Gene therapy for arrhythmogenic right ventricular cardiomyopathy

Abstract

Compositions and methods for preventing and treating cardiac arrhythmias. A method for preventing or treating arrhythmogenic right ventricular myopathy (ARVC), comprising administering to a subject in need thereof a composition comprising a prophylactically or therapeutically effective amount of the plakophilin-2 (PKP2) gene. The composition further comprises an adenovirus-associated vector (AAV) for delivering the PKP-2 gene. In an embodiment, the present invention provides that the AAV is a cardioactive AAV serotype and contains a cardiac-specific promoter. A method for treating a cardiovascular disease characterized by abnormal cardiac intercellular junction complexes, comprising administering to a subject in need thereof a composition comprising a prophylactically or therapeutically effective amount of the plakophilin-2 (PKP2) gene.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 173,527, filed April 12, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Incorporation by reference of sequence listing The contents of the text file named "24978-0711_SeqList_ST25", created on April 12, 2022 and measuring 89KB, are incorporated by reference in their entirety into this specification.

[0003] government funding This invention was made with Government support under No. HL142251 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0004] Incorporation by reference of sequence listing The contents of a text file named "XXXX", created on XXX and measuring XXXKB in size, are incorporated herein by reference in their entirety.

[0005] The present invention relates to gene therapy for arrhythmogenic right ventricular cardiomyopathy (ARVC). [Background technology]

[0006] To date, there are no effective treatments or cures for arrhythmogenic right ventricular cardiomyopathy (ARVC), as well as randomized clinical trials of ARVC-specific treatment modalities, screening regimens, or medications (1). Current approaches are targeted at palliation of symptoms and are centered on lifestyle modifications (avoiding competitive sports that may cause sudden cardiac death) and pharmacological interventions (antiarrhythmic drugs, beta-blockers) (2, 3). These approaches may progress to more invasive procedures, including implantable cardioverter defibrillators (ICDs), cardiac catheter ablation, or heart transplantation, if patients become unresponsive or intolerant to pharmacological therapy (2, 3). ICDs have frequent device / lead-related complications, catheter ablation is prone to recurrence due to the development of new arrhythmic sites, and heart transplantation has a 23% mortality rate at 10 years after surgery (3). These factors highlight the critical need to identify new preventive and therapeutic strategies that can target the molecular triggers of ARVC (cell-cell junction disruption) as a means to prevent or halt disease progression. Summary of the Invention

[0007] The present disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising, in a 5' to 3' direction, a) a first AAV ITR sequence; b) a promoter sequence; c) a transgene nucleic acid molecule, the transgene nucleic acid molecule comprising a nucleic acid sequence encoding a plakophilin-2 (PKP2) polypeptide; d) a post-transcriptional regulatory element; e) a polyA sequence; and f) a second AAV ITR sequence.

[0008] The disclosure provides an rAAV vector comprising a nucleic acid sequence set forth in SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:30.

[0009] In some embodiments, the PKP2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:13.

[0010] In some embodiments, the nucleic acid sequence encoding the PKP2 polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:14.

[0011] In some embodiments, the first AAV ITR sequence comprises a nucleic acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:25. In some embodiments, the second AAV ITR sequence comprises a nucleic acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:25.

[0012] In some aspects, the promoter sequence is a cardiac-specific promoter sequence.

[0013] In some aspects, the promoter sequence comprises a Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, an H1 promoter, a CAG promoter, a hybrid chicken beta-actin promoter, a MeCP2 promoter, an EF1 promoter, a ubiquitous chicken beta-actin hybrid (CBh) promoter, a U1a promoter, a U1b promoter, a MeCP2 promoter, a MeP418 promoter, a MeP426 promoter, a minimal MeCP2 promoter, a VMD2 promoter, a mRho promoter, an EFla promoter, a Ubc promoter, a human beta-actin promoter, a TRE promoter, an Ac5 promoter, a polyhedrin promoter, a CaMKIIa promoter, a Gal1 promoter, a TEF1 promoter, a GDS promoter, an ADH1 promoter, a Ubi promoter, or an alpha-1-antitrypsin (hAAT) promoter. In some embodiments, the promoter sequence comprises a cardiac troponin T (cTnT) promoter sequence. In some embodiments, the cTnT promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO:2.

[0014] In some embodiments, the poly A sequence comprises a rabbit beta-globin poly A sequence. In some embodiments, the rabbit beta-globin poly A sequence comprises the nucleic acid sequence set forth in SEQ ID NO:6.

[0015] In some aspects, the post-transcriptional regulatory element is an oPRE post-transcriptional regulatory element. In some aspects, the oPRE post-transcriptional regulatory element comprises the nucleic acid sequence set forth in SEQ ID NO:5, SEQ ID NO:27, or SEQ ID NO:28. The disclosure provides an rAAV vector according to any one of the preceding claims, comprising, in a 5' to 3' direction: a) a first AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:7; b) a promoter sequence comprising the nucleic acid sequence set forth in SEQ ID NO:2; c) a transgene nucleic acid molecule, wherein the transgene nucleic acid molecule comprises a nucleic acid sequence encoding a PKP2 polypeptide, wherein the nucleic acid sequence encoding the PKP2 polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO:4; d) a post-transcriptional regulatory element comprising the nucleic acid sequence set forth in SEQ ID NO:5; e) a polyA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:6; and f) a second AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:8.

[0016] The present disclosure provides an rAAV viral vector comprising: (i) an AAV capsid protein; and (ii) an rAAV vector described in any one of the preceding claims.

[0017] In some embodiments, the AAV capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein, or an AAVrh10 capsid protein. In some embodiments, the AAV capsid protein is an AAV9 or AAVrh10 capsid protein.

[0018] The present disclosure provides a pharmaceutical composition comprising: a) an rAAV viral vector of any embodiment of the present disclosure and at least one pharma- ceutically acceptable excipient and / or additive.

