprises an intron element 3’ of a promoter. In certain embodiments, a nucleic acid sequence comprises one intron element. In certain embodiments, a nucleic acid sequence comprises two intron elements. In certain embodiments, a nucleic acid sequence comprises more than two intron elements. In certain embodiments, one or more intron elements each comprise a P-globin intron element or a MVM intron element.

[0144] In certain embodiments, a nucleic acid sequence provided herein comprises a P- globin intron element. In certain embodiments, a P-globin intron element comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 3. In certain embodiments, a P-globin intron element is 3’ of a promoter (e.g., a hAAT promoter).

[0145] In certain embodiments, a nucleic acid sequence provided herein comprises a MVM intron element. In certain embodiments, a MVM intron element comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12. In certain embodiments, a MVM intron element is 3’ of a promoter (e.g., a hAlb promoter).

Exemplary TREs

[0146] Any one or more of a TREs described herein can be combined in any order to drive efficient transcription.

[0147] In certain embodiments, a nucleic acid sequence provided herein comprises a TRE comprising a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 1, 2, 3, 10, 11, 12, 27, 28, 29, 30, 44, 45, 46, 47, 48, and 49.

[0148] In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 1. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 2. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 3. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 12. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 27. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 28. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 29. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 30. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 44. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 45. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 46. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 47. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 48. In certain embodiments, a TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 49.

[0149] In certain embodiments, a nucleic acid sequence provided herein comprises a first TRE and a second TRE. In certain embodiments, a first TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 1, 2, 3, 10, 1 1 , 12, 27, 28, 29, and 30. In certain embodiments, a second TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 1, 2, 3, 10, 11, 12, 27, 28, 29, and 30. In certain embodiments, a first TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 1, 2, 3, 27, 28, and 29 and a second TRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 10, 11, 12, and 30. In certain embodiments, a first TRE and a second TRE are the same. In certain embodiments, a first TRE and a second TRE are different. In certain embodiments, a first TRE or a second TRE are liver- specific.

Post-transcriptional Regulatory Element

[0150] In certain embodiments, a nucleic acid sequence provided herein comprises a post- transcriptional regulatory element (PRE). In certain embodiments, a PRE is operably linked to one or more coding sequences, i.e., to control expression of an RNA or polypeptide encoded by a coding sequence. In certain embodiments, a PRE comprises a poly adenylation signal sequence. In certain embodiments, a PRE comprises a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE). In certain embodiments, a PRE is identical or substantially identical to a native PRE for a coding sequence. In certain embodiments, a PRE is an exogenous PRE. In certain embodiments, a PRE comprises a Kozak consensus sequence. In certain embodiments, a PRE comprises a polycistronic element. Polyadenylation Sequences

[0151] In certain embodiments, a PRE comprises a polyadcnylation signal (poly(A)) sequence. In certain embodiments, a polynucleotide sequence provided herein may comprise at least one poly(A) sequence. Most nascent eukaryotic mRNA possesses a poly(A) tail at its 3’ end which is added during a complex process that includes cleavage of a primary transcript and a coupled polyadenylation reaction (see, e.g., Proudfoot et al., Cell 108:501-512, 2002, which is hereby incorporated herein by reference in its entirety). A poly (A) tail can confer RNA stability and transferability (see, e.g., Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994). In certain embodiments, a poly(A) sequence is positioned 3’ to a coding sequence.

[0152] In certain embodiments, polyadenylation refers to a covalent linkage of a polyadenylyl moiety, or its modified variant, to an RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are poly adenylated at a 3’ end. In certain embodiments, a 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to pre-mRNA through enzymatic action, polyadenylate polymerase. In higher eukaryotes, a poly(A) tail is added onto transcripts that contain a specific sequence, a polyadenylation signal. In certain embodiments, a poly(A) tail and a protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of mRNA from a cell’s nucleus, and translation. Polyadenylation occurs in a cell nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, an mRNA chain is cleaved through action of an endonuclease complex associated with RNA polymerase. A cleavage site is usually characterized by the presence of a base sequence AAUAAA near a given cleavage site. After an mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site.

[0153] In certain embodiments, a poly(A) signal sequence is a sequence that triggers endonuclease cleavage of an mRNA and addition of a series of adenosines to the 3’ end of a cleaved mRNA. A “poly(A)” portion refers to a series of adenosines attached by poly adenylation to an mRNA. In certain embodiments of for the present disclosure, such as, e.g., transient expression, a polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.

[0154] There are several poly(A) signal sequences that can be used, including those derived from bovine growth hormone (bgh) (Woychik et al., Proc. Natl. Acad. Sci. U.S.A. 8 l(13):3944-3948, 1984; U.S. Patent No. 5,122,458; Yew et al., Human Gene Ther. 8(5):575-584, 1997; Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332, 2001; Wu et al., Mol. Ther. 16(2):280-289, 2008; Gray et al., Human Gene Ther. 22:1143-1153, 2011; Choi et al., Mol. Brain 7 : 17, 2014, each of which is incorporated in its entirety herein by reference), mouse-P-globin, mouse-a-globin (Orkin et al., EMBO J. 4(2):453-456, 1985; Thein et al., Blood 71(2):313-319, 1988, each which is incorporated in its entirety herein by reference), human collagen, polyoma virus (Batt et al., Mol. Cell Biol. 15(9):4783-4790, 1995, each of which is incorporated in its entirety herein by reference), Herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy-chain gene polyadenylation signal (US 2006/0040354, which is incorporated in its entirety herein by reference), human growth hormone (hGH) (Szymanski et al., Mol. Therapy 15(7): 1340- 1347, 2007; Ostegaard et al., Proc. Natl. Acad. Sci. U.S.A. 102(8):2952-2957, 2005, each of which is incorporated in its entirety herein by reference), synthetic polyA (Levitt et al., Genes Dev. 3(7): 1019-1025, 1989; Yew et al., Human Gene Ther. 8(5):575-584, 1997; Ostegaard et al., Proc. Natl. Acad. Sci. U.S.A. 102(8):2952-2957, 2005; Choi et al., Mol. Brain 7:17, 2014, each of which is incorporated in its entirety herein by reference), HIV-1 upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6): 1167-1173, 2007, each of which is incorporated in its entirety herein by reference), adenovirus (L3) upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6): 1167- 1173, 2007, which is incorporated in its entirety herein by reference), hTHGB upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6): 1167-1173, 2007), hC2 upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6): 1167-1173, 2007), a SV40 poly(A) signal sequence, such as a SV40 late or early poly(A) signal sequence (Schek et al., Mol. Cell Biol. 12(12):5386-5393, 1992; Choi et al., Mol. Brain 7:17, 2014; Schambach et al., Mol. Ther. 15(6): 1167- 1173, 2007, each of which is incorporated in its entirety herein by reference).

[0155] In certain embodiments, a nucleic acid sequence provided herein comprises a bovine growth hormone polyadenylation (BGHpA) sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 22 or 23.

[0156] In certain embodiments, a nucleic acid sequence provided herein comprises a simian virus 40 polyadenylation (SV40pA) sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 8 or 9.

Additional Regulatory Sequences

[0157] In certain embodiments, a PRE comprises a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE). In certain embodiments, a nucleic acid sequence provided herein comprises one or more WPRE sequences.

[0158] In certain embodiments, a nucleic acid sequence provided herein comprises a WPRE having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, a least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7.

Kozak Sequences

[0159] In certain embodiments, a PRE comprises a Kozak consensus sequence. In certain embodiments, a nucleic acid sequence provided herein comprises one or more Kozak sequences. In certain embodiments, a natural 5’ UTR includes a sequence that plays a role in translation initiation. For example, in certain embodiments, they harbor signatures like Kozak sequences, which can be involved in a process by which a ribosome initiates translation of many genes. Kozak sequences generally have a consensus sequence CCR(A/G)CCAUGG, where R is a purine (A or G) three bases upstream of a start codon (AUG), which is followed by another “G”. In certain embodiments, Kozak sequences may be included in synthetic or additional sequence elements, such as cloning sites. [0160] In certain embodiments, a nucleic acid sequence provided herein comprises a Kozak consensus sequence. In certain embodiments, a Kozak consensus sequence comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to a nucleotide sequence GCCACCATG. In certain embodiments, a Kozak consensus sequence is 5’ of a coding sequence.

Polycistronic Element

[0161] In certain embodiments, a PRE comprises a polycistronic element. In certain embodiments, a nucleic acid sequence provided herein comprises a polycistronic element. In certain embodiments, a polycistronic element comprises a nucleotide sequence that encodes for an internal ribosome entry site (IRES). An IRES is an element that promotes direct internal ribosome entry to an initiation codon, such as ATG, of a protein coding region, thereby leading to capindependent translation of a gene. Various internal ribosome entry sites include, without limitation, an IRES obtainable from viral or cellular mRNA sources, e.g., immunoglobulin heavy-chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0162] In certain embodiments, a polycistronic element comprises a nucleotide sequence that encodes for a 2A sequence or element. A 2A sequence refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Various 2A sequences include, without limitation, those found in members of the Picomaviridae virus family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO), Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A sequences derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.

Exemplary PREs

[0163] Any one or more of PREs described herein can be combined in any order to control expression of an RNA or polypeptide encoded by a coding sequence.

[0164] In certain embodiments, a nucleic acid sequence provided herein comprises a PRE comprising a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NOs; 7, 8, 9, 22, 23, 31, and 32. In certain embodiments, a PRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 7. In certain embodiments, a PRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 8. In certain embodiments, a PRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 9. In certain embodiments, a PRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 22. In certain embodiments, a PRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23. In certain embodiments, a PRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31. In certain embodiments, a PRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 32.

[0165] In certain embodiments, a nucleic acid sequence provided herein comprises a first PRE and a second PRE. In certain embodiments, a first PRE comprises a nucleotide having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NOs: 7, 8, 9, 22, 23, 31, and 32. In certain embodiments, a second PRE comprises a nucleotide sequence having at least 70% sequence identity (e.g., at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NOs: 7, 8, 9, 22, 23, 31, and 32. In certain embodiments, a first PRE and a second PRE are the same. In certain embodiments, a first PRE and a second PRE are different. [0166] In certain embodiments, a nucleic acid sequence comprises a PRE 5’ of a coding sequence. In certain embodiments, a nucleic acid sequence provided herein comprises a PRE 3’ of a coding sequence.

Untranslated Regions (UTRs)

[0167] In certain embodiments, a nucleic acid sequence provided herein can include one or more untranslated regions. In certain embodiments, a nucleic acid sequence provided herein can include a 5’ UTR and/or a 3’ UTR. In certain embodiments, if more than one UTR is present, UTRs may come from a single gene or more than one gene.

[0168] An untranslated region (UTR) of a gene is transcribed but not translated. In certain embodiments, a 5’ UTR starts at a transcription start site and continues to a start codon but does not include that stall codon. In certain embodiments, a 3’ UTR starts immediately following a stop codon and continues until a transcriptional termination signal. Without wishing to be bound by any particular theory, UTRs may contribute to the stability of nucleic acid molecule and may contribute to translation. In certain embodiments, regulatory features of a UTR can be incorporated into any technologies (e.g., a polynucleotide sequence, a composition, a kit, and a method) as described herein to, e.g., enhance stability of an encoded polypeptide.

[0169] For example, in certain embodiments, a 5’ UTR is included in any a polynucleotide sequence described herein. Non-limiting examples of 5’ UTRs including those from the following genes: albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as a mRNA. In certain embodiments, a 5’ UTR forms a secondary structure, e.g., a second structure that is involved in elongation factor binding.

[0170] In certain embodiments, a 5’ UTR from an mRNA that is transcribed by a cell can be included in any technologies (e.g., a polynucleotide sequence, a composition, a kit, and/or a method) described herein.

[0171] Generally, 3’ UTRs have stretches of adenosines and uridines embedded in them. These AU-rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, AU-rich elements (AREs) can be separated into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol. 15:2010-2018, 1995, each of which is incorporated in its entirety herein by reference): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A) (U/A) nonamcrs. GM-CSF and TNF-alpha mRNAs arc examples that contain class II AREs. Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif. Two well-studied examples of this class are c-Jun and myogenin mRNAs.

[0172] Most proteins binding to AREs destabilize a messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase stability of mRNA. HuR binds to AREs of all three classes. Engineering HuR specific binding sites into a 3’ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of a message in vivo.

Staffer Sequence

[0173] In certain embodiments, a nucleic acid sequence provided herein comprises one or more stuff er sequences. In certain embodiments, a nucleic acid sequence provided herein comprises an expression cassette and further comprise one or more stuffer sequences. Stuffer sequences may be employed to maintain the size of a nucleic acid (e.g., a nucleic acid comprised within a vector) within appropriate limits for efficient DNA packaging (e.g., viral packaging of a vector), and as such may be employed to increase an efficiency of DNA packaging.

[0174] For example, AAV preferentially packages genomes that are approximately the same size of a native AAV genome. However, some expression cassettes intended to be packaged within AAV can be substantially smaller than the size of a native AAV genome, and may result in the packaging of unwanted nucleic acid sequences. Hence, to increase an efficiency of AAV packaging, stuffer sequences may be employed, linked to an expression cassette intended to be packaged, to result in a vector genome that is closer to the packaging capacity of AAV.

[0175] A stuffer sequence may have an effect on a function of a nucleic acid, and will accordingly select the most suitable stuffer sequence for use. Further, stuffer sequences can be designed to minimize any adverse effects in the context of gene therapy, e.g., by selecting stuffer sequences from regions of a genome that have minimal impact if integration occurs, and regions of a genome that have minimal risk of initiating unexpected transcription. In certain embodiments, stuffer sequences are from regions of the human genome. Stuffer sequences can be derived from, for example, a natural non-coding sequence (e.g., an intron sequence, an intergenic sequence, etc.), a synthetic non-coding sequence, and fragments and combinations thereof.

[0176] In certain embodiments, a stuffer sequence is located 5' to a coding sequence disclosed herein. In certain embodiments, a stuffer sequence is located 5' to a polynucleotide sequence comprising a transcriptional regulatory element, a post-transcriptional regulatory clement, and/or a coding sequence disclosed herein. In certain embodiments, a stuffcr sequence is located 5' to an expression cassette disclosed herein (e.g., an expression cassette comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and a coding sequence disclosed herein).