[0019] The present disclosure provides a method for treating a subject having a disease and / or disorder in which the PKP2 gene is involved, the method comprising administering to the subject at least one therapeutically effective amount of an rAAV viral vector of any embodiment of the present disclosure or a pharmaceutical composition of any embodiment of the present disclosure.

[0020] In some aspects, the disease and / or disorder in which the PKP2 gene is implicated is a cardiovascular disease characterized by abnormal cardiac intercellular junction complexes.

[0021] In some aspects, the disease and / or disorder in which the PKP2 gene is implicated is arrhythmogenic right ventricular cardiomyopathy (ARVC).

[0022] In some embodiments, the effective amount improves electrical and structural cardiac integrity in the subject. In some embodiments, the effective amount rescues and reconstructs intercellular junction proteins in the subject. In some embodiments, the effective amount improves cardiac function in the subject. In some embodiments, the effective amount preserves electrical and structural integrity in the subject and prevents ARVC.

[0023] In some embodiments, the rAAV viral vector or pharmaceutical composition comprises about 1.0×10 12 vg / kg ~ approx. 2.5×10 14 In some embodiments, the rAAV viral vector or pharmaceutical composition is administered to a subject at a dose ranging from about 1.0×10 12 vg / kg ~ approx. 5.0×10 13 The subject is administered a dose in the range of 100 to 200 mg / kg.

[0024] In some aspects, the rAAV viral vector or pharmaceutical composition is administered to a subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraaurally, intraocularly or periocularly, orally, rectally, transmucosally, by inhalation, transdermal, parenterally, subcutaneously, intradermally, intramuscularly, intracisternally, intraneurally, intrathoracically, topically, intralymphatically, intracisternally, or intraneurally.

[0025] In some aspects, the rAAV viral vector of any one of the embodiments of the present disclosure or the pharmaceutical composition of any of the embodiments of the present disclosure for use in treating a disease and / or disorder involving the PKP2 gene in a subject in need thereof.

[0026] In some aspects, the disease and / or disorder in which the PKP2 gene is involved is ARVC.

[0027] In some embodiments, the rAAV viral vector or pharmaceutical composition comprises about 1.0×10 12 vg / kg ~ approx. 2.5×10 14 In some embodiments, the rAAV viral vector or pharmaceutical composition is for administration to a subject at a dose ranging from about 1.0×1012 vg / kg ~ approx. 5.0×10 13 It is intended for administration to a subject at doses in the range of vg / kg.

[0028] In some embodiments, the rAAV viral vector or pharmaceutical composition is for intravenous, intrathecal, intracerebral, intraventricular, intranasal, intratracheal, intraaural, intraocular or periocular, oral, rectal, transmucosal, inhalation, transdermal, parenteral, subcutaneous, intradermal, intramuscular, intracisternal, intraneural, intrathoracic, topical, intralymphatic, intracisternal, or intraneural administration to a subject. In some embodiments, the rAAV viral vector or pharmaceutical composition is for intravenous administration. [Brief description of the drawings]