[0177] In certain embodiments, a staffer sequence is located 3' to a coding sequence disclosed herein. In certain embodiments, a staffer sequence is located 3' to a sequence comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and/or a coding sequence disclosed herein. In certain embodiments, a staffer sequence is located 3' to an expression cassette disclosed herein (e.g., an expression cassette comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and a coding sequence disclosed herein).

[0178] In certain embodiments, a first staffer sequence is located 5' to a coding sequence disclosed herein, and a second staffer sequence is located 3' to a coding sequence disclosed herein. In certain embodiments, a first staffer sequence is located 5' to a polynucleotide sequence comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and/or a coding sequence disclosed herein, and a second staffer sequence is located 3' to a polynucleotide sequence comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and/or a coding sequence disclosed herein. In certain embodiments, a first staffer sequence is located 5' to an expression cassette disclosed herein (e.g., an expression cassette comprising a transcriptional regulatory element, a post-transcriptional regulatory element, and a coding sequence disclosed herein), and a second staffer sequence is located 3' to an expression cassette (e.g., an expression cassette comprising a transcriptional regulatory element, a post- transcriptional regulatory element, and a coding sequence disclosed herein).

[0179] In certain embodiments, a 5' staffer sequence is positioned 5' to one or more TREs. In certain embodiments, a 3' staffer sequence positioned 3' to one or more PREs. In certain embodiments, a 5' staffer sequence positioned 5' to one or more TREs and a 3' staffer sequence positioned 3' to one or more PREs.

[0180] In certain embodiments, a staffer sequence is located within a 5' and 3' ITR nucleotide sequence of a vector genome. In certain embodiments, a stuffer sequence is located 3' to a 5' ITR nucleotide sequence and 5' to a 3' ITR nucleotide sequence of a vector genome. [0181] In certain embodiments, a staffer sequence is located outside a 5' and 3' ITR nucleotide sequence of a vector genome. Where a stuff er sequence is located outside a 5' and 3' ITR nucleotide sequence of a vector genome, this may allow for enhanced AAV purity of packaging. In such embodiments, a stuff er sequence outside of a 5' and 3' ITR nucleotide sequence of a vector genome may enhance viral purity and reduce off-target incorporation of plasmid backbone sequences, for example, by making such plasmid backbone sequences larger than the packaging capacity of AAV (i.e., larger than 4.7 kbp). In certain embodiments, a stuffer sequence is located 5' to a 5' ITR nucleotide sequence and/or 3' to a 3' ITR nucleotide sequence of a vector genome.

Exemplary Polynucleotides

[0182] Exemplary embodiments of nucleic acid sequences provided herein are described below and in the Examples.

[0183] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising (a) a first nucleic acid comprising a first transcriptional regulatory element (TRE) and a first coding sequence, and (b) a second nucleic acid comprising a second TRE and a second coding sequence. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising (a) a first TRE operatively linked to a first coding sequence, wherein a first TRE is or comprises: (1) a promoter, (2) one or more enhancer elements, or (3) a promoter and one or more enhancer elements, and/or (b) a second TRE operatively linked to a second coding sequence, wherein the second TRE is or comprises: (1) a promoter, (2) one or more enhancer elements, or (3) a promoter and one or more enhancer elements.

[0184] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter and one or more ApoE enhancer elements. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha I -antitrypsin (hAAT) promoter and one ApoE enhancer element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter and two ApoE enhancer elements. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1-antitrypsin (hAAT) promoter and three ApoE enhancer elements.

[0185] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1-antitrypsin (hAAT) promoter, one or more ApoE enhancer elements, and a P-globin intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, one ApoE enhancer element, and a P-globin intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, two ApoE enhancer elements, and a P-globin intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, three ApoE enhancer elements, and a P-globin intron element.

[0186] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, and a P-globin intron element.

[0187] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human albumin (hAlb) promoter and one or more ApoE enhancer elements. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human albumin (hAlb) promoter and one ApoE enhancer element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human albumin (hAlb) promoter and two ApoE enhancer elements. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human albumin (hAlb) promoter and three ApoE enhancer elements. [0188] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a MVM intron element, a human albumin (hAlb) promoter, and one or more ApoE enhancer elements. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a MVM intron element, a human albumin (hAlb) promoter, and one ApoE enhancer element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a MVM intron element, a human albumin (hAlb) promoter, and two ApoE enhancer elements. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a MVM intron element, a human albumin (hAlb) promoter, and three ApoE enhancer elements.

[0189] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a MVM intron element, and a human albumin (hAlb) promoter.

[0190] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a MVM intron element, a human albumin (hAlb) promoter, and a human AFP enhancer element. [0191] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human albumin (hAlb) promoter, and a human AFP enhancer clement.

[0192] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, one or more ApoE enhancer elements, and a human albumin (hAlb) promoter. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, one ApoE enhancer element, and a human albumin (hAlb) promoter. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1-antitrypsin (hAAT) promoter, two ApoE enhancer elements, and a human albumin (hAlb) promoter. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1-antitrypsin (hAAT) promoter, three ApoE enhancer elements, and a human albumin (hAlb) promoter.

[0193] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a P-globin intron element, a human alpha 1-antitrypsin (hAAT) promoter, one or more ApoE enhancer elements, and a human albumin (hAlb) promoter. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a P-globin intron element, a human alpha 1 -antitrypsin (hAAT) promoter, one ApoE enhancer element, and a human albumin (hAlb) promoter. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a P-globin intron element, a human alpha 1-antitrypsin (hAAT) promoter, two ApoE enhancer elements, and a human albumin (hAlb) promoter. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a P-globin intron element, a human alpha 1-antitrypsin (hAAT) promoter, three ApoE enhancer elements, and a human albumin (hAlb) promoter.

[0194] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1-antitrypsin (hAAT) promoter, one or more ApoE enhancer elements, a human albumin (hAlb) promoter, and a MVM intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, one ApoE enhancer element, a human albumin (hAlb) promoter, and a MVM intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, two ApoE enhancer elements, a human albumin (hAlb) promoter, and a MVM intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a human alpha 1 -antitrypsin (hAAT) promoter, three ApoE enhancer elements, a human albumin (hAlb) promoter, and a MVM intron element.

[0195] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a P-globin intron element, a human alpha 1 -antitrypsin (hAAT) promoter, one or more ApoE enhancer elements, a human albumin (hAlb) promoter, and a MVM intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a P- globin intron element, a human alpha 1 -antitrypsin (hAAT) promoter, one ApoE enhancer element, a human albumin (hAlb) promoter, and a MVM intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a P-globin intron element, a human alpha 1 -antitrypsin (hAAT) promoter, two ApoE enhancer elements, a human albumin (hAlb) promoter, and a MVM intron element. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a P-globin intron element, a human alpha 1- antitrypsin (hAAT) promoter, three ApoE enhancer elements, a human albumin (hAlb) promoter, and a MVM intron element.

[0196] In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 78. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 79. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 80. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 81. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 82. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 83. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 84. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 85. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 86. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 87. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 88. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 89. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 90. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 91. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 92. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 93. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 94. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 95. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 96. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 97. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 98. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 99. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 100. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 101. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 102. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 103. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 104. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 105. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 106. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 107. In certain embodiments, the present disclosure provides an isolated polynucleotide comprising a nucleic acid sequence having at least 80% sequence identity (e.g., at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 108.

[0197] In certain embodiments, the present disclosure provides isolated polynucleotides comprising at least a first nucleic acid comprising a first transcriptional regulatory element (TRE) and a first coding sequence and a second nucleic acid comprising a second TRE and a second coding sequence. In certain embodiments, a first nucleic acid comprises, from 5’ to 3’, a first TRE comprising a human alpha 1 -antitrypsin (hAAT) promoter and one or more ApoE enhancer elements, and a first coding sequence. In certain embodiments, a second nucleic acid comprises a second TRE comprising a human albumin (hAlb) promoter, and a second coding sequence. In certain embodiments, a first nucleic acid and a second nucleic acid are in the same orientation in a polynucleotide. In certain embodiments, a first nucleic acid and a second nucleic acid are separated by a polycistronic element. In certain embodiments, a polycistronic element is an IRES or 2A sequence.

[0198] In certain embodiments, a polynucleotide comprises, from 5’ to 3’: a first TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 27; a first coding sequence; a first PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31; a second TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 30; a second coding sequence; and a second PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 22. [0199] In certain embodiments, a first nucleic acid and a second nucleic acid are in the opposite orientation in a polynucleotide.

[0200] In certain embodiments, a polynucleotide comprises, from 5’ to 3’: a second PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23; a second coding sequence; a second TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11; a first TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 28; a first coding sequence; and a first PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31.

[0201] In certain embodiments, a polynucleotide comprises, from 5’ to 3’: a second PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23; a second coding sequence; a second TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11; a first TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ TD NO: 29; a first coding sequence; and a first PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31.

[0202] In certain embodiments, a polynucleotide comprises, from 5’ to 3: a second PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23; a second coding sequence; a second TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 11; a first TRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 27; a first coding sequence; and a first PRE comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 31 or 32.

[0203] In certain embodiments, the present disclosure provides isolated polynucleotides comprising one or more ApoE enhancer elements and one or both of a hAlb promoter and a hAAT promoter. In certain embodiments, a polynucleotide comprises, from 5’ to 3’, a hAlb promoter and one or more ApoE enhancer elements. In certain embodiments, a polynucleotide comprises, from 5’ to 3’, one or more ApoE enhancer elements and a hAAT promoter. In certain embodiments, a polynucleotide comprises, from 5’ to 3’, a hAlb promoter, one or more ApoE enhancer elements, and a hAAT promoter.

[0204] In certain embodiments, the present disclosure provides isolated polynucleotides comprising, from 5’ to 3’, a first coding sequence encoding a growth factor or receptor thereof, a first TRE, a second TRE, and a second coding sequence encoding a growth factor or receptor thereof. In certain embodiments, a polynucleotide further comprises a first post-transcriptional regulatory element (PRE). In certain embodiments, a first PRE is 5’ of a first coding sequence. In certain embodiments, a polynucleotide further comprises a second PRE, optionally wherein a second PRE is 3’ of a second coding sequence. In certain embodiments, a first PRE and/or second PRE each comprise a polyadenylation sequence and/or a WPRE sequence.

[0205] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22.

[0206] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 24, SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:

10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25.

[0207] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:

11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 22.

[0208] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 24, SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25.

[0209] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23, SEQ ID NO: 19, SEQ ID NO: 16, SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 121, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 8.

[0210] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 24, SEQ ID NO: 23, SEQ ID NO: 19, SEQ ID NO: 16, SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 121, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 25.

[0211] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23, SEQ ID NO: 19, SEQ ID NO: 16, SEQ ID NO: 15, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 8.

[0212] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 24, SEQ ID NO: 23, SEQ ID NO: 19, SEQ ID NO: 16, SEQ ID NO: 15, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 25.

[0213] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23, SEQ ID NO:

19, SEQ ID NO: 16, SEQ ID NO: 15, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 8.

[0214] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 24, SEQ ID NO: 23, SEQ ID NO: 19, SEQ ID NO: 16, SEQ ID NO: 15, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 25.

[0215] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23, SEQ ID NO:

20, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 8.

[0216] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 24, SEQ ID NO: 23, SEQ ID NO: 20, SEQ ID NO: 17, SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 11 , SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 25.

[0217] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 23, SEQ ID NO: 21, SEQ ID NO: 18, SEQ ID NO: 14, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 9.

[0218] In certain embodiments, the present disclosure provides isolated polynucleotides comprising from 5’ to 3’: a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 24, SEQ ID NO: 23, SEQ ID NO: 21, SEQ ID NO: 18, SEQ ID NO: 14, SEQ ID NO: 12, SEQ ID NO: 11, SEQ ID NO: 122, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 25.

[0219] In certain embodiments, the present disclosure provides isolated polynucleotides comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 44-61.

[0220] In certain embodiments, the present disclosure provides isolated polynucleotides comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 55-68 and 109-119. [0221] Among other things, the present disclosure provides a polynucleotide that is the complement of any of the nucleic acid sequences described herein. [0222] Among other things, the present disclosure provides a polynucleotide that is the reverse complement of any of the nucleic acid sequences described herein.

Vectors

[0223] Among other things, the present disclosure provides a vector comprising a nucleic acid sequence (e.g., a polynucleotide) described herein. Suitable vectors, include, without limitation, plasmids, viruses, cosmids, artificial chromosomes, linear DNA, and mRNA. In certain embodiments, a vector is a plasmid, a viral vector or a DNA minimal vector. In certain embodiments, a vector is an expression vector. In certain embodiments, a viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, or a lentiviral vector. In certain embodiments, a viral vector is an AAV vector.

[0224] Vectors (e.g., expression vectors) can be introduced into cells for propagation of the vector and/or for expression of a polypeptide encoded by the vector. Accordingly, among other things, the instant disclosure provides a recombinant cell comprising a nucleic acid sequence (e.g., a polynucleotide) or a vector (e.g., an expression vector) described herein.

[0225] Suitable vectors, include, without limitation, plasmids, minimal vectors (e.g., minicircles, Nanoplasmids™, doggybones, MIDGE vectors, and the like), viruses, cosmids, artificial chromosomes, linear DNA, and mRNA. In certain embodiments, a vector is a DNA plasmid or a DNA minimal vector. Any DNA plasmid or DNA minimal vector that can accommodate the necessary vector elements can be used for the vector. Suitable DNA minimal vectors include, without limitation, linear covalently closed DNA (e.g., ministring DNA), linear covalently closed dumbbell shaped DNA (e.g., doggybone DNA, dumbbell DNA), minicircles, Nanoplasmids™, minimalistic immunologically defined gene expression (MIDGE) vectors. DNA minimal vectors and their methods of production are described in, e.g., U.S. Patent Application Nos. 20100233814, 20120282283, 20130216562, 20150218565, 20150218586, 20160008488, 20160215296, 20160355827, 20190185924, 20200277624, and 20210010021, all of which are herein incorporated by reference in their entireties.

[0226] A variety of host cells and expression vector systems can be utilized. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. sublilis) transformed with, e.g., recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing capsid protein coding sequences; yeast (e.g., Saccharomyces Pichid) transformed with, e.g., recombinant yeast expression vectors containing capsid protein coding sequences; insect cell systems infected with, e.g., recombinant virus expression vectors (e.g., baculovirus) containing capsid protein coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with, e.g., recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with, e.g., recombinant plasmid expression vectors (e.g., Ti plasmid) containing capsid protein coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, Rl.l, B-W, L- M, BSC1, BSC40, YB/20 and BMT10 cells) harboring, e.g., recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia vims 7.5K promoter). In certain embodiments, suitable cells are human cells, e.g., human cell lines. In certain embodiments, a mammalian expression vector is pOptiVEC™ or pcDNA3.3. In certain embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), are suitable. For example, mammalian cells such as CHO or HEK293 cells, together with a vector containing the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system that can be used in conjunction with nucleic acid sequences described herein.

[0227] In bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended. For example, when a large quantity of polypeptide is to be produced, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794); pIN vectors (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509); and the like, all of which are herein incorporated by reference in their entireties. For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that a cloned target gene product can be released from the GST moiety. [0228] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV), for example, can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence can be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

[0229] In mammalian host cells, a number of viral-based expression systems can be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the capsid protein molecule in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12): 3655-9, which is herein incorporated by reference in its entirety). Specific initiation signals can also be required for efficient translation of inserted capsid protein coding sequences. These signals include an ATG initiation codon and adjacent sequences. Furthermore, an initiation codon must be in phase with a reading frame of a desired coding sequence to ensure translation of an entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. An efficiency of expression can be enhanced by an inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al. (1987) Methods Enzymol. 153: 516-544, which is herein incorporated by reference in its entirety).

[0230] In addition, a host cell strain can be chosen which modulates an expression of inserted sequences, or modifies and processes a gene product in a specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for a function of a protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure a correct modification and processing of a foreign protein expressed. To this end, eukaryotic host cells which possess cellular machinery for proper processing of a primary transcript, glycosylation, and phosphorylation of a gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HcpG2, SP210, Rl.l, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells.

[0231] For long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines which stably express a capsid protein described herein can be engineered.

[0232] In certain embodiments, rather than using expression vectors which contain viral origins of replication, host cells can be transformed with a polynucleotide (e.g., DNA or RNA) controlled by appropriate transcriptional regulatory elements (e.g., promoter, enhancer, sequences, transcription terminators, poly adenylation sites, etc.), and a selectable marker. Following the introduction of polynucleotide, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. A selectable marker in a recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express a capsid protein described herein or a fragment thereof.

[0233] A number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler M et al. (1977) Cell 11(1): 223-32); hypoxanthineguanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034); and adenine phosphoribosyltransferase (Lowy I et al. (1980) Cell 22(3): 817-23) genes in tk-, hgprt- or aprt-cells, respectively, all of which are herein incorporated by reference in their entireties. Also, antimetabolite resistance can be used as a basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al. (1980) PNAS 77(6): 3567-70; O’Hare K et al. (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Feigner PE (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al. (1984) Gene 30(1-3): 147- 56), all of which are herein incorporated by reference in their entireties. Methods can be utilized to select the desired recombinant clone and such methods are described, for example, in Ausubel FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbere-Garapin F et al. (1981) J Mol Biol 150: 1-14, all of which are herein incorporated by reference in their entireties.

III. Recombinant Adeno-Associated Virus (rAAV) Genomes and Compositions

[0234] Among other things, the present disclosure provides recombinant adeno-associated virus (rAAV) genomes comprising a nucleic acid sequence (e.g., a polynucleotide) described herein.

[0235] In certain embodiments, a rAAV genome further comprises a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence 5' of a polynucleotide, and a 3’ inverted terminal repeat (3’ ITR) nucleotide sequence 3’ of a polynucleotide. ITR sequences from any AAV serotype or variant thereof can be used in a rAAV genome described herein. 5' and 3' ITR can be from an AAV of the same serotype or from AAVs of different serotypes. In certain embodiments, a 5’ ITR nucleotide sequence has at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 24. In certain embodiments, a 3’ ITR nucleotide sequence has at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 25 or 26.

[0236] In certain embodiments, a rAAV genome is a single- stranded rAAV genome. In certain embodiments, a rAAV genome is a self-complementary rAAV genome.

[0237] In certain embodiments, a rAAV genome comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 55-68 and 78-119.

[0238] Among other things, the present disclosure provides a rAAV particle comprising a capsid comprising an AAV capsid protein and an rAAV genome as described herein. [0239] A capsid protein can be used in rAAV particles described herein, including, without limitation, a capsid protein from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. A capsid protein can be from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof.

[0240] In certain embodiments, a capsid protein comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 69-71.

[0241] Among other things, the present disclosure provides compositions, e.g., pharmaceutical compositions, comprising a nucleic acid, polynucleotide, vector, rAAV genome, and/or rAAV particle as described herein. In certain embodiments, compositions comprise a pharmaceutically acceptable excipient, adjuvant, diluent, vehicle or carrier, or a combination thereof. A “pharmaceutically acceptable carrier” includes any material which, when combined with an active ingredient of a composition, allows the ingredient to retain biological activity and without causing disruptive physiological reactions, such as an unintended immune reaction. Pharmaceutically acceptable carriers include water, phosphate buffered saline, emulsions such as oil/water emulsion, and wetting agents. Compositions comprising such carriers are formulated by methods such as those set forth in Remington’s Pharmaceutical Sciences, current Ed., Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Ding Delivery Systems (1999) H. C. Ansel et al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., 3rd ed. Amer. Pharmaceutical Assoc.

IV. Adeno-Associated Virus Packaging Systems

[0242] Among other things, the present disclosure provides packaging systems for recombinant preparation of a recombinant adeno-associated virus (rAAV) particle. Such packaging systems generally comprise: a first nucleotide encoding one or more AAV Rep proteins; a second nucleotide encoding an AAV capsid protein as described herein; and a third nucleotide sequence comprising any of rAAV genome sequences as described herein, wherein a packaging system is operative in a cell for enclosing a transfer genome in a capsid to form an AAV.

[0243] In certain embodiments, a packaging system comprises a first vector comprising a first nucleotide sequence encoding one or more AAV Rep proteins and a second nucleotide sequence encoding an AAV capsid protein, and a second vector comprising a third nucleotide sequence comprising a rAAV genome. As used in the context of a packaging system as described herein, a “vector” refers to a nucleic acid molecule that is a vehicle for introducing nucleic acids into a cell (e.g., a plasmid, a virus, a cosmid, an artificial chromosome, etc.). In certain embodiments, a packaging system can further comprise a fourth nucleotide sequence comprising one or more helper virus genes. In certain embodiments, a fourth nucleotide sequence comprises adenoviral E2, E4, and VA genes. In certain embodiments, a packaging system can further comprise a third vector (e.g., a helper virus vector), comprising a fourth nucleotide sequence. A third vector may be an independent third vector, integral with a first vector, or integral with a second vector.

[0244] In certain embodiments, a packaging system comprises a first vector comprising a first nucleotide sequence encoding one or more AAV Rep proteins, a second nucleotide sequence encoding one or more recombinant AAV capsid protein, and a third nucleotide sequence comprising any of the rAAV genome sequences as described herein, wherein a packaging system is operative in a cell for enclosing a transfer genome in a capsid to form an AAV. In certain embodiments, a packaging system can further comprise a fourth nucleotide sequence comprising one or more helper virus genes. In certain embodiments, a fourth nucleotide sequence comprises adenoviral E2, E4, and VA genes. In certain embodiments, a packaging system can further comprise a second vector (e.g., a helper virus vector), comprising a fourth nucleotide sequence. A second vector may be an independent second vector, integral with a first vector.

[0245] Any AAV Rep protein can be employed in packaging systems described herein. In certain embodiments, a Rep nucleotide sequence encodes an AAV2 Rep protein. Suitable AAV2 Rep proteins may include, without limitation, Rep 78/68 or Rep 68/52.

[0246] In certain embodiments, a helper virus comprising an adenovirus, a herpes virus (including herpes simplex vims (HSV)), a poxvirus (such as vaccinia virus), a cytomegalovirus (CMV), or a baculovirus. In certain embodiments, where a helper vims is adenovirus, an adenovims genome comprises one or more adenovims RNA genes comprising El, E2, E4 or VA. In certain embodiments, where a helper virus is adenovirus, an adenovirus genome comprises one or more adenovirus RNA genes comprising E2, E4, or VA. In certain embodiments, where a helper vims is HSV, a HSV genome comprises one or more of HSV genes comprising UL5/8/52, ICPO, ICP4, ICP22, or UL30/UL42.

[0247] In certain embodiments, vectors (e.g., first, second, and/or third vectors) provided herein are contained within one or more plasmids.

[0248] In certain embodiments, a first, second, and/or third vectors are contained within one or more recombinant helper viruses. In certain embodiments, a first vector and a third vector are contained within a recombinant helper vims. In certain embodiments, a second vector and a third vector are contained within a recombinant helper virus.

[0249] Among other things, the present disclosure provides a method for recombinant preparation of an AAV as described herein, wherein the method comprises transfecting or transducing a cell with a packaging system as described herein under conditions operative for enclosing a rAAV genome in a capsid to form a rAAV particle as described herein. Exemplary methods for recombinant preparation of a rAAV particle include transient transfection e.g., with one or more transfection plasmids containing a first, and a second, and optionally a third vector as described herein), viral infection (e.g. with one or more recombinant helper viruses, such as a adenovirus, poxvirus (such as vaccinia virus), herpes virus (including HSV, cytomegalovirus, or baculovims, containing a first, and a second, and optionally a third vector as described herein)), and stable producer cell line transfection or infection (e.g., with a stable producer cell, such as a mammalian or insect cell, containing a Rep nucleotide sequence encoding one or more AAV Rep proteins and/or a Cap nucleotide sequence encoding one or more AAV capsid proteins, and with a rAAV genome as described herein being delivered in a form of a plasmid or a recombinant helper virus).

[0250] Accordingly, the instant disclosure provides a packaging system for preparation of a rAAV particle, wherein a packaging system comprises: a first nucleotide sequence encoding one or more AAV Rep proteins; a second nucleotide sequence encoding a capsid protein of any one of the A A Vs described herein; a third nucleotide sequence comprising an rAAV genome sequence of any one of the AAVs described herein; and optionally a fourth nucleotide sequence comprising one or more helper virus genes (e.g., adenoviral E2, E4, and VA genes). V. Methods

[0251] Among other things, the present disclosure provides a method comprising introducing into a cell a polynucleotide as described herein, a vector as described herein, a rAAV genome as described herein, a rAAV particle as described herein, or a pharmaceutical composition as described herein. In certain embodiments, the method comprises transducing a cell with a rAAV particle as described herein.

[0252] Accordingly, the present disclosure provides methods for transducing a cell. The methods generally comprise contacting the cell with a rAAV particle disclosed herein under conditions whereby the cell is transduced. A rAAV particle disclosed herein can be used to transduce cells in vitro, in vivo and ex vivo.

[0253] Among other things, the present disclosure provides methods for delivering a coding sequence into a cell. The methods generally comprise comprising contacting the cell with a rAAV particle disclosed herein under conditions whereby the cell is transduced, and a coding sequence is expressed.

[0254] A rAAV particle disclosed herein can comprise a coding sequence under the control of a TRE. Accordingly, in certain embodiments, the present disclosure provides methods for expressing a coding sequence in a cell, the method generally comprising contacting the cell with such a rAAV particle under conditions whereby the cell is transduced, and a coding sequence is expressed. A coding sequence can encode a polypeptide and/or an RNA molecule, as described herein. Accordingly, in certain embodiments, the present disclosure provides methods for producing a polypeptide and/or an RNA molecule in a cell, the method generally comprising contacting the cell with such a rAAV particle under conditions whereby the cell is transduced and the polypeptide and/or an RNA molecule is produced. In certain embodiments, the method comprises expressing a first coding sequence and a second coding sequence in a cell, comprising introducing into the cell a polynucleotide as described herein, a vector as described herein, a rAAV genome as described herein, or a rAAV particle as described herein.

[0255] In certain embodiments, a cell is in a subject and the polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein is administered to the subject. In certain embodiments, the method comprises expressing a first coding sequence and a second coding sequence in a subject, comprising administering to the subject an effective amount of a polynucleotide as described herein, a vector as described herein, a rAAV genome as described herein, a rAAV particle as described herein, or a pharmaceutical composition as described herein. A polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein can be administered to a subject by all suitable routes, including, without limitation, intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally. [0256] In certain embodiments, a subject is a member of any mammalian or nonmammalian species. Suitable subjects include, without limitation, humans, non-human primates, canines, felines, ungulates (e.g., equine, bovine, swine (e.g., pig)), avians, rodents (e.g., rats, mice), and other subjects. In certain embodiments, a subject is human. In certain embodiments, a subject is canine. In certain embodiments, a subject is feline. In certain embodiments, a subject is equine. [0257] Among other things, the present disclosure provides a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition as described herein for use in medicine. Among other things, the present disclosure provides a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition as described herein for use as therapy. Among other things, the present disclosure provides a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition as described herein for use as a medicament.

[0258] Among other things, the present disclosure provides methods of treating an arrhythmogenic cardiomyopathy (ACM) in a subject. Arrhythmogenic cardiomyopathy is an inherited heart muscle disorder characterized by redistribution of junctional proteins, arrhythmias, and progressive myocardial injury. Pathological features of ACM include loss of myocytes and fibrofatty replacement of right ventricular myocardium. Biventricular involvement is often observed. Arrhythmogenic cardiomyopathy is a cell junction cardiomyopathy, typically caused by genetic abnormalities of cardiac desmosomes, which results in the detachment of myocytes and affects intracellular signal transduction.