[0029] [Figure 1A] It shows that adenovirus technology can stably express PKP2 protein. Figure 1A shows Western blot analysis of mutant (MUT) and wild-type (WT) PKP2 protein after adenovirus transduction of neonatal cardiomyocytes at the indicated multiplicity of infection (MOI). FLAG antibody recognizes PKP2, and GAPDH serves as a loading control. [Figure 1B] We show that the adenovirus technique can stably express PKP2 protein. Figure 1B shows a schematic diagram of early intraperitoneal injection of AAV9 PKP2 at postnatal day 2 (P2) in wild-type (WT) mice, and analysis of the heart 4 weeks later. [Figure 1C] This shows that adenovirus technology can stably express PKP2 protein. Figure 1C shows Western blot analysis of PKP2 protein after AAV9 PKP2 injection on postnatal day 2 (P2). PKP2 antibody recognizes endogenous and transduced PKP2, FLAG antibody recognizes only transduced PKP2, and GAPDH serves as a loading control. [Diagram 2]We show that increasing doses of PKP2 protein in vitro remodel cardiac cell-cell junctions. Western blot analysis of desmosome (PKP2, DSP, DSG2, JUP), fascia-adherens (N-Cad), and gap junction (CX43) proteins after wild-type (WT) or mutant (MUT) PKP2 adenoviral transduction of PKP2 homozygous mutant neonatal cardiomyocytes (Hom CM). GAPDH serves as a loading control. [Figure 3A] Figure 3A shows that early AAV9 PKP2 administration restores cardiac cell-cell junctions and improves cardiac morphology. Figure 3A shows a schematic diagram of early intraperitoneal injection of AAV9 PKP2 at postnatal day 2 (P2) in PKP2 homozygous mutant (PKP2 Hom) mice and analysis of the hearts 4 weeks later. [Figure 3B] Figure 3B shows that early AAV9 PKP2 administration restores cardiac cell-cell junctions and improves cardiac morphology. Figure 3B shows Western blot analysis of control (Ctrl), AAV9-PKP2-treated PKP2 Hom hearts, and PKP2 Hom hearts without viral treatment for desmosomal proteins (PKP2, DSP, DSG2, JUP), adhesion fascia protein (N-CAD), and gap junction protein (CX43). GAPDH serves as a loading control. [Figure 3C] Figure 3C shows that early AAV9 PKP2 administration restores cardiac cell-cell junctions and improves cardiac morphology. Heart weight (HW) to body weight (BW) ratio analysis of control (Ctrl), untreated PKP2 homozygous mutant (Hom) hearts, and PKP2 Hom hearts treated with AAV9-PKP2. Median with standard deviation. n=5Ctrl, n=5Hom, n=3Hom-AAV9 PKP2. One-way ANOVA with Tukey post-hoc test. **, p<0.01. [Figure 4A]Early AAV9 PKP2 administration prevents cardiac mechanical and electrical dysfunction. Figure 4A shows representative short-axis cardiac magnetic resonance images of control (Ctrl), AAV9 GFP-treated PKP2 homozygous mutant (Hom), and AAV9 PKP2-treated PKP2 Hom hearts. [Figure 4B] Figure 4B shows that early AAV9 PKP2 administration prevents cardiac mechanical and electrical dysfunction. Figure 4B shows quantification of heart rate, ejection fraction (EF), end-diastolic volume (EDV), and end-systolic volume (ESV) in the left ventricle (LV) and right ventricle (RV). Median with standard deviation. n=5 Ctrl, n=5 Hom-AAV9 GFP, n=6 Hom-AAV9 PKP2. Two-way ANOVA with Tukey post-hoc test. ****, p<0.0001. **, p<0.01. *, p<0.05. [Figure 4C] Early AAV9 PKP2 administration prevents cardiac mechanical and electrical dysfunction. Figure 4C shows representative composite surface electrocardiograms of control (Ctrl), AAV9 GFP-treated PKP2 homozygous mutant (Hom), and AAV9 PKP2-treated PKP2 Hom hearts. Scale bar = 20 ms. [Figure 4D] Figure 4D shows that early AAV9 PKP2 administration prevents cardiac mechanical and electrical dysfunction. Quantification of heart rate, PR interval, and QRS interval. Median with standard deviation. n=5 Ctrl, n=5 Hom-AAV9 GFP, n=4 Hom-AAV9 PKP2. One-way ANOVA with Tukey post-hoc test. *, p<0.05. [Figure 4E] Early AAV9 PKP2 administration prevents cardiac mechanical and electrical dysfunction. Figure 4E shows representative surface electrocardiograms showing electrical activity over time. Premature ventricular contractions (PVCs) are indicated by arrows. Quantification of the percentage of mice with PVCs in each condition. [Figure 5A]We show that early AAV9 PKP2 administration preserves cardiac morphology and prevents pathogenic tissue remodeling. Figure 5A shows representative cardiac histological sections from control (Ctrl), AAV9 GFP-treated PKP2 homozygous mutant (Hom), and AAV9 PKP2-treated PKP2 Hom hearts stained with hematoxylin and eosin. Scale bar = 1 mm. [Figure 5B] We show that early AAV9 PKP2 administration preserves cardiac morphology and prevents pathogenic tissue remodeling. Figure 5B shows representative cardiac histological sections from Ctrl, Hom-AAV9 GFP, and Hom-AAV9 PKP2 in the left ventricle (LV) and right ventricle (RV) stained with Masson's Trichrome for fibrosis. Scale bar = 100 μM. [Figure 5C] Figure 5C shows that early AAV9 PKP2 administration preserves cardiac morphology and prevents pathogenic tissue remodeling. RNA-based reverse transcription quantitative PCR analysis of the profibrotic gene collagen type I alpha 1 (Col1a1) from Ctrl, Hom-AAV9 GFP, and Hom-AAV9 PKP2 hearts. Median with standard deviation. n=4 Ctrl, n=4 Hom-AAV9 GFP, n=6 Hom-AAV9 PKP2. One-way ANOVA with Tukey post-hoc test. *, p<0.05. [Figure 6A] Figure 6A shows that early AAV9 PKP2 administration improves survival and provides durable cardioprotection. Figure 6A shows survival analysis of Ctrl, PKP2 homozygous mutant (Hom), and PKP2 Hom-AAV9 PKP2 mice. [Figure 6B] Figure 6B shows representative short-axis cardiac magnetic resonance images of 6-month-old Ctrl and PKP2 Hom-AAV9 PKP2 hearts, showing that early AAV9 PKP2 administration improves survival and provides durable cardioprotection. [Figure 6C]Early AAV9 PKP2 administration improves survival and provides durable cardioprotection. Figure 6C shows left ventricular (LV) and right ventricular (RV) ejection fraction (EF) quantification at 6 months of age. Median with standard deviation. n=2 Ctrl, n=4 Hom-AAV9 PKP2. Two-way ANOVA with Tukey post-hoc test. [Figure 6D] Early AAV9 PKP2 administration improves survival and provides durable cardioprotection. Figure 6D shows representative composite surface electrocardiograms of 6-month-old Ctrl and PKP2 Hom-AAV9 PKP2 hearts. Scale bar = 10 ms. [Figure 6E] Figure 6E shows that early AAV9 PKP2 administration improves survival and provides durable cardioprotection. Quantification of heart rate, PR interval, and QRS interval. Median with standard deviation. n=2 Ctrl, n=4 Hom-AAV9 PKP2. Unpaired t-test. [Figure 6F] Figure 6F shows that early AAV9 PKP2 administration improves survival and provides durable cardioprotection. Western blot analysis of Ctrl and PKP2 Hom-AAV9 PKP2 hearts for desmosomal proteins (PKP2, DSP, DSG2, JUP), adhesion fascia protein (N-CAD), and gap junction protein (CX43). GAPDH serves as a loading control. [Figure 6G] Figure 6G shows that early AAV9 PKP2 administration improves survival and provides durable cardioprotection. Serum analysis of alkaline phosphatase (ALP) and alanine aminotransferase (ALT) liver enzyme levels. Median with standard deviation. n=5 Ctrl, n=6 Hom-AAV9 PKP2. Unpaired t-test. [Figure 7A] Late administration of AAV9 PKP2 improves cell junction protein levels and mechanistic function. Figure 7A shows a schematic of retro-orbital injection of AAV9 PKP2 in PKP2 homozygous mutant (PKP2 Hom) mice (disease hallmarks present) at 4 weeks and cardiac analysis 2 weeks later. [Figure 7B] Late administration of AAV9 PKP2 improves cell-cell junction protein levels and mechanical function. Figure 7B shows Western blot analysis of Ctrl, PKP2 Hom-AAV9 GFP, and PKP2 Hom-AAV9 PKP2 hearts for desmosomal proteins (PKP2, DSP, DSG2, JUP), adhesion fascia protein (N-CAD), and gap junction protein (CX43). GAPDH serves as a loading control. [Figure 7C] Figure 7C shows representative short-axis cardiac magnetic resonance images of PKP2 Hom-AAV9 GFP and PKP2 Hom-AAV9 PKP2 hearts at both end diastole and end systole 2 weeks after injection. [Figure 7D] Figure 7D shows that late administration of AAV9 PKP2 improves cell-cell junction protein levels and mechanical function. Left ventricular (LV) and right ventricular (RV) ejection fraction (EF) quantification 2 weeks after injection. Median with standard deviation. n=6 Hom-AAV9 GFP, n=6 Hom-AAV9 PKP2. Two-way ANOVA with Tukey post-hoc test. **, p<0.01. *, p<0.05. [Figure 7E] Figure 7E shows survival analysis of Ctrl, PKP2 Hom-AAV9 GFP, and PKP2 Hom-AAV9 PKP2 mice. Log-rank test. ***, p<0.001. [Figure 8A]We show that adenovirus technology can stably express human PKP2 protein in adult mouse heart and avoid ARVC disease outcomes in PKP2 Hom mice. Western blot analysis of AAV9-hPKP2 or AAVrh10-PKP2 delivered to the heart of adult wild-type control mice. Study 1 with AAV9-hPKP2 had a duration of 10 days before evaluating expression in mouse heart. Study 2 with AAVrh10-hPKP2 had a duration of 21 days before evaluating expression in mouse heart and liver. Expression of PKP2 and DSP was evaluated using beta-actin and GAPDH, which served as a loading control. [Figure 8B] We show that adenovirus technology can stably express human PKP2 protein in adult mouse hearts and avoid ARVC disease outcomes in PKP2 Hom mice. Four-week survival curves after early administration (postnatal day 2 (P2)) of formulations and hPKP2 (via AAV9 and AAVrhlO) in PKP2 Hom mice are shown (deipct). [Figure 8C] We show that adenovirus technology can stably express human PKP2 protein in adult mouse hearts and avoid ARVC disease outcomes in PKP2 Hom mice. We show surface ECG analysis of formulations and hPKP2 (via AAV9 and AAVrhlO) and bar graph analysis of ectopic beats / premature ventricular contractions (PVCs) in PKP2 Hom mice after early administration (P2). [Figure 8D] We show that adenovirus technology can stably express human PKP2 protein in adult mouse hearts and avoid ARVC disease outcomes in PKP2 Hom mice. Representative cardiac short-axis views of magnetic resonance images at end diastole from wild-type control untreated mice, formulation-treated PKP2 Hom, AAV9-hPKP2-treated PKP2 Hom, and AAVrh10-hPKP2-treated PKP2 Hom are shown. The dimensions of the right ventricle (RV) and left ventricle (LV) of each group at end diastole are outlined. [Figure 8E]We show that adenovirus technology can stably express human PKP2 protein in adult mouse hearts and avoid ARVC disease outcomes in PKP2 Hom mice. Western blot analysis of PKP2 and cell-cell junction proteins (desmoplakin (DSP), desmoglein-2 (DSG2), connexin 43 (Cx43), N-cadherin (NCAD), plakoglobin (JUP)) in hearts from wild-type control untreated mice, formulation-treated PKP2 Hom, AAV9-hPKP2-treated PKP2 Hom, and AAVrh10-hPKP2-treated PKP2 Hom is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] All publications, patents, and patent applications mentioned in this application are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0031] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the invention, exemplary methods, devices, and materials are described herein.