[0259] Arrhythmogenic cardiomyopathy is a genetically heterogeneous disorder, for which there are several genes in which mutations are known to be causative. Genes in which mutations have been reported to cause ACM include: junction plakoglobin (JUP); desmoplakin (DSP) plakophilin-2 (PKP2); desmoglein-2 (DSG2); desmocollin-2 (DSC2); transmembrane protein 43 (TMEM43); lamin A/C (LMNA); desmin (DES); alpha-T-catenin (CTNNA3); phospholamban (PLN); transforming growth factor 3 (TGFB3); titin (TTN); sodium voltage-gated channel alpha subunit 5 (SCN5A; Na_v1.5); and cadherin C (CDH2). [0260] While the original ACM disease phenotype was characterized hy predominant right ventricle (RV) involvement (ARVC), with minor and late left ventricle (LV) disease, clinical variants characterized by early and greater LV involvement, which may parallel (e.g., biventricular ACM) or exceed (e.g., left-dominant ACM, or ALVC) the severity of RV involvement, have been increasingly reported.

[0261] There is not a single gold standard method for the diagnosis of ACM, and as such, the best strategy consists of combining multiple sources of clinical information, such as genetic, electrocardiographic, arrhythmic, morpho-functional, and histopathological findings. Cunent international criteria for ARVC and ALVC diagnosis include assessing morpho-functional ventricular abnormalities (i.e., global and regional ventricular dilatation and systolic dysfunction), structural myocardial abnormalities (i.e., for existence of fibrous or fibro-fatty myocardial replacement), echocardiographic abnormalities (e.g., depolarization and repolarization abnormalities), ventricular arrhythmias, and/or family history and molecular genetics. Phenotypic variants of ACM such as ARVC, biventricular ACM, and ALVC, are diagnosed based on a scoring system for fulfilling the above-mentioned criteria.

[0262] In certain embodiments, methods of treating ACM in a subject provided herein comprise administering to the subject an effective amount of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein. In certain embodiments, a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a first coding sequence encoding a fibroblast growth factor 21 (FGF21) and a second coding sequence encoding a soluble transforming growth factor beta receptor 2 (sTGFpR2). In certain embodiments, the polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a first coding sequence encoding a sTGFpR2 and a second coding sequence encoding a FGF21.

[0263] In certain embodiments, an ACM can be arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), or biventricular arrhythmogenic cardiomyopathy. In certain embodiments, an ACM is ARVC. In certain embodiments, an ACM is secondary to mutation in the desmoplakin (DSP) gene (DSP ACM). In certain embodiments, a subject is a mammal. In certain embodiments, a subject is a human. In certain embodiments, a subject is a canine. [0264] Among other things, the present disclosure provides methods for treating a mitral valve disease (MVD) in a subject. Various methods for diagnosing MVD include, without limitation, via echocardiograms, electrocardiograms, chest x-rays, cardiac magnetic resonance imaging, exercise tests, stress tests, and/or cardiac catheterization. A staging system for MVD generally refers to four basic groups: Stage A - at risk: risk factors for MVD are present; Stage B - progressive: MVD is mild or moderate, and there are no heart valve symptoms; Stage C - asymptomatic severe: MVD is severe, and there are no heart valve symptoms; and Stage D - symptomatic severe: MVD is severe and is causing symptoms. Stage B can be further subdivided into Stage Bl and Stage B2; Stage Bl is diagnosed when a heart murmur is detected but there is no radiographic or echocardiographic evidence of cardiac remodeling or remodeling that is not severe enough to meet current clinical trial criteria for treatment; Stage B2 is diagnosed when a heart murmur is detected and there is radiographic or echocardiographic evidence of cardiac remodeling that is severe enough to meet current clinical trial criteria for treatment.

[0265] In certain embodiments, methods of treating MVD in a subject provided herein comprise administering to the subject an effective amount of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein. In certain embodiments, a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a first coding sequence encoding a fibroblast growth factor 21 (FGF21) and a second coding sequence encoding a soluble transforming growth factor beta receptor 2 (sTGFpR2). In certain embodiments, a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a first coding sequence encoding a sTGFpR2 and a second coding sequence encoding a FGF21.

[0266] In certain embodiments, a method for treating a MVD in a subject further comprises administering to the subject an effective amount of one or more additional therapeutics to treat MVD. Such additional therapeutics to treat MVD may include, without limitation, diuretics, blood thinners (i.e., anticoagulants), and blood pressure medications. For example, in dogs, pimobendan is used in the management of heart failure due to MMVD. Pimobendan is often used in conjunction with an ACE inhibitor such as enalapril or benazepril. In certain embodiments, a method for treating MVD in a subject further comprises administering to the subject an effective amount of pimobendan. Other additional therapeutics for treating MVD include furosemide, spironolactone (i.e., an aldosterone antagonist), and an angiotensin-converting enzyme (ACE) inhibitor. [0267] In certain embodiments, the additional therapeutic to treat MVD (e.g., pimobendan) is administered at the same time as the gene therapy. In certain embodiments, the additional therapeutic to treat MVD (e.g., pimobendan) is administered at a different time as the gene therapy. [0268] In certain embodiments, the mitral valve disease comprises one or more disease or condition. A disease or condition can be myxomatous mitral valve disease, mitral valve stenosis, mitral valve prolapse, or mitral valve regurgitation.

[0269] Among other things, the present disclosure provides methods for treating familial partial lipodystrophy (FPL) in a subject. Familial partial lipodystrophy (FPL) is a rare genetic disorder that results in selective, progressive loss of body fat from various areas of the body. Individuals with FPL typically have reduced subcutaneous fat in the arms and legs, and they may or may not experience loss of body fat in the head and trunk regions. Individuals with FPL may also experience excess subcutaneous fat in other regions of the body, in particular, the neck, face, and intra- abdominal regions. The prevalence of FPL is estimated to be one in a million people; however, many cases may go misdiagnosed or undiagnosed.

[0270] FPL encompasses several subtypes differentiated by the underlying genetic mutation. The specific symptoms, severity, and prognosis can vary greatly between each type of FPL. FPL is caused by mutations of specific genes, and the various subtypes are characterized by the underlying genetic mutation.

[0271] The most common form of FPL is FPL type 2 (also known as Dunnigan lipodystrophy), in which affected individuals experience progressive loss of body fat in the arms, legs, and trunk, at around the time of puberty. Fat may accumulate in other areas of the body including the face, neck, and upper back between the shoulder blades. Individuals with FPL experience insulin resistance which may be associated with acanthosis nigricans, hepatomegaly (enlarged liver) in the form of steatosis which may lead to cirrhosis and liver dysfunction, glucose intolerance, hypertriglyceridemia which may result in pancreatitis, diabetes, coronary artery disease and other types of atherosclerotic vascular disease, and muscular dystrophy. Some women with FPL may develop polycystic ovary syndrome. In rare cases, individuals with FPL have a specific mutation in the LMNA gene; such individuals have increased risk of developing cardiomyopathy which can result in congestive heart failure and cardiac arrhythmias.

[0272] Other types of FPL include FPL type 1 (also known as Kobberling lidodystrophy), characterized by fat loss that is generally confined to the arms and legs; FPL type 3, caused by a genetic mutation in PPARG, is generally milder than FPL type 2; FPL type 4, caused by a genetic mutation in PLIN1, is characterized by lipodystrophy most prominent in the lower limbs and buttocks; FPL type 5, caused by a genetic mutation in AKT2, is characterized by lipodystrophy most prominently affecting arms and legs; and autosomal recessive FPL (also known as FPL type 6), caused by a genetic mutation in CIDEC. FPL has also been associated with a genetic mutation in AGPAT2.

[0273] Diagnosis of FPL is based upon the identification of characteristic symptoms coupled with clinical testing, and molecular genetic testing to detect mutations in genes that cause FPL.

[0274] In certain embodiments, the methods of treating FPL in a subject provided herein comprise administering to the subject an effective amount of a polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition described herein. In certain embodiments, the polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a first coding sequence encoding a fibroblast growth factor 21 (FGF21) and a second coding sequence encoding a soluble transforming growth factor beta receptor 2 (sTGF0R2). In certain embodiments, the polynucleotide, vector, rAAV genome, rAAV particle, or pharmaceutical composition comprises a first coding sequence encoding a sTGFpR2 and a second coding sequence encoding a FGF21.

[0275] In certain embodiments, a method for treating FPL in a subject further comprises administering to the subject an effective amount of one or more additional therapeutics to treat FPL (e.g., a non-gene therapy therapeutic to treat FPL). Such additional therapeutics to treat FPL may include, without limitation, fibric acid and derivatives thereof, statins, n-3 polyunsaturated fatty acids, hyperglycemic drugs such as metformin and sulfonylureas, insulin, anti -hypertensives, and/or metreleptin.

[0276] In certain embodiments, the one or more additional therapeutics to treat FPL is administered at the same time as the gene therapy. In certain embodiments, the one or more additional therapeutics to treat FPL is administered at a different time as the gene therapy.

[0277] In certain embodiments, a subject is diagnosed with autosomal recessive FPL (also known as FPL type 6), FPL type 1 (also known as Kobberling lipodystrophy), FPL type 2 (also known as Dunnigan lipodystrophy), FPL type 3, FPL type 4, or FPL type 5. In certain embodiments, a subject comprises a genetic mutation in LMNA, PPARG, PLIN1, AKT2. CIDEC, and/or A GPA T2. In certain embodiments, a subject is diagnosed with FPL type 2 and/or comprises a genetic mutation in LMNA. In certain embodiments, the subject is diagnosed with FPL type 2 and comprises a genetic mutation in LMNA.

VI. Examples

[0278] The following examples are offered by way of illustration, and not by way of limitation.

Example 1: Bicistronic Constructs

[0279] Exemplary dual promoter bicistronic vectors were designed, and an ability of these vectors to effect efficient and sustained expression of two different coding sequences was determined. Vectors were designed to compare unidirectional or bidirectional expression of two exemplary genes of interest, FGF21 and a fusion protein comprising a soluble TGFPR2 extracellular domain and IgG Fc (sTGFpR2-Fc). Genetic elements in vectors are set forth in Tables 1 and 2, below. SEQ ID NOs for each element are shown.

Table 1. Genetic elements in vectors p581 and p582. Table 2. Genetic elements in vectors p583, p584, p589, p608, and p695.

[0280] Unidirectional dual promoter vectors p581 and p582 comprise two tandem liverspecific promoters to drive expression of FGF21 and sTGFpR2-Fc.

[0281] The p581 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element; a TRE comprising 3 ApoE enhancer elements, a human AAT promoter, and an intron clement comprising a P-globin intron; a coding sequence encoding a caninc FGF21 polypeptide; a PRE comprising a WPRE sequence and a SV40 polyadenylation sequence; a TRE comprising an AFP enhancer element, a human albumin promoter, and an intron element comprising a MVM intron; a coding sequence encoding a soluble canine TGFPR2 polypeptide linked to a human TGFPR2 signal peptide and a canine FC polypeptide; a PRE comprising a bGH polyadenylation sequence; and a 3’ ITR element.

[0282] The p582 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element; a TRE comprising 3 ApoE enhancer elements, a human AAT promoter, and an intron element comprising a P-globin intron; a coding sequence encoding a canine FGF21 polypeptide; a PRE comprising a WPRE sequence and a SV40 polyadenylation sequence; a TRE comprising an AFP enhancer element, a human albumin promoter, and an intron element comprising a MVM intron; a coding sequence encoding a soluble canine TGFPR2 polypeptide linked to a canine AAT signal peptide and a canine FC polypeptide; a PRE comprising a bGH poly adenylation sequence; and a 3’ ITR element.

[0283] Bidirectional dual promoter vectors p583, p584, p589, p608, and p695 comprise a liver- specific bidirectional promoter system to drive expression of FGF21 and sTGFpR2-Fc.

[0284] The p583 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element; a PRE comprising a bGH polyadenylation sequence; a coding sequence encoding a soluble canine TGF0R2 polypeptide linked to a human TGFPR2 signal peptide and a canine FC polypeptide; a TRE comprising an intron element comprising a MVM intron, a human albumin promoter, 2 ApoE enhancer elements, a human AAT promoter, and an intron element comprising a P-globin intron; a coding sequence encoding a canine FGF21 polypeptide; a PRE comprising a WPRE sequence and a SV40 polyadenylation sequence; and a 3’ ITR element.

[0285] The p584 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element; a PRE comprising a bGH poly adenylation sequence; a coding sequence encoding a soluble canine TGFPR2 polypeptide linked to a canine AAT signal peptide and a canine FC polypeptide; a TRE comprising an intron element comprising a MVM intron, a human albumin promoter, 1 ApoE enhancer element, a human AAT promoter, and an intron element comprising a P-globin intron; a coding sequence encoding a canine FGF21 polypeptide; a PRE comprising a WPRE sequence and a SV40 polyadenylation sequence; and a 3’ ITR element.

[0286] The p589 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element; a PRE comprising a bGH polyadenylation sequence; a coding sequence encoding a soluble canine TGFPR2 polypeptide linked to a canine AAT signal peptide and a canine FC polypeptide; a TRE comprising an intron element comprising a MVM intron, a human albumin promoter, 3 ApoE enhancer elements, a human AAT promoter, and an intron element comprising a P-globin intron; a coding sequence encoding a canine FGF21 polypeptide; a PRE comprising a WPRE sequence and a SV40 polyadenylation sequence; and a 3’ ITR element.

[0287] The p608 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element; a PRE comprising a bGH poly adenylation sequence; a coding sequence encoding a soluble mouse TGFPR2 polypeptide linked to a human TGFPR2 signal peptide and an IgG2 FC polypeptide; a TRE comprising an intron element comprising a MVM intron, a human albumin promoter, 3 ApoE enhancer elements, a human AAT promoter, and an intron element comprising a P-globin intron; a coding sequence encoding a canine FGF21 polypeptide; a PRE comprising a WPRE sequence and a SV40 polyadcnylation sequence; and a 3’ ITR clement.

[0288] The p695 vector comprises, 5’ to 3’, the following genetic elements: a 5’ ITR element; a PRE comprising a bGH polyadcnylation sequence; a coding sequence encoding a soluble human TGFPR2 polypeptide linked to a human TGFPR2 signal peptide and an IgGl FC polypeptide; a TRE comprising an intron element comprising a MVM intron, a human albumin promoter, 3 ApoE enhancer elements, a human AAT promoter, and an intron element comprising a P-globin intron; a coding sequence encoding a canine FGF21 polypeptide; a PRE comprising a WPRE sequence and a SV40 polyadcnylation sequence; and a 3’ ITR element.