[0032] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques may be implemented in accordance with the teachings of Molecular Cloning: A Laboratory Manual, 2 nded. (Sambrook et al., 1989), Oligonucleotide Synthesis (MJ Gait, ed., 1984), Animal Cell Culture (RIFreshney, ed., 1987), Methods in Enzymology (Academic Press, Inc.), Current Protocols in Molecular Biology (FMAusubel et al., eds., 1987, and regular updates), PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994), Remington, The Science and Practice of Pharmacy, 20 th ed., (Lippincott, Williams & Wilkins 2003), and Remington, The Science and Practice of Pharmacy, 22. th ed., (Pharmaceutical Press and Philadelphia College of Pharmacy at University of the Sciences 2012), among others.

[0033] The present invention provides compositions and methods for preventing and treating cardiac arrhythmias. In some aspects, the present invention provides a method for preventing or treating arrhythmogenic right ventricular cardiomyopathy (ARVC), comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a composition comprising the plakophilin-2 (PKP2) gene.

[0034] In some embodiments, the present invention provides that the composition further comprises an adenovirus-associated vector (AAV) for delivering the PKP2 gene. In some embodiments, the present invention provides that the AAV is a cardioactive AAV serotype and contains a cardiac-specific promoter.

[0035] The present disclosure provides, inter alia, isolated polynucleotides, recombinant adeno-associated virus (rAAV) vectors, and rAAV viral vectors, including transgene nucleic acid molecules that include a nucleic acid sequence encoding a plakophilin-2 (PKP2) polypeptide. The present disclosure also provides methods for producing these isolated polynucleotides, rAAV vectors, and rAAV viral vectors, and their use to deliver transgenes to treat or prevent diseases or disorders, including diseases associated with loss and / or improper function of the PKP2 gene.