Example 2: Pilot Experiments in Mice

[0289] Vectors p581, p582, p583, p584, and p589 as described in Example 1 were packaged in AAV8 capsid (AAV8-p581, AAV8-p582, AAV8-p583, AAV8-p584, and AAV8- p589, respectively) and administered to C57BL/6J mice to assay coding sequence expression. As control, a single promoter bicistronic vector (comprising 3 ApoE enhancer elements, a human AAT promoter, and an intron element comprising a P-globin intron operably linked to drive expression of cFGF21 and csTGF R2-Fc separated by a 2A element) packaged in AAV8, was used. As another control, a cocktail composition comprising a first rAAV vector containing cFGF21 coding sequence, and a second rAAV vector containing csTGFpR2-Fc coding sequence, each packaged in AAV8, was used.

[0290] Expression levels of canine FGF21 (cFGF21) and canine sTGFpR2-Fc (csTGF R2-Fc) in plasma of C57BL/6J mice administered recombinant AAV8 were determined by standard ELISA using antibodies to TGFPR2 and FGF21, respectively. Plasma samples were collected at 2 weeks, 4 weeks, and 6 weeks after administration of AAV8-p581, AAV8-p582, AAV8-p583, AAV8-p584, and AAV8-p589. FIGS. 1A and IB show expression levels of cFGF21 and of csTGF R2-Fc in plasma of mice at 6 weeks post-administration, respectively. It was observed that p581 and p589 induced low expression of both cFGF21 and csTGF R2-Fc; p582 induced high expression of csTGF R2-Fc and lower expression of cFGF21; and p583 and p584 both induced high expression of cFGF21 and moderate levels of csTGFpR2-Fc.

Example 3: Pilot Experiments in Canines

[0291] Expression of cFGF21 and csTGF R2-Fc effected by AAV8-p583, AAV8-p584, and AAV8-p589 was tested in canines. Recombinant AAV8 were injected into adult beagles (3 subjects per rAAV tested, for a total of 9 subjects). Expression levels of cFGF21 and csTGFpR2- Fc of administered canines were determined by standard EEISA using antibodies to TGFPR2 and FGF21, respectively, in plasma samples collected at 2 weeks, 4 weeks, 6 weeks, and 8 weeks postadministration (FIGS. 2A-2D).

[0292] As shown in FIGS. 2A and 2C, administration of AAV8-p583 resulted in consistently high average expression of both cFGF21 and csTGFpR2-Fc amongst administered canines, and administration of AAV8-p584 induced lower expression of both cFGF21 and csTGFpR2-Fc. As shown in FIGS. 2B and 2D, administration of AAV8-p589 resulted in a canine subject (#10001) having high plasma expression levels of both coding sequences.

Example 4: Expression of sTGFpR2 and FGF21 in Mouse Model of ARVC

[0293] PKP2cKO mice were generated as described in Cerrone et al. (2017) Nature Communications 8: 106. PKP2cKO mice have cardiomyocyte-specific, tamoxifen-induced knockout of desmosomal protein plakophilin-2 (PKP2). PKP2 knockout induced PKP2cKO mice have previously been shown to develop progressive right-ventricular dominant arrhythmogenic cardiomyopathy. A general study design in this Example for using PKP2cKO mice to investigate efficacy of sTGF R2, FGF21, and bigenic sTGF R2/FGF21 gene therapy was to administer a gene therapy at 1.5E13 vg/kg to PKP2cKO mice (day -7), followed by tamoxifen-induced knockout seven days later (day 0). 21 days after knockout induction, mice were subjected to isoproterenol (ISO) challenge and electrocardiography. 28 days after knockout induction, the mice were subjected to echocardiography and were then sacrificed, and terminal blood was collected.

[0294] The p608 vector was designed with the same vector promoter elements as p589, to express mouse FGF21 (mFGF21) and mouse sTGFpR2-Fc (msTGFpR2-Fc). Vector p6O8 was packaged in AAV8 capsid (AAV8-p608) and administered to C57BE/6J mice. Expression levels of mFGF21 and msTGFpR2-Fc in C57BL/6J mice administered with AAV8-p6O8 were determined by standard ELISA using antibodies to TGFPR2 and FGF21, respectively, in plasma samples collected at 2 weeks, 4 weeks, 6 weeks and 8 weeks post-administration. Results shown in FIGS. 3A and 3B, indicate successful expression and detection of both coding sequences.

[0295] In a separate experiment, four-week-old mice were administered, via single intravenous retro-orbital or tail vein injection, AAV8-p608, or as controls: an rAAV vector containing mFGF21 coding sequence, or a rAAV vector containing msTGFpR2-Fc coding sequence, each packaged in AAV8 (AAV8-mFGF21 and AAV8-msTGFpR2-Fc, respectively). AAV8-p6O8, AAV8-mFGF21 , and AAV8-msTGFpR2-Fc were each administered at a dose of 1.5E13 vg/kg. Control wild type (WT) and PKP2cK0 animals were administered vehicle only. mFGF21 and msTGF|3R2-Fc expression was measured by ELIS As using antibodies to mouse FGF21 and TGFPR2, respectively. As shown in FIG. 4A and FIG. 4B, expression levels of mFGF21 and msTGFpR2-Fc were confirmed in PKP2cK0 mice administered AAV8-p608, AAV8-mFGF21 , and AAV8-msTGFpR2-Fc.

[0296] Cardiac structure and function of PKP2cKO mice treated with AAV8-p6O8 was assessed. Fractional shortening (FS) and ejection fraction was measured by standard M-mode echocardiography. As shown in FIGS. 5A-5C, administration of AAV8-p608 resulted in enhanced preservation of left ventricle systolic function measures as a function of left ventricle ejection fraction (LVEF; FIG. 5A), enhanced preservation of fractional shortening (FS; FIG. 5B), and significant reduction in right ventricle dilatation (FIG. 5C). Taken together, administration of AAV8-p6O8 significantly improved cardiac structure and function of PKP2cKO mice.

[0297] Arrhythmia burden was also assessed in PKP2cKO mice treated with AAV8-p608. Arrhythmia burden, assessed by number of premature ventricular contractions (PVCs) is a clinically meaningful measurement in arrhythmogenic cardiomyopathy: it is a predictor of survival (correlates with mortality and life-threatening ventricular arrhythmias in arrhythmogenic right ventricular cardiomyopathy), it is a key driver of symptoms, and it is directly linked to quality of life - even single PVCs can trigger anxiety and post-traumatic stress disorder in patients. As shown in FIG. 6, PKP2cKO mice administered AAV8-p6O8 were observed to have a reduction in the percentage of cases with PVCs greater than 100 in 30 minutes following ISO challenge.

[0298] In order to investigate progression of cardiac fibrosis, hearts of mice were obtained after sacrifice (28 days after induction of knockout), fixed, embedded, and sectioned according to standard techniques. Sections were then stained with Masson’s Trichrome Staining and analyzed for cardiac fibrosis by assessing the percentage of collagen present, indicative of fibrosis. FIG. 7A and FIG. 7B shows level of fibrosis detected in the left ventricle (LV) and right ventricle (RV) of PKP2cKO hearts of mice that received AAV8-p608 or vehicle as control.

Example 5: Treatment of Patients with Arrhythmogenic Right Ventricular Cardiomyopathy

[0299] Arrhythmogenic right ventricular cardiomyopathy (ARVC) - previously referred to as arrhythmogenic right ventricular dysplasia (ARVD), is an inherited, rare, myocardial disorder affecting the right ventricle and, in some cases, also the left ventricle. ARVD/C is caused by mutations in genes that encode dcsmosomal proteins. These proteins arc involved with ccll-to-ccll adhesion, and disruptions in these proteins account for 17% of all sudden cardiac deaths in young populations.

[0300] This prophetic Example describes a method of treating human patients with ARVC using a non-replicating, recombinant adeno-associated virus (rAAV) vector as described herein. The p695 vector was designed to be analogous to p608, but for expression of human FGF21 (hFGF21) and human sTGFpR2-Fc (hsTGFpR2-Fc). Vector p695 will be packaged in AAV8 capsid (AAV8-p695) for administration into human patients and is expected to promote expression of hFGF21 and hsTGFpR2-Fc in patient cells.

[0301] Without being bound by any particular theory, treatment with the AAV8-p695 is expected to delay progression of complications, symptoms, and cardiac tissue changes in ARVC patients. The number and severity of arrhythmias should decrease, the inflammatory environment will be reduced leading to less structural changes and a decrease in fibrofatty tissue replacement of cardiac tissue. Also, heart function is expected to remain normal and not deteriorate.

[0302] Selection of patients will include diagnosis of ARVC according to standard diagnostic procedures. Standard diagnosis is based on a scoring system taking into account right ventricle structural and functional abnormalities (dilatation, akinesia, dyskinesia, aneurysms) detected by echocardiography, MRI and angiography; electrocardiographic features (inverted T waves in right precordial leads, epsilon waves and late potentials by signal averaged ECG (SAECG), left bundle branch block ventricular tachycardia, >500 ventricular' extrasystoles per 24 h); tissue characterization at endomyocardial biopsy (fibro-fatty replacement of myocardium); and family history. Contrast enhanced MRI substantially enhances the diagnostic sensitivity, particularly in left ventricle variants, while electro anatomic mapping is superior in detecting early RV involvement. Differential diagnosis includes idiopathic RV outflow tract tachycardia, myocarditis, sarcoidosis and congenital heart diseases (see these terms). Although prenatal diagnosis through amniocentesis is feasible, it is subject to ethical and legal considerations.

[0303] Production of AAV8-p695 for administration to patients will be done by standard transient triple plasmid transfection methods. The plasmids used for transfection will contain rep/cap genes, adenovirus-derived helper plasmid suppling genes needed for replication, and the recombinant AAV plasmid containing the genes of interest (GOT; e.g., hFGF21 and hsTGFpR2- Fc).

[0304] AAV8-p695 will be administered to patients as a single dose intravenous injection for systemic delivery.

Example 6: Treatment of Patients with DSP ACM

[0305] This Example describes a first-in-human phase 1/2, prospective, open-label, sequential dose escalation trial of AAV8-p695 in participants with arrhythmogenic cardiomyopathy (ACM) secondary to mutation in the desmoplakin (DSP) gene (DSP ACM) who are at high risk for ventricular arrhythmias and sudden cardiac death. AAV8-p695 is vector p695 as described above, packaged in AAV8 capsid.

[0306] A minimum of 3, up to a maximum of 5, participants will be treated in each of 3 dose cohorts for a total of 9-15 evaluable participants. The objectives of the trial include the assessment of safety and tolerability of AAV8-p695 in participants with ACM secondary to a DSP variant, and to evaluate the expression of the transgene protein and pharmacodynamics of AAV8- p695 on underlying disease in order to identify the dose(s) and inform the design of a follow-on clinical trial. This trial will establish the initial safety and tolerability profile of the clinical drug product, drive dose selection for the next trial, identify the best efficacy outcome measures and endpoints to use for the registration trial(s), and provide the context for further discussions with regulators on the full development plan.

[0307] Inclusion Criteria: diagnosis and main criteria for inclusion into the trial include:

- Males and females age > 18 years at time of Day 1 (treatment).

- Signed the informed consent form and release of medical information form.

- Anti-AAV8 total antibody ELISA luminescent signal < a specific cut point [or neutralizing antibody inhibition of viral vector by <50% at a specific serum dilution] .

- A clinical diagnosis of ACM

- Documentation of a single pathogenic or likely pathogenic mutation in the desmoplakin gene categorized as associated or probably associated with ACM and confirmed by an accredited genetic testing laboratory. Pathogenicity of variants will be adjudicated by the trial genetic core laboratory or registered genetic counselor according to the joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association of Molecular- Pathology with ACM-specific modifications. - High risk for ventricular arrhythmias and/or sudden cardiac death defined by an average of > 500 PVCs in a 24-hour period, assessed over a 7-day ambulatory ECG recording with a minimum of at least a 48-hour recording, within 3 months prior to enrollment.

- Functioning, clinically indicated ICD with remote interrogation capability and historical interrogation data covering the preceding 12 months prior to treatment. Of note, if combined CRT- implantable cardioverter-defibrillator (CRT-D) is clinically indicated, it must have been implanted > 3 months prior to screening echocardiogram.

- History of ambulatory ECG monitoring over at least a 6-month period with > 3 recordings of > 48-hours duration and approximately 3 months apart from each other with the data available to serve as a lead-in baseline for this trial. If an intervention had taken place (e.g., catheter ablation) and/or substantial changes in the ACM therapeutic regimen or lifestyle (i.e., increase in exercise) were made between the historical ECG monitoring and screening for this trial, the 24-hour average PVC burden must be within ± 10% of the screening PVC count; otherwise, the historical ECG monitoring will need to be repeated.

- Stable, optimized ACM therapeutic regimen for > 30 days prior to Screening, including beta blocker, as tolerated, anti-arrhythmic agent(s) as indicated, catheter ablation, restricted exercise, heart failure guideline directed medical therapy, as indicated, and intention to maintain stable medication regimen throughout trial period in the judgement of the investigator.

- Clinically stable regarding both overall medical condition and comorbidities as well as underlying disease within 30 days prior to enrollment in the judgement of the investigator.

- Current and up-to-date with FDA-approved or authorized CDC recommended immunizations (e.g., annual influenza, meningococcal and meningococcal B, COVID- 19, etc.) at least 14 days prior to the screening period.

- Ability to reliably comply with protocol assessments in the judgement of the investigator.

- All male participants regardless of fertility status or the fertility status of their partner must agree to use a condom and spermicide during any sexual relations for 6 months following administration of investigational medicine product to protect their partner from potential viral shedding.

- All participants regardless of fertility status or the fertility status of their partner must agree to have any male partner use a condom and spermicide during any sexual relations for 6 months following administration of investigational medicine product to protect their partner from potential viral shedding. - All participants capable of procreation must agree to use adequate contraception for 6 months following administration of investigational medicinal product to avoid pregnancy (defined as oral or injectable contraceptives, intrauterine devices, surgical sterilization in addition to/or a combination of a condom and spermicide).

- Agree to not donate sperm or oocytes for 6 months following administration of investigational medicinal product.

[0308] Exclusion Criteria: criteria for exclusion from the trial include:

- Left ventricular ejection fraction < 40 % as assessed by the Echocardiography Core Laboratory.