[0036] The present disclosure provides an rAAV vector or rAAV viral vector comprising a nucleic acid sequence encoding plakophilin-2 (PKP2) for scaffolding and remodeling cardiac intercellular junction complexes and alleviating both electrical and structural abnormalities in ARVC. Published studies have not demonstrated the sufficiency of a single gene (desmosome or otherwise) for remodeling cardiac intercellular junction complexes and preventing the development of ARVC disease. Among other things, the present disclosure demonstrates that AAV-mediated delivery of PKP2 in neonatal cardiomyocytes carrying prevalent human PKP2 mutations can rescue the loss of cardiac intercellular junction proteins that drive cardiac electrical and structural abnormalities in this model. Furthermore, the present disclosure shows that AAV-mediated delivery of PKP2 in PKP2 mutant neonatal cardiomyocytes carrying prevalent human PKP2 mutations can similarly rescue the loss of cardiac intercellular junction proteins, suggesting that loss of PKP2 protein dosage is a key driver of cardiac structural and electrical defects in this model. The present disclosure further demonstrates that early-stage administration of AAV-mediated PKP2 gene therapy in neonatal mice carrying this widespread human PKP2 mutation was sufficient to prevent postnatal degradation of cardiac intercellular junction complexes, prevent the onset of adult ARVC disease (preserving cardiac electrical and mechanical function), and significantly improve mouse lifespan.The present disclosure further shows that late-stage administration of adeno-associated virus-mediated PKP2 gene therapy in adult mice carrying this widespread human PKP2 mutation was sufficient to rescue and reconstruct intercellular junction proteins, improve cardiac function, and prevent death.This data all together highlights that PKP2 serves as an efficient molecular scaffold capable of reconstructing cardiac intercellular junctions, and that PKP2 gene therapy, when administered prophylactically or at a later stage in disease progression, can serve as a valuable therapeutic option for ARVC patients.

[0037] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae. Adeno-associated viruses are single-stranded DNA viruses that grow intracellularly, with certain functions provided by a coinfecting helper virus. General information and discussion of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol1, pp.169-228, and Berns, 1990, Virology, pp.1743-1764, Raven Press, (New York). It is well known that the various serotypes are very closely related, both structurally and functionally, even at the genetic level, so it is fully expected that the same principles described in these discussions will be applicable to additional AAV serotypes characterized after the publication date of the discussions. (See, e.g., Blacklowe, 1988, pp. 165-174 in Parvoviruses and Human Disease, J.R. Puttson, ed., and Rose, Comprehensive Virology 3:1-61 (1974).) For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes, and they all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by extensive cross-hybridization between serotypes along the length of the genome, and heteroduplex analysis revealing the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes can infect cells from a variety of tissue types. At least eleven consecutive AAV serotypes are known in the art.Non-limiting exemplary serotypes useful in the methods disclosed herein include any of eleven serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAVPHP.B. AAV particles comprise, consist essentially of, or consist of three major viral proteins: VP1, VP2, and VP3. In some embodiments, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, or AAVrh10.

[0038] Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, and AAVrh10). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids that contain the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03, AAV-KP-1 (described in detail in Kerun et al. JCI Insight, 2019;4(22):e131610), and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018;26(1):289-303).

[0039] Structure and function of AAV AAV is a replication-deficient parvovirus whose single-stranded DNA genome contains two 145-nucleotide inverted terminal repeats (ITRs) and is approximately 4.7 kb in length. There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829, the AAV-5 genome is provided in GenBank Accession No. AF085716, the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively, and the AAV-9 genome is described in Gao et al. al. J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928. U.S. Pat. No. 9,434,928 also provides the sequences of capsid proteins and self-complementary genomes. In one embodiment, the AAV genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), joined by differential splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately responsible for replicating the viral genome.

[0040] The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of three related capsid proteins. More specifically, after a single mRNA from which each of the VP1, VP2, and VP3 proteins is translated is transcribed, it can be spliced ​​in two different ways: either a longer or a shorter intron can be excised, resulting in the formation of two pools of mRNA: a 2.3 kb-long and a 2.6 kb-long mRNA pool. In many cases, the long intron is preferred, and thus the 2.3 kb-long mRNA can be referred to as the major splice variant. This form lacks the first AUG codon at which synthesis of the VP1 protein begins, resulting in a reduction in the overall level of VP1 protein synthesis. The first AUG codon remaining in the major splice variant is the start codon for the VP3 protein. However, upstream of that codon in the same open reading frame is an ACG sequence (encoding threonine) surrounded by an optimal Kozak (translation initiation) sequence. In some embodiments, the Kozak sequence is set forth in SEQ ID NO:3.This contributes to the low level synthesis of the VP2 protein, which, like VP1, is actually a VP3 protein that contains additional N-terminal residues, each of which is incorporated herein by reference; Becerra SP et al., (December 1985) "Direct mapping of adeno-associated viral capsid proteins B and C: a possible ACG initiation codon". Proceedings of the National Academy of Sciences of the United States of America. 82(23):7919-23; Cassinotti P et al., (November 1988) "Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced ​​mRNA coding for viral capsid protein 1". Virology. 167(1):176-84; Muralidhar S et al., (January 1994) "Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codon". codons: effects on regulation of synthesis and biological activity”. Journal of Virology. 68(1):170-6, and Trempe JP, Carter BJ (September 1988) “Alternate mRNA splicing is required for synthesis of adeno-associated viral VP1 capsid protein”. Journal of Virology. 62(9):3356-63. A single consensus polyA site is located at map position 95 of the AAV genome.The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0041] Each VP1 protein contains a VP1 portion, a VP2 portion, and a VP3 portion. The VP1 portion is the N-terminal portion of the VP1 protein that is unique to the VP1 protein. The VP2 portion is an amino acid sequence present in the VP1 protein that is also found in the N-terminal portion of the VP2 protein. The VP3 portion and the VP3 protein have the same sequence. The VP3 portion is the C-terminal portion of the VP1 protein that is shared with the VP1 and VP2 proteins.

[0042] The VP3 protein can be further divided into separate variable surface regions I-IX (VR-I-IX). As described in DiMatta et al., "Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9," J. Virol., Vol. 86(12):6947-6958, June 2012, the contents of which are incorporated herein by reference, each of the variable surface regions (VRs) can comprise or contain specific amino acid sequences that, either alone or in combination with specific amino acid sequences of each of the other VRs, can confer a unique infectious serotype to the particular serotype (e.g., reduced antigenicity, improved transduction, and / or tissue-specific propensity compared to other AAV serotypes).

[0043] AAV has unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection of humans and other animals is painless and asymptomatic. Moreover, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and essentially persist for the life of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted as cloned DNA into plasmids, making the construction of recombinant genomes feasible. Moreover, because signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA to generate AAV vectors. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is a very stable and abundant virus. It easily survives the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making low-temperature storage of AAV less important. AAV can even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0044] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000); and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997), muscle is highly vascularized, so recombinant AAV transduction results in the appearance of the transgene product in the systemic circulation after intramuscular injection. Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. The recombinant AAV (rAAV) genome of the present invention comprises, consists essentially of, or consists of a nucleic acid molecule encoding a therapeutic protein (e.g., PKP2) and one or more AAV ITRs flanking the nucleic acid molecule. The production of pseudotyped rAAV is disclosed, for example, in WO2001 / 083692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0045] Isolated polynucleotide comprising a transgene sequence The present disclosure provides an isolated polynucleotide comprising at least one transgene nucleic acid molecule.

[0046] In some embodiments, the transgene nucleic acid molecule may comprise a nucleic acid sequence encoding a PKP2 polypeptide, or at least a fragment thereof. As will be understood by those skilled in the art, PKP2 is encoded by the PKP2 gene in the human genome. Thus, the transgene nucleic acid molecule may comprise, consist essentially of, or consist of a PKP2 sequence or any fragment thereof. In some embodiments, the transgene nucleic acid molecule may comprise a nucleic acid sequence encoding a biological equivalent of a PKP2 polypeptide. In some embodiments, the PKP2 polypeptide may be any isoform of PKP2 known in the art. In some embodiments, the PKP2 isoform may be the PKP2 2a isoform. In some embodiments, the PKP2 isoform may be the PKP2 2b isoform.

[0047] As used herein, and unless otherwise specified, a plakophilin-2 (PKP2) gene as described herein refers to a nucleic acid sequence encoding a functional PKP2 protein. The gene or encoded protein may be naturally occurring or may be modified, but retains its therapeutic activity as described herein. The gene or encoded protein may have the nucleotide sequence or amino acid sequence of an isolated naturally occurring PKP2 gene or protein in a mammal, including from a human, as is well known in the published literature.

[0048] The term "PKP2" refers to the plakophilin-2 full-length protein, and functional fragments thereof, including amino acid sequences that include segments of the full-length domain with 100% sequence identity and variations thereof of at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. Variations in amino acid sequence are contemplated as encompassed by the present disclosure, provided that the variations in amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. Certain percentages between 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity, etc. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those made within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartate, glutamate, (2) basic amino acids are lysine, arginine, histidine, (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include: (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine.For example, it is reasonable to expect that isolated replacement of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar replacement of amino acids with structurally related amino acids, specifically when the replacement does not involve amino acids within framework sites, will not have a significant effect on the binding or properties of the resulting molecule. Whether an amino acid change results in a functional PKP2 protein can be easily determined by assaying the specific activity of the protein derivative. Fragments or analogs of PKP2 proteins can be easily prepared by those skilled in the art. Preferred amino and carboxy termini of fragments or analogs occur near the boundaries of functional domains. Sequences can be modified for improved therapeutic activity.

[0049] The term "PKP2 gene" refers to a full-length nucleotide sequence, DNA, or RNA encoding plakophilin 2 protein, including a nucleotide sequence that comprises a segment of the full-length nucleotide sequence and its variations at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% with 100% sequence identity, or a functional fragment thereof. Fragments include nucleic acid sequences, DNA, or RNA that comprise a segment of the full-length gene and its variations at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% with 100% sequence identity. Variations in gene sequence are contemplated as encompassed by the present disclosure, provided that the variations in nucleic acid sequence maintain at least 75%, or at least 80%, 90%, 95%, or 99% identity. Certain percentages include between 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity, etc. Sequences can be modified for improved therapeutic activity and optimized for delivery, such as using adeno-associated virus (AAV) or other well-known gene delivery vector systems.

[0050] In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:13, or a fragment thereof. In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to at least a portion of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:13, or a fragment thereof.

[0051] In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the amino acid sequence set forth in SEQ ID NO: 1, or a fragment thereof. In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to at least a portion of the amino acid sequence set forth in SEQ ID NO: 1, or a fragment thereof.

[0052] In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the amino acid sequence set forth in SEQ ID NO: 13, or a fragment thereof. In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to at least a portion of the amino acid sequence set forth in SEQ ID NO: 13, or a fragment thereof.

[0053] In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to any one of the nucleic acid sequences set forth in SEQ ID NO: 4 or SEQ ID NO: 14. In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14. A nucleic acid sequence encoding a PKP2 polypeptide may be referred to as a PKP2 sequence.

[0054] In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to any one of the nucleic acid sequences set forth in SEQ ID NO: 4. In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 4.

[0055] In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to any one of the nucleic acid sequences set forth in SEQ ID NO: 14. In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 14.

[0056] Codon Optimization In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide may be a codon-optimized nucleic acid sequence encoding a PKP2 polypeptide. A codon-optimized nucleic acid sequence encoding a PKP2 polypeptide may comprise, consist essentially of, or consist of a nucleic acid sequence that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (or any percentage in between) or less identical to a wild-type human nucleic acid sequence encoding a PKP2 polypeptide.

[0057] In some embodiments, the codon-optimized nucleic acid sequence encoding a PKP2 polypeptide may not contain a donor splice site. In some embodiments, the codon-optimized nucleic acid sequence encoding a PKP2 polypeptide may contain about 1, or about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 or less donor splice sites. In some embodiments, the codon-optimized nucleic acid sequence encoding a PKP2 polypeptide contains at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 fewer donor splice sites compared to a wild-type human nucleic acid sequence encoding a PKP2 polypeptide. Without wishing to be bound by theory, removal of donor splice sites in a codon-optimized nucleic acid sequence may unexpectedly and unpredictably increase the expression of a PKP2 polypeptide in vivo, since cryptic splicing is prevented. Furthermore, cryptic splicing can vary between different subjects, meaning that expression levels of PKP2 polypeptides containing donor splice sites can vary unpredictably between different subjects. Such unpredictability is unacceptable in the context of human therapy.

[0058] In some embodiments, the codon-optimized nucleic acid sequence encoding a PKP2 polypeptide may have a GC content that is different from the GC content of a wild-type human nucleic acid sequence encoding a PKP2 polypeptide. In some embodiments, the GC content of the codon-optimized nucleic acid sequence encoding a PKP2 polypeptide is more evenly distributed throughout the nucleic acid sequence compared to the wild-type human nucleic acid sequence encoding a PKP2 polypeptide. Without wishing to be bound by theory, by distributing the GC content more evenly throughout the nucleic acid sequence, the codon-optimized nucleic acid sequence exhibits a more uniform melting temperature ("Tm") throughout the length of the transcript. The uniformity of the melting temperature unexpectedly results in increased expression of the codon-optimized nucleic acid in a human subject, since transcription and / or translation of the nucleic acid sequence occurs with less stalling of the polymerase and / or ribosome.

[0059] In some embodiments, the codon-optimized nucleic acid sequence encoding a PKP2 polypeptide may have fewer inhibitory microRNA target binding sites compared to a wild-type human nucleic acid sequence encoding a PKP2 polypeptide. In some embodiments, the codon-optimized nucleic acid sequence encoding a PKP2 polypeptide may have at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten, or at least ten fewer inhibitory microRNA target binding sites compared to a wild-type human nucleic acid sequence encoding a PKP2 polypeptide. Without wishing to be bound by theory, by having fewer inhibitory microRNA target binding sites, the codon-optimized nucleic acid sequence encoding a PKP2 polypeptide unexpectedly exhibits increased expression in a human subject.

[0060] In some aspects, a codon-optimized nucleic acid sequence encoding a PKP2 polypeptide exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 50%, at least a 75%, at least a 100%, at least a 200%, at least a 300%, at least a 500%, or at least a 1000% increase in expression in a human subject compared to a wild-type or non-codon-optimized nucleic acid sequence encoding a PKP2 polypeptide.

[0061] AAV vectors In some aspects, the isolated polynucleotide comprising at least one transgene nucleic acid molecule described herein can be a recombinant AAV (rAAV) vector.

[0062] As used herein, the term "vector" refers to a nucleic acid that comprises, consists essentially of, or consists of an intact replicon, such that when the vector is placed in a cell, it can be replicated, for example, by a process of transfection, infection, or transformation. Once inside the cell, it is understood in the art that the vector can be replicated as an extrachromosomal (episomal) element or can be integrated into a host cell chromosome. A vector can include nucleic acids derived from retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papovaviruses, or modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acids include the use of naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising, consisting essentially of, or consisting of DNA condensed with cationic polymers such as heterologous polylysine, defined length oligopeptides, and polyethyleneimine, optionally contained in liposomes; and ternary complexes comprising, consisting essentially of, or consisting of viruses and polylysine-DNA.

[0063] With respect to general recombinant techniques, vectors containing both a promoter and a cloning site to which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the cloned transgene to eliminate extra, potentially inappropriate alternative translation initiation codons or other sequences that may disrupt or reduce expression at either the transcriptional or translational levels. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to enhance expression.

[0064] As used herein, "rAAV vector" refers to a vector that comprises, consists essentially of, or consists of one or more transgene sequences and one or more AAV inverted terminal repeats (ITRs). Such AAV vectors, when present in a host cell that provides the functionality of the rep and cap gene products, can be replicated and packaged into infectious viral particles, for example, by transfection of the host cell. In some embodiments, the AAV vector contains a promoter, at least one nucleic acid that can encode at least one protein or RNA, and / or enhancer and / or terminator within the flanking ITRs that are packaged into infectious AAV particles. The encapsidated nucleic acid portion can be referred to as the AAV vector genome. Plasmids containing rAAV vectors can also contain elements for manufacturing purposes, such as antibiotic resistance genes, origin of replication sequences, etc., but these are not encapsidated and therefore do not form part of the AAV particle.

[0065] In some embodiments, the rAAV vector may include at least one transgene nucleic acid molecule. In some embodiments, the rAAV vector may include at least one AAV inverted terminal repeat (ITR) sequence. In some embodiments, the rAAV vector may include at least one promoter sequence. In some embodiments, the rAAV vector may include at least one enhancer sequence. In some embodiments, the rAAV vector may include at least one post-transcriptional regulatory element. In some embodiments, the rAAV vector may include at least one polyA sequence. In some embodiments, the rAAV vector may include at least one reporter protein. In some embodiments, the rAAV vector may include a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence. In some embodiments, the rAAV vector may include, in the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence.

[0066] In some embodiments, the rAAV vector can include a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, a post-transcriptional regulatory element, a polyA sequence, and a second AAV ITR sequence. In some embodiments, the rAAV vector can include, from the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, a post-transcriptional regulatory element, a polyA sequence, and a second AAV ITR sequence.

[0067] In some embodiments, the rAAV vector may comprise two or more transgene nucleic acid molecules. In some embodiments, the rAAV vector may comprise at least two transgene nucleic acid molecules, such that the rAAV vector comprises a first transgene nucleic acid molecule and at least a second transgene nucleic acid molecule. In some embodiments, the first and at least a second transgene nucleic acid molecule may comprise the same nucleic acid sequence. In some embodiments, the first and at least a second transgene nucleic acid molecule may comprise different nucleic acid sequences. In some embodiments, the first and at least a second transgene nucleic acid sequence may be adjacent to each other.

[0068] In some embodiments, the rAAV vector may include two or more promoter sequences. In some embodiments, the rAAV vector may include at least two promoter sequences, such that the rAAV vector includes a first promoter sequence and at least a second promoter sequence. In some embodiments, the first and at least a second promoter sequence may include the same sequence. In some embodiments, the first and at least a second promoter sequence may include different sequences. In some embodiments, the first and at least a second promoter sequence may be adjacent to each other. In some embodiments, where the rAAV vector also includes a first transgene nucleic acid molecule and at least a second transgene nucleic acid molecule, the first promoter may be located upstream (5') of the first transgene nucleic acid molecule, and the at least a second promoter may be located between the first transgene nucleic acid molecule and the at least a second transgene nucleic acid molecule, such that the at least a second promoter is downstream (3') of the first transgene nucleic acid molecule and upstream (5') of the at least a second transgene nucleic acid molecule.