- Severe RV dysfunction as assessed by screening echocardiogram and confirmed by the Echocardiography Core Laboratory in accordance with American Society of Echocardiography guidelines, taking into account visual assessment by multiple views and quantitative assessment of at least one of the following: tricuspid lateral annular systolic velocity wave (S’) derived from Doppler tissue imaging, fractional area change (FAC), RV index of myocardial performance or TAPSE. Extreme height or body surface area will also be considered in the assessment of RV function.

- Athlete participating in competitive or very frequent high endurance sports < 18 months prior to screening, against the advice of the American Heart Association/America College of Cardiology Arrhythmias and Conduction Defects Task Force or Sport Cardiology Section of the European Associate of Preventive Cardiology. (Such a participant must have undergone a detraining protocol > 18 months prior to screening and have proof of a stable baseline PVC count.)

- Liver function tests (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase and total bilirubin) > 1.5 X upper limit of normal (ULN) or known intrinsic liver disease (e.g., cirrhosis, chronic hepatitis B or hepatitis C virus infection).

- Current or likely need for hemodialysis within 12 months following screening, or current glomerular filtration rate (GFR) < 40 mL/minute/1.73 m2 estimated by Modification of Diet in Renal Disease (MDRD) formula for calculating the GFR MDRD calculation.

- Bleeding diathesis or thrombocytopenia defined as platelet count < 75,000 platelets/pL.

- Diagnosis of, or treatment for, any cancer within the 5 years prior to screening except for Grade 1 prostate cancer, basal cell carcinoma or carcinomas in situ where surgical excision was considered curative. (Past medical history of cancer is not exclusionary as long as the subject has been disease free for at least 5 years since the time of diagnosis and treatment). - Pregnant or lactating/breastfeeding at time of screening.

- History of skin sensitivity to medical adhesives that could impact compliance with 7-day ECG monitor compliance.

- Received live virus vaccine within a specific number of weeks or inactive vaccine within a specific number of weeks prior to Day 1 or expected to receive any vaccination within 3 months after Day 1.

- Previous recipient of a gene therapy, cell-based therapy or CRISPR/Cas9 or other form of gene editing therapy at any time prior to screening.

- Received an investigational intervention or participated in another clinical study within 30 days or within 5 half-lives of the drug prior to screening. Exception may be made if the individual is enrolled in a non-therapeutic observational study (registry), the observational portion of a therapeutic study where the sponsoring authority authorizes enrollment.

- Recent history of psychiatric disease (including drug or alcohol abuse) that is likely to impair subject’s ability to comply with protocol-mandated procedures, in the opinion of the investigator.

- Other unstable or uncontrolled comorbidity.

- Other concurrent medical condition(s) that, while not explicitly excluded by the protocol, could jeopardize the safety of the participant or objectives of the study, in the judgement of the investigator.

[0309] Concomitant Corticosteroid Prophylactic Therapy: Starling one day prior to Day 1 , systemic corticosteroid therapy equivalent to oral prednisolone at 1 mg/kg of body weight per day will be initiated for a total of 30 days. At the end of the 30-day period, liver function by clinical examination and laboratory testing will be performed, and if unremarkable, the corticosteroid dose may be tapered gradually over the next 28 days while monitoring liver function by weekly laboratory testing. If liver function abnormalities persist or appear during the taper, continue systemic corticosteroids at the original dose until findings become unremarkable, and then taper the dose gradually over the next 28 days or longer if needed. Consult with the sponsor if liver function abnormalities continue to persist > 2 x upper limit of normal (ULN) after the 30-day period of systemic corticosteroids.

[0310] Criteria for Evaluation: Safety: Adverse events (AEs), concomitant medications, laboratory tests (complete blood count [CBC] with white blood cell [WBC] differential and platelets, basic metabolic and comprehensive hepatic serum chemistry panels), Enzyme-linked ImmunoSpot (ELISpot), laboratory evidence of complement activation, anti-drug antibody (FGF21), anti-AAV8 total antibody ELISA [or NAb titer], urinalysis, urine pregnancy test, physical examination including weight and vital signs, interrogation of ICD, 12-lead electrocardiogram (ECG), clinical events, endomyocardial biopsy and conditions of interest in gene therapy trials. Activity: Ambulatory and stress-induced ECG recording, 12-lead ECG, ICD interrogation (including actigraphy), clinical events, echocardiography, NYHA Class and HF Stage, quality of life as assessed by ACM-QoL, CDS, FSAS, and KCCQ.

* * *

[0311] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0312] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

SEQUENCE LISTING

The following table provides a listing of the nucleic acid and amino acid sequences represented by the various SEQ ID NOs used throughout the specification.

Ill

EXEMPLARY EMBODIMENTS

Embodiment 1. An isolated polynucleotide comprising:

(a) a first nucleic acid comprising, from 5’ to 3’: a first transcriptional regulatory element (TRE) comprising a human alpha 1- antitrypsin (hAAT) promoter and one or more ApoE enhancer elements; and a first coding sequence, and

(b) a second nucleic acid comprising, from 5’ to 3’ : a second TRE comprising a human albumin (hAlb) promoter; and a second coding sequence.

Embodiment 2. An isolated polynucleotide comprising from 5’ to 3’:

(a) a human albumin (hAlb) promoter and one or more ApoE enhancer elements;

(b) one or more ApoE enhancer elements and a human alpha 1 -antitrypsin (hAAT) promoter; or

(c) a hAlb promoter, one or more ApoE enhancer elements, and a hAAT promoter.

Embodiment 3. The polynucleotide of embodiment 1 or 2, wherein the hAAT promoter comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

Embodiment 4. The polynucleotide of any one of the preceding embodiments, wherein the hAlb promoter comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

Embodiment 5. The polynucleotide of any one of the preceding embodiments, comprising two ApoE enhancer elements. Embodiment 6. The polynucleotide of any one of the preceding embodiments, comprising three ApoE enhancer elements.

Embodiment 7. The polynucleotide of any one of the preceding embodiments, wherein the one or more ApoE enhancer elements each comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

Embodiment 8. The polynucleotide of any one of the preceding embodiments, further comprising one or more intron elements.

Embodiment 9. The polynucleotide of any one of the preceding embodiments, wherein the one or more intron elements each comprise a P-globin intron element or a MVM intron element.

Embodiment 10. The polynucleotide of any one of the preceding embodiments, further comprising a P-globin intron element, optionally wherein the P-globin intron element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3.

Embodiment 11. The polynucleotide of embodiment 10, wherein the P-globin intron element is 3’ of the hAAT promoter.

Embodiment 12. The polynucleotide of any one of the preceding embodiments, further comprising a MVM intron element, optionally wherein the MVM intron element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. Embodiment 13. The polynucleotide of embodiment 12, wherein the MVM intron element is 3’ of the hAlb promoter.

Embodiment 14. The polynucleotide of embodiment 1, wherein the second TRE further comprises a human AFP enhancer element, optionally wherein the human AFP enhancer element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

Embodiment 15. The polynucleotide of embodiment 1, wherein the first TRE comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 27-29.

Embodiment 16. The polynucleotide of embodiment 1, wherein the second TRE comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or 11.

Embodiment 17. The polynucleotide of any one of the preceding embodiments, further comprising one or more post-transcriptional regulatory elements (PREs).

Embodiment 18. The polynucleotide of embodiment 17, wherein the one or more PREs each comprise a polyadenylation sequence and/or a WPRE sequence.

Embodiment 19. The polynucleotide of embodiment 18 , wherein the PRE comprises a bovine growth hormone poly adenylation (BGHpA) sequence, optionally wherein the BGHpA sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 or 23. Embodiment 20. The polynucleotide of embodiment 18, wherein the PRE comprises a simian virus 40 poly adenylation (SV40pA) sequence and a WPRE sequence, optionally wherein the SV40pA sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 9 and/or the WPRE sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

Embodiment 21. The polynucleotide of embodiment 1, further comprising a Kozak consensus sequence 5’ to the first coding sequence and/or a Kozak consensus sequence 5’ to the second coding sequence.

Embodiment 22. The polynucleotide of embodiment 1, wherein the first nucleic acid and the second nucleic acid are in the same orientation in the polynucleotide, optionally wherein the first nucleic acid and the second nucleic acid are separated by a polycistronic element.

Embodiment 23. The polynucleotide of embodiment 22, wherein the polycistronic element is an IRES or 2A sequence.

Embodiment 24. The polynucleotide of embodiment 22 or 23, comprising from 5’ to 3’:

(a) the first TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27;

(b) the first coding sequence;

(c) the first PRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31; (d) the second TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30;

(e) the second coding sequence; and

(f) the second PRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22.

Embodiment 25. The polynucleotide of embodiment 1, wherein the first nucleic acid and the second nucleic acid are in the opposite orientation in the polynucleotide.

Embodiment 26. The polynucleotide of embodiment 25, comprising from 5’ to 3’ :

(a) the second PRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23;

(b) the second coding sequence;

(c) the second TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11;

(d) the first TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28;

(e) the first coding sequence; and

(f) the first PRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31.

Embodiment 27. The polynucleotide of embodiment 25, comprising from 5’ to 3”;

(b) the second coding sequence;

(d) the first TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29;

(e) the first coding sequence; and

Embodiment 28. The polynucleotide of embodiment 25, comprising from 5’ to 3’ :

(b) the second coding sequence;

(d) the first TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity SEQ ID NO: 27;

(e) the first coding sequence; and

(f) the first PRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31 or 32.

Embodiment 29. The polynucleotide of any one of the preceding embodiments, wherein the first coding sequence and/or the second coding sequence each encode a miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptazyme, RNA aptamer, IncRNA, ribozyme or mRNA.

Embodiment 30. The polynucleotide of any one of the preceding embodiments, wherein the first coding sequence and/or the second coding sequence each encode one or more polypeptides.

Embodiment 31. The polynucleotide of any one of the preceding embodiments, wherein the first coding sequence and/or the second coding sequence each encode a growth factor or receptor thereof.

Embodiment 32. The polynucleotide of any one of the preceding embodiments, wherein the first coding sequence encodes a growth factor.

Embodiment 33. The polynucleotide of any one of the preceding embodiments, wherein the first coding sequence encodes fibroblast growth factor 21 (FGF21). Embodiment 34. The polynucleotide of any one of the preceding embodiments, wherein the first coding sequence encodes a canine FGF21, a mouse FGF21, or a human FGF21.

Embodiment 35. The polynucleotide of any one of embodiments 31-34, wherein the first coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 4-6.

Embodiment 36. The polynucleotide of any one of the preceding embodiments, wherein the second coding sequence encodes a growth factor receptor.

Embodiment 37. The polynucleotide of any one of the preceding embodiments, wherein the second coding sequence encodes a transforming growth factor beta receptor 2 (TGPFR2).

Embodiment 38. The polynucleotide of any one of the preceding embodiments, wherein the second coding sequence encodes a canine TG0FR2, a mouse TGPFR2, or a human TGPFR2.

Embodiment 39. The polynucleotide of any one of embodiments 31-38, wherein the second coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-18.

Embodiment 40. The polynucleotide of any one of the preceding embodiments, wherein the second coding sequence encodes a soluble growth factor receptor.

Embodiment 41. The polynucleotide of any one of the preceding embodiments, wherein the second coding sequence encodes a soluble transforming growth factor beta receptor 2 (sTGpFR2). Embodiment 42. The polynucleotide of any one of embodiments 31-41 , wherein the growth factor receptor is linked to an Fc region, optionally wherein the Fc region is a canine Fc region, an IgG2 Fc region, or an IgGl Fc region.

Embodiment 43. The polynucleotide of any one of embodiments 31-42, wherein the second coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33-35.

Embodiment 44. The polynucleotide of any one of embodiments 31-43, wherein the growth factor receptor is linked to a signal peptide, optionally wherein the signal peptide is a human TGFPR2 signal peptide or a canine AAT signal peptide.

Embodiment 45. The polynucleotide of embodiment 44, wherein the growth factor receptor is soluble transforming growth factor beta receptor 2 (sTGpFR2).

Embodiment 46. The polynucleotide of embodiment 44 or 45, wherein the second coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 36-39.

Embodiment 47. The polynucleotide of any one of embodiments 31-46, wherein the growth factor receptor comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 40-43.

Embodiment 48. The polynucleotide of any one of the preceding embodiments, wherein the first coding sequence and/or the second coding sequence does not encode a functional antibody. Embodiment 49. An isolated polynucleotide comprising from 5’ to 3’: a first coding sequence encoding a growth factor or receptor thereof; a first transcriptional regulatory element (TRE); a second TRE; and a second coding sequence encoding a growth factor or receptor thereof.

Embodiment 50. The polynucleotide of embodiment 49, wherein the first TRE and the second TRE are the same or different.

Embodiment 51. The polynucleotide of embodiment 49 or 50, wherein the first TRE and/or the second TRE are liver- specific.

Embodiment 52. The polynucleotide of embodiment 51, wherein the liver-specific TRE comprises one or more elements selected from the group consisting of: an ApoA-I promoter, an ApoA-II promoter, an ApoA-IV promoter, an ApoB promoter, an ApoC-I promoter, an ApoC-II promoter, an ApoC-III promoter, an ApoE promoter, an albumin promoter, an a-fetoprotein promoter, a phosphoenolpyruvate carboxykinase 1 (PCK1) promoter, a phosphoenolpyruvate carboxykinase 2 (PCK2) promoter, a transthyretin (TTR) promoter, an a- antitrypsin (AAT or SERPINA1) promoter, a hemopexin promoter, an alcohol dehydrogenase 6 promoter, a cholesterol 7 alpha-hydroxylase promoter, a factor IX promoter, and an a-microglobulin promoter.

Embodiment 53. The polynucleotide of embodiment 49 or 50, wherein the first TRE and/or the second TRE are muscle-specific.

Embodiment 54. The polynucleotide of embodiment 53, wherein the muscle- specific TRE comprises one or more elements selected from the group consisting of: a human skeletal muscle a-actin (HSA) promoter, a muscle creatine kinase (MCK) promoter, a MHCK7 promoter, a dMCK promoter, a tMCK promoter, a CK6 promoter, a CK8 promoter, a CK8e promoter, a human desmin (DES) promoter or variant thereof, a cardiac troponin T (cTnT) promoter, a myosin light-chain (MLC2v) promoter, a human a-myosin heavy chain gene (aMHC) promoter, a MLC promoter, a human troponin T (TNNI1 ) promoter, a AUSEx3 promoter, a SPcA5- 12 promoter, a SP-301 promoter, a MH promoter, and a Sk-CRM4/DES promoter.

Embodiment 55. The polynucleotide of any one of embodiments 49-54, wherein in the first TRE and/or second TRE each comprise an intron element.

Embodiment 56. The polynucleotide of any one of embodiments 49-55, wherein the polynucleotide further comprises a first post-transcriptional regulatory element (PRE), optionally wherein the first PRE is 5’ of the first coding sequence.

Embodiment 57. The polynucleotide of any one of embodiments 49-56, wherein the polynucleotide further comprises a second PRE, optionally wherein the second PRE is 3’ of the second coding sequence.

Embodiment 58. The polynucleotide of embodiment 56 or 57, wherein the first PRE and/or second PRE each comprise a polyadenylation sequence and/or a WPRE sequence.

Embodiment 59. The polynucleotide of any one of embodiments 49-58, wherein the first coding sequence and/or second coding sequence encodes a growth factor receptor, optionally wherein the growth factor receptor is a soluble growth factor receptor.

Embodiment 60. The polynucleotide of any one of embodiments 49-59, wherein the first coding sequence and/or second coding sequence encodes a soluble transforming growth factor beta receptor 2 (sTGpFR2).

Embodiment 61. The polynucleotide of embodiment 60, wherein the sTGpFR2 is linked to an Fc region.

Embodiment 62. The polynucleotide of any one of embodiments 49-61, wherein the growth factor is linked to a signal peptide. Embodiment 63. The polynucleotide of any one of embodiments 49-62, wherein the first coding sequence and/or second coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-18 or 33-39.

Embodiment 64. The polynucleotide of any one of embodiments 49-63, wherein the first coding sequence and/or second coding sequence encodes a growth factor, optionally wherein the growth factor is fibroblast growth factor 21 (FGF21).

Embodiment 65. The polynucleotide of any one of embodiments 49-64, wherein the first coding sequence and/or second coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 4-6.

Embodiment 66. An isolated polynucleotide comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 44-61.

Embodiment 67. A polynucleotide that is the complement of the polynucleotide of any one of the preceding embodiments.

Embodiment 68. A polynucleotide that is the reverse complement of the polynucleotide of any one of the preceding embodiments.

Embodiment 69. A vector comprising the polynucleotide of any one of the preceding embodiments. Embodiment 70. The vector of embodiment 69, which is a plasmid, a viral vector, or a DNA minimal vector.

Embodiment 71. The vector of embodiment 69 or 70, which is an expression vector.

Embodiment 72. The vector of embodiment 70 or 71, wherein the viral vector is selected from the group consisting of an adenoviral vector, an adeno-associated virus (AAV) vector, and a lentiviral vector.

Embodiment 73. The vector of any one of embodiments 69-72, wherein the vector is an AAV vector.

Embodiment 74. A recombinant adeno-associated virus (rAAV) genome comprising a polynucleotide of any one of embodiments 1-69.

Embodiment 75. The rAAV genome of embodiment 74, further comprising a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence, and a 3’ inverted terminal repeat (3’ ITR) nucleotide sequence.

Embodiment 76. The rAAV genome of embodiment 75, wherein the 5’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and/or the 3’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25 or 26.

Embodiment 77. The rAAV genome of any one of embodiments 74-76, comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 62-68. Embodiment 78. A recombinant AAV particle comprising an AAV capsid and the rAAV genome of any one of embodiments 74-77.

Embodiment 79. The rAAV particle of embodiment 78, wherein the AAV capsid protein is derived from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof.

Embodiment 80. The rAAV particle of embodiment 78 or 79, wherein the AAV capsid protein comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69, 70, and/or 71.

Embodiment 81. A pharmaceutical composition comprising a polynucleotide of any one of embodiments 1-69, a vector of any one of embodiments 70-73, a rAAV genome of any one of embodiments 74-77, or an rAAV particle of any one of embodiments 78-80.

Embodiment 82. The polynucleotide of any one of embodiments 1-69, the vector of any one of embodiments 70-73, the rAAV genome of any one of embodiments 74-77, the rAAV particle of any one of embodiments 78-80, or the pharmaceutical composition of embodiment 81, for use in medicine, for use as therapy, or for use as a medicament.

Embodiment 83. A packaging system for preparation of an rAAV particle, wherein the packaging system comprises:

(a) a first nucleotide sequence encoding one or more AAV Rep proteins;

(b) a second nucleotide sequence encoding an AAV capsid protein; and

(c) a third nucleotide sequence comprising the rAAV genome sequence of any one of embodiments 74-77. Embodiment 84. The packaging system of embodiment 83, wherein the packaging system comprises a first vector comprising the first nucleotide sequence and the second nucleotide sequence, and a second vector comprising the third nucleotide sequence.

Embodiment 85. The packaging system of embodiment 84, further comprising a fourth nucleotide sequence comprising one or more helper virus genes, optionally wherein the fourth nucleotide sequence is comprised within a third vector.

Embodiment 86. The packaging system of embodiment 85, wherein the fourth nucleotide sequence comprises one or more genes from a virus selected from the group consisting of adenovirus, herpesvirus, vaccinia virus, and cytomegalovirus (CMV).

Embodiment 87. The packaging system of embodiment 85 or 86, wherein the first vector, second vector, and/or the third vector is a plasmid.

Embodiment 88. A method for recombinant preparation of an rAAV, the method comprising introducing the packaging system of any one of embodiments 83-87 into a cell under conditions whereby the rAAV particle is produced.

Embodiment 89. A method comprising introducing into a cell the polynucleotide of any one of embodiments 1-69, the vector of any one of embodiments 70-73, the rAAV genome of any one of embodiments 74-77, the rAAV particle of any one of embodiments 78-80, or the pharmaceutical composition of embodiment 81.

Embodiment 90. A method of expressing a first coding sequence and a second coding sequence in a cell, comprising introducing into the cell the polynucleotide of any one of embodiments 1-69, the vector of any one of embodiments 70-73, the rAAV genome of any one of embodiments 74-77, or the rAAV particle of any one of embodiments 78-80.

Embodiment 91. A method of expressing a first coding sequence and a second coding sequence in a subject, comprising administering to the subject an effective amount of the polynucleotide of any one of embodiments 1-69, the vector of any one of embodiments 70-73, the rAAV genome of any one of embodiments 74-77, the rAAV particle of any one of embodiments 78-80, or the pharmaceutical composition of embodiment 81.

Embodiment 92. A method of treating an arrhythmogenic cardiomyopathy (ACM) in a subject, comprising administering to the subject an effective amount of the polynucleotide of any one of embodiments 1-69, the vector of any one of embodiments 70-73, the rAAV genome of any one of embodiments 74-77, the rAAV particle of any one of embodiments 78-80, or the pharmaceutical composition of embodiment 81, wherein:

(a) the first coding sequence encodes a fibroblast growth factor 21 (FGF21) and the second coding sequence encodes a soluble transforming growth factor beta receptor 2 (sTGFpR2); or

(b) the first coding sequence encodes a sTGFpR2 and the second coding sequence encodes a FGF21.

Embodiment 93. The method of embodiment 92, wherein the ACM is selected from the group consisting of arrhythmogenic right ventricular' cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), and biventricular arrhythmogenic cardiomyopathy.

Embodiment 94. The method of embodiment 92 or 93, wherein the ACM is ARVC.

Embodiment 95. The method of embodiment 92 or 93, wherein the ACM is secondary to mutation in the desmoplakin (DSP) gene (DSP ACM)

Embodiment 96. The method of any one of embodiments 92-95, wherein the subject is a mammal.

Embodiment 97. The method of any one of embodiments 92-95, wherein the subject is selected from the group consisting of a human, a non-human primate, a canine, a feline, an equine, a bovine, a swine, an avian, and a rodent. Embodiment 98. The method of any one of embodiments 92-95, wherein the subject is a caninc.

Embodiment 99. The method of any one of embodiments 92-95, wherein the subject is a human.

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide comprising:

(a) a first nucleic acid comprising a first transcriptional regulatory element (TRE) and a first coding sequence, and

(b) a second nucleic acid comprising a second TRE and a second coding sequence.

2. The isolated polynucleotide of claim 1 comprising:

(a) the first TRE operatively linked to the first coding sequence, wherein the first TRE is or comprises:

(1) a promoter,

(2) one or more enhancer elements, or

(3) a promoter and one or more enhancer elements; and/or

(b) the second TRE operatively linked to the second coding sequence, wherein the second TRE is or comprises:

(1) a promoter,

(2) one or more enhancer elements, or

(3) a promoter and one or more enhancer elements.

3. The isolated polynucleotide of claim 2, wherein the promoter is or comprises a human albumin (hAlb) promoter or a human alpha 1 -antitrypsin (hAAT) promoter.

4. The isolated polynucleotide of claim 2 or 3, wherein the promoter comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2.

5. The isolated polynucleotide of claim 2 or 3, wherein the promoter comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

6. The isolated polynucleotide of any one of claims 2-5, wherein the one or more enhancer elements each comprise an apolipoprotein E (ApoE) enhancer clement or an alpha-fctoprotcin (AFP) enhancer element.

7. The isolated polynucleotide of claim 6, wherein the one or more enhancer elements comprise at least two ApoE enhancer elements.

8. The isolated polynucleotide of claim 6 or 7, wherein the one or more enhancer elements comprise at least three ApoE enhancer elements.

9. The polynucleotide of any one of claims 2-8, wherein the one or more enhancer elements each comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1.

10. The polynucleotide of any one of claims 2-6, wherein the one or more enhancer elements each comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10.

11. The isolated polynucleotide of any one of the preceding claims, further comprising one or more intron elements.

12. The isolated polynucleotide of claim 11, wherein the one or more intron elements each comprise a P-globin intron element or a MVM intron element.

13. The isolated polynucleotide of claim 12, wherein the P-globin intron element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3.

14. The isolated polynucleotide of claim 12 or 13, wherein the P-globin intron element is 3’ of the first and/or second TRE.

15. The isolated polynucleotide of claim 12, wherein the MVM intron element comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12.

16. The isolated polynucleotide of claim 12 or 15, wherein the MVM intron element is 3’ of the first and/or second TRE.

17. The isolated polynucleotide of any one of claims 2-16, wherein the first TRE comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 27-29.

18. The isolated polynucleotide of any one of claims 2-17, wherein the second TRE comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or 11.

19. The isolated polynucleotide of any one of the preceding claims, wherein the first nucleic acid further comprises a first post-transcriptional regulatory element (PRE).

20. The isolated polynucleotide of any one of the preceding claims, wherein the second nucleic acid further comprises a PRE.

21. The isolated polynucleotide of claim 19 or 20 comprising:

(a) a first PRE comprising:

(1) a poly adenylation sequence,

(2) a WPRE sequence, or (3) a polyadenylation sequence and a WPRE sequence; and/or

(b) a second PRE comprising:

(1) a poly adenylation sequence,

(2) a WPRE sequence, or

(3) a polyadenylation sequence and a WPRE sequence.

22. The isolated polynucleotide of claim 21, wherein the poly adenylation sequence is or comprises a bovine growth hormone polyadenylation (BGHpA) sequence or a simian virus 40 polyadenylation (SV40pA) sequence.

23. The isolated polynucleotide of claim 21 or 22, wherein the poly adenylation sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22 or 23.

24. The isolated polynucleotide of claim 21 or 22, wherein the polyadenylation sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or 9.

25. The isolated polynucleotide of any one of claims 21-24, wherein the WPRE sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

26. The isolated polynucleotide of any one of the preceding claims, further comprising a Kozak consensus sequence 5’ to the first coding sequence and/or a Kozak consensus sequence 5’ to the second coding sequence.

27. The isolated polynucleotide of any one of the preceding claims, wherein the first nucleic acid and the second nucleic acid arc in the same orientation in the polynucleotide, optionally wherein the first nucleic acid and the second nucleic acid are separated by a polycistronic element.

28. The isolated polynucleotide of claim 27, wherein the polycistronic element is an IRES or 2A sequence.

29. The isolated polynucleotide of claim 27 or 28, comprising from 5’ to 3’:

(a) the first TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 27,

(b) the first coding sequence,

(c) the first PRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31,

(d) the second TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30,

(e) the second coding sequence, and

30. The isolated polynucleotide of any one of claims 1-25, wherein the first nucleic acid and the second nucleic acid are in the opposite orientation in the polynucleotide.

3E The isolated polynucleotide of claim 30, comprising from 5’ to 3’: (a) the second PRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23,

(b) the second coding sequence,

(c) the second TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11,

(d) the first TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28,

(e) the first coding sequence, and

32. The isolated polynucleotide of claim 30, comprising from 5’ to 3’:

(a) the second PRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23,

(b) the second coding sequence,

(d) the first TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29,

(e) the first coding sequence, and

33. The isolated polynucleotide of claim 25, comprising from 5’ to 3’:

(b) the second coding sequence,

(d) the first TRE comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity SEQ ID NO: 27,

(e) the first coding sequence, and

34. The isolated polynucleotide of any one of the preceding claims, wherein the first coding sequence and/or the second coding sequence each encode a miRNA, a shRNA, a siRNA, an antisense RNA, a gRNA, an antagomir, a miRNA sponge, an RNA aptazyme, an RNA aptamer, a IncRNA, a ribozyme or a mRNA.

35. The polynucleotide of any one of the preceding claims, wherein the first coding sequence and/or the second coding sequence each encode one or more polypeptides.

36. The isolated polynucleotide of any one of the preceding claims, wherein the first coding sequence and/or the second coding sequence each encode a growth factor or receptor thereof.

37. The isolated polynucleotide of any one of the preceding claims, wherein the first coding sequence encodes a growth factor.

38. The isolated polynucleotide of any one of the preceding claims, wherein the first coding sequence encodes fibroblast growth factor 21 (FGF21).

39. The isolated polynucleotide of any one of the preceding claims, wherein the first coding sequence encodes a canine FGF21, a mouse FGF21, or a human FGF21.

40. The isolated polynucleotide of any one of the preceding claims, wherein the first coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 4-6.