[0069] Any of the aforementioned rAAV vectors may further comprise at least one enhancer. The at least one enhancer may be located anywhere within the rAAV vector. In some embodiments, the at least one enhancer may be located immediately upstream (5') of the promoter. Thus, the rAAV vector may comprise, in a 5' to 3' direction, a first AAV ITR sequence, an enhancer, a promoter sequence, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence. In some embodiments, the at least one enhancer may be located immediately downstream (3') of the promoter. Thus, the rAAV vector may comprise, in a 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, an enhancer, a transgene nucleic acid molecule, a polyA sequence, and a second AAV ITR sequence. In some embodiments, the at least one enhancer may be located immediately downstream of the transgene nucleic acid molecule. Thus, a rAAV vector can include, from the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, a transgene nucleic acid molecule, an enhancer, a polyA sequence, and a second AAV ITR sequence.

[0070] In some aspects, the rAAV vectors of the disclosure comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence set forth in SEQ ID NO:9, or a fragment thereof.

[0071] In some aspects, the rAAV vectors of the present disclosure comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence set forth in SEQ ID NO:18, or a fragment thereof.

[0072] In some aspects, the rAAV vectors of the disclosure comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence set forth in SEQ ID NO:21, or a fragment thereof.

[0073] In some embodiments, the rAAV vectors of the disclosure comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the nucleic acid sequence set forth in SEQ ID NO:22, or a fragment thereof.

[0074] In some aspects, the rAAV vectors of the disclosure comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence set forth in SEQ ID NO:23, or a fragment thereof.

[0075] In some aspects, the rAAV vectors of the disclosure comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence set forth in SEQ ID NO:24, or a fragment thereof. In some aspects, the rAAV vectors of the disclosure comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence set forth in SEQ ID NO:26, or a fragment thereof. In some aspects, the rAAV vectors of the disclosure comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence set forth in SEQ ID NO:29, or a fragment thereof. In some aspects, the rAAV vectors of the present disclosure comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to the nucleic acid sequence set forth in SEQ ID NO:30, or a fragment thereof.

[0076] AAV ITR sequences In some aspects, the AAV ITR sequence can include any AAV ITR sequence known in the art, hi some aspects, the AAV ITR sequence can be an AAV1 ITR sequence, an AAV2 ITR sequence, an AAV4 ITR sequence, an AAV5 ITR sequence, an AAV6 ITR sequence, an AAV7 ITR sequence, an AAV8 ITR sequence, an AAV9 ITR sequence, an AAV10 ITR sequence, an AAV11 ITR sequence, an AAV12 ITR sequence, an AAV13 ITR sequence, an AAVrh74 ITR sequence, or an AAVrh10 ITR sequence.

[0077] Thus, in some aspects, the AAV ITR sequence may comprise, consist essentially of, or consist of an AAV1 ITR sequence, an AAV2 ITR sequence, an AAV4 ITR sequence, an AAV5 ITR sequence, an AAV6 ITR sequence, an AAV7 ITR sequence, an AAV8 ITR sequence, an AAV9 ITR sequence, an AAV10 ITR sequence, an AAV11 ITR sequence, an AAV12 ITR sequence, an AAV13 ITR sequence, an AAVrh74 ITR sequence, or an AAVrh10 ITR sequence.

[0078] In some aspects, the rAAV vectors of the disclosure may comprise, consist essentially of, or consist of AAV2 ITR sequences. In some aspects, the rAAV vectors of the disclosure may comprise, consist essentially of, or consist of AAV2 ITR sequences or modified AAV2 ITR sequences.

[0079] In some embodiments, the AAV2 ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:7.

[0080] In some embodiments, the AAV2 ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:8.

[0081] In some embodiments, the AAV2 ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:15.

[0082] In some embodiments, the AAV2 ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:16.

[0083] In some embodiments, the AAV2 ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:19.

[0084] In some embodiments, the AAV2 ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:20.

[0085] In some embodiments, the AAV2 ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:25.

[0086] In some embodiments, the first AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:7, and the second AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:8.

[0087] In some embodiments, the first AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:8, and the second AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:7.

[0088] In some embodiments, the first AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:25, and the second AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:8.

[0089] In some embodiments, the first AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:8, and the second AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:25.

[0090] In some embodiments, the first AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:15, and the second AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:16.

[0091] In some embodiments, the first AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:16, and the second AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:15.

[0092] In some embodiments, the first AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:19, and the second AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:20.

[0093] In some embodiments, the first AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:20, and the second AAV ITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:19.

[0094] In some aspects, the ITR sequences of the present disclosure may be in any order, such as in a forward orientation, or may be inverted in a reverse orientation. In some aspects, the ITR sequences of the present disclosure may include mutations, deletions, insertions, or rearrangements of one or more nucleotides in the nucleic acid sequence encoding the ITR sequence.

[0095] Promoter sequences and enhancers The terms "promoter" and "promoter sequence" as used herein refer to a control sequence, which is a region of a polynucleotide sequence at which the initiation and rate of transcription of a coding sequence, such as a gene or transgene, is controlled. A promoter can be, for example, constitutive, inducible, repressible, or tissue-specific. A promoter can contain genetic elements to which molecules such as regulatory proteins and RNA polymerases and transcription factors can bind. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, the H1 promoter, the ubiquitous chicken β-actin hybrid (CBh) promoter, the small nuclear RNA (U1a or U1b) promoter, the MeCP2 promoter, the MeP418 promoter, the MeP426 promoter, the minimal MeCP2 promoter, the VMD2 promoter, the mRho promoter, or the EF1 promoter.

[0096] Additional non-limiting exemplary promoters provided herein include, but are not limited to, EFla, Ubc, human β-actin, CAG, TRE, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, Ubi