41. The isolated polynucleotide of any one of the preceding claims, wherein the second coding sequence encodes a growth factor receptor.

42. The isolated polynucleotide of any one of the preceding claims, wherein the second coding sequence encodes a transforming growth factor beta receptor 2 (TGPFR2).

43. The isolated polynucleotide of any one of the preceding claims, wherein the second coding sequence encodes a caninc TG0FR2, a mouse TGPFR2, or a human TGPFR2.

44. The isolated polynucleotide of any one of the preceding claims, wherein the second coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-18.

45. The isolated polynucleotide of any one of claims 1-40, wherein the second coding sequence encodes a soluble growth factor receptor.

46. The isolated polynucleotide of claim 45, wherein the second coding sequence encodes a soluble transforming growth factor beta receptor 2 (sTGpFR2), optionally wherein the second coding sequence encodes a canine sTGpFR2, a mouse sTGpFR2, or a human sTGpFR2.

47. The isolated polynucleotide of any one of claims 31-46, wherein the growth factor or receptor thereof is linked to an Fc region, optionally wherein the Fc region is a canine Fc region, an IgG2 Fc region, or an IgGl Fc region.

48. The isolated polynucleotide of any one of claims 45-47, wherein the second coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 33-35.

49. The isolated polynucleotide of any one of claims 36-48, wherein the growth factor or receptor thereof is linked to a signal peptide, optionally wherein the signal peptide is a human TGFPR2 signal peptide or a canine AAT signal peptide.

50. The isolated polynucleotide of claim 49, wherein the growth factor receptor is soluble transforming growth factor beta receptor 2 (sTGpFR2).

51. The isolated polynucleotide of claim 49 or 50, wherein the second coding sequence comprises a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 36-39.

52. The isolated polynucleotide of any one of claims 36-51 , wherein the growth factor receptor comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 40-43.

53. The isolated polynucleotide of any one of the preceding claims, wherein the first coding sequence and/or the second coding sequence does not encode a functional antibody.

54. An isolated polynucleotide comprising:

(a) a first nucleic acid comprising:

(1) a first coding sequence encoding a first growth factor or receptor thereof, and

(2) a first transcriptional regulatory element (TRE); and

(b) a second nucleic acid comprising:

(1) a second TRE, and

(2) a second coding sequence encoding a second growth factor or receptor thereof.

55. The polynucleotide of claim 54, wherein the first TRE and the second TRE are the same or different.

56. The isolated polynucleotide of claim 54 or 55, wherein the first TRE and/or the second TRE arc liver- specific.

57. The isolated polynucleotide of claim 56, wherein the liver- specific TRE comprises one or more elements comprising an ApoA-I promoter, an ApoA-II promoter, an ApoA-IV promoter, an ApoB promoter, an ApoC-I promoter, an ApoC-II promoter, an ApoC-III promoter, an ApoE promoter, an albumin promoter, an a-fetoprotein promoter, a phosphoenolpyruvate carboxykinase 1 (PCK1) promoter, a phosphoenolpyruvate carboxykinase 2 (PCK2) promoter, a transthyretin (TTR) promoter, an a-antitrypsin (AAT or SERPINA1) promoter, a hemopexin promoter, an alcohol dehydrogenase 6 promoter, a cholesterol 7alpha-hydroxylase promoter, a factor IX promoter, or an a-microglobulin promoter.

58. The isolated polynucleotide of claim 54 or 55, wherein the first TRE and/or the second TRE are muscle-specific.

59. The isolated polynucleotide of claim 58, wherein the muscle- specific TRE comprises one or more elements comprising a human skeletal muscle a-actin (HSA) promoter, a muscle creatine kinase (MCK) promoter, a MHCK7 promoter, a dMCK promoter, a tMCK promoter, a CK6 promoter, a CK8 promoter, a CK8e promoter, a human desmin (DES) promoter or variant thereof, a cardiac troponin T (cTnT) promoter, a myosin light-chain (MLC2v) promoter, a human a-myosin heavy chain gene (aMHC) promoter, a MLC promoter, a human troponin I (TNNI1) promoter, a AUSEx3 promoter, a SPcA5-12 promoter, a SP-301 promoter, a MH promoter, or a Sk- CRM4/DES promoter.

60. The isolated polynucleotide of any one of claims 54-59, wherein the first TRE and/or second TRE each comprise an intron element.

61. The isolated polynucleotide of any one of claims 54-60, wherein the polynucleotide further comprises a first post-transcriptional regulatory element (PRE), optionally wherein the first PRE is 5’ of the first coding sequence.

62. The isolated polynucleotide of any one of claims 54-60, wherein the polynucleotide further comprises a second PRE, optionally wherein the second PRE is 3’ of the second coding sequence.

63. The isolated polynucleotide of claim 61 or 62, wherein the first PRE and/or second PRE each comprise a polyadenylation sequence and/or a WPRE sequence.

64. The isolated polynucleotide of any one of claims 54-63, wherein the first coding sequence and/or second coding sequence each encode a soluble growth factor receptor.

65. The isolated polynucleotide of any one of claims 54-64, wherein the first and/or second growth factor or receptor thereof is or comprises a soluble transforming growth factor beta receptor 2 (STG0FR2).

66. The isolated polynucleotide of claim 65, wherein the STGPFR2 is linked to an Fc region.

67. The isolated polynucleotide of any one of claims 54-66, wherein the first and/or second growth factor or receptor thereof is linked to a signal peptide.

68. The isolated polynucleotide of any one of claims 54-67, wherein the first coding sequence and/or second coding sequence comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-18 or 33-39.

69. The isolated polynucleotide of any one of claims 54-64 wherein the first and/or second growth factor or receptor thereof is or comprises a fibroblast growth factor 21 (FGF21).

70. The isolated polynucleotide of any one of claims 54-69, wherein the first coding sequence and/or second coding sequence comprise a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 4-6.

71. An isolated polynucleotide comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 44-61.

72. A polynucleotide that is the complement of the isolated polynucleotide of any one of the preceding claims.

73. A polynucleotide that is the reverse complement of the isolated polynucleotide of any one of claims 1-71.

74. A vector comprising the isolated polynucleotide of any one of claims 1-71 or the polynucleotide of claim 72 or 73.

75. The vector of claim 74, wherein the vector is or comprises a plasmid, a viral vector, or a DNA minimal vector.

76. The vector of claim 74 or 75, wherein the vector is or comprises is an expression vector.

77. The vector of claim 75 or 76, wherein the vector is or comprises an adenoviral vector, an adeno-associated virus (AAV) vector, or a lentiviral vector.

78. The vector of any one of claims 74-77, wherein the vector is an AAV vector.

79. A recombinant adeno-associated virus (rAAV) genome comprising the isolated polynucleotide of any one of claims 1-71 or the polynucleotide of claim 72 or 73.

80. The rAAV genome of claim 79, further comprising a 5’ inverted terminal repeat (5’ ITR) nucleotide sequence, and a 3’ inverted terminal repeat (3’ ITR) nucleotide sequence.

81. The rAAV genome of claim 80, wherein:

(a) the 5’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24, and/or

(b) the 3’ ITR nucleotide sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25 or 26.

82. The rAAV genome of any one of claims 79-81, comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 62-68.

83. An rAAV particle comprising:

(a) the rAAV genome of any one of claims 79-82, and

(b) an AAV capsid protein.

84. The rAAV particle of claim 83, wherein the AAV capsid protein is derived from a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.l, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, or AAV5 capsid protein, or an engineered variant thereof.

85. The rAAV particle of claim 83 or 84, wherein the AAV capsid protein comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 69, 70, and/or 71.

86. A pharmaceutical composition comprising the isolated polynucleotide of any one of claims 1-71, the polynucleotide of claim 72 or 73, the vector of any one of claims 74-78, the rAAV genome of any one of claims 79-82, or the rAAV particle of any one of claims 83-85.

87. The isolated polynucleotide of any one of claims 1-71, the polynucleotide of claim 72 or 73, the vector of any one of claims 74-78, the rAAV genome of any one of claims 79-82, or the rAAV particle of any one of claims 83-85, or the pharmaceutical composition of claim 86, for use in medicine, for use as therapy, or for use as a medicament.

88. A packaging system for preparation of an rAAV, wherein the packaging system comprises:

(a) a first nucleotide sequence encoding one or more AAV Rep proteins;

(b) a second nucleotide sequence encoding an AAV capsid protein; and

(c) a third nucleotide sequence comprising the rAAV genome sequence of any one of claims 79-82.

89. The packaging system of claim 88, wherein the packaging system comprises a first vector comprising the first nucleotide sequence and the second nucleotide sequence, and a second vector comprising the third nucleotide sequence.

90. The packaging system of claim 89, further comprising a fourth nucleotide sequence comprising one or more helper virus genes, optionally wherein the fourth nucleotide sequence is comprised within a third vector.

91. The packaging system of claim 90, wherein the fourth nucleotide sequence comprises one or more genes from a virus comprising an adenovirus, a herpesvirus, a vaccinia virus, and a cytomegalovirus (CMV).

92. The packaging system of claim 90 or 91, wherein the first vector, second vector, and/or the third vector is a plasmid.

93. A method for recombinant preparation of an rAAV particle, the method comprising introducing the packaging system of any one of claims 83-87 into a cell under conditions whereby the rAAV particle is produced.

94. A method comprising introducing into a cell:

(a) the polynucleotide of any one of claims 1-71,

(b) the polynucleotide of claim 72 or 73

(c) the vector of any one of claims 74-78,

(d) the rAAV genome of any one of claims 79-82,

(e) the rAAV particle of any one of claims 83-85, or

(f) the pharmaceutical composition of claim 86.

95. A method of expressing a first coding sequence and a second coding sequence in a cell, comprising introducing into the cell:

(a) the polynucleotide of any one of claims 1-71,

(b) the polynucleotide of claim 72 or 73

(c) the vector of any one of claims 74-78,

(d) the rAAV genome of any one of claims 79-82,

(e) the rAAV particle of any one of claims 83-85, or

(f) the pharmaceutical composition of claim 86.

96. A method of expressing a first coding sequence and a second coding sequence in a subject, comprising administering to the subject an effective amount of:

(a) the polynucleotide of any one of claims 1-71,

(b) the polynucleotide of claim 72 or 73

(c) the vector of any one of claims 74-78,

(d) the rAAV genome of any one of claims 79-82,

(e) the rAAV particle of any one of claims 83-85, or

(f) the pharmaceutical composition of claim 86.

97. A method comprising introducing the isolated polynucleotide of any one of claims 1-71 , the polynucleotide of claim 72 or 73, the vector of any one of claims 74-78, the rAAV genome of any one of claims 79-82, the rAAV particle of any one of claims 83-85, or the pharmaceutical composition of claim 86, wherein:

98. The method of claim 97, wherein the method is a method of treatment of a subject.

99. The method of claim 97 or 98, wherein the introducing comprises administering to the subject a therapeutically effective amount of the isolated polynucleotide of any one of claims 1- 71, the polynucleotide of claim 72 or 73, the vector of any one of claims 74-78, the rAAV genome of any one of claims 79-82, the rAAV particle of any one of claims 83-85, or the pharmaceutical composition of claim 86.

100. The method of any one of claims 97-99, wherein the method is a method of treating arrhythmogenic cardiomyopathy (ACM).

101. The method of claim 100, wherein the ACM is arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), or biventricular arrhythmogenic cardiomyopathy.

102. The method of claim 100 or 101, wherein the ACM is ARVC.

103. The method of any one of claims 101-102, wherein the ACM is secondary to a mutation in a desmoplakin (DSP) gene (DSP ACM).

104. The method of any one of claims 98-103, wherein the subject is a mammal.

105. The method of any one of claims 98- 104, wherein the subject is a human, a non-human primate, a caninc, a feline, an equine, a bovine, a swine, an avian, or a rodent.

106. The method of any one of claims 98-105, wherein the subject is a canine.

107. The method of any one of claims 98-105, wherein the subject is a human.

108. Use of the isolated polynucleotide of any one of claims 1-71, the polynucleotide of claim

72 or 73, the vector of any one of claims 74-78, the rAAV genome of any one of claims 79-82, the rAAV particle of any one of claims 83-85, or the pharmaceutical composition of claim 86 in the manufacture of a medicament.

109. Use of the isolated polynucleotide of any one of claims 1-71, the polynucleotide of claim 72 or 73, the vector of any one of claims 74-78, the rAAV genome of any one of claims 79-82, the rAAV particle of any one of claims 83-85, or the pharmaceutical composition of claim 86 in the manufacture of a medicament for treatment of ACM.

110. The method of claim 109, wherein the ACM is arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), or biventricular arrhythmogenic cardiomyopathy.

111. The method of claim 109 or 110, wherein the ACM is ARVC.

112. The method of any one of claims 109- 111, wherein the ACM is secondary to a mutation in a desmoplakin (DSP) gene (DSP ACM).

113. The isolated polynucleotide of any one of claims 1-71, the polynucleotide of claim 72 or 73, the vector of any one of claims 74-78, the rAAV genome of any one of claims 79-82, the rAAV particle of any one of claims 83-85, or the pharmaceutical composition of claim 86 for use as a medicament.

114. The isolated polynucleotide of any one of claims 1-71 , the polynucleotide of claim 72 or 73, the vector of any one of claims 74-78, the rAAV genome of any one of claims 79-82, the rAAV particle of any one of claims 83-85, or the pharmaceutical composition of claim 86 for use as a medicament as a treatment for ACM.

115. The method of claim 114, wherein the ACM is arrhythmogenic right ventricular cardiomyopathy (ARVC), arrhythmogenic left ventricular cardiomyopathy (ALVC), or biventricular arrhythmogenic cardiomyopathy.

116. The method of claim 114 or 115, wherein the ACM is ARVC.

117. The method of any one of claims 114- 116, wherein the ACM is secondary to a mutation in a desmoplakin (DSP) gene (DSP ACM).

118. A kit comprising:

(a) the polynucleotide of any one of claims 1-71,

(b) the polynucleotide of claim 72 or 73

(c) the vector of any one of claims 74-78,

(d) the rAAV genome of any one of claims 79-82,

(e) the rAAV particle of any one of claims 83-85, or

(f) the pharmaceutical composition of claim 86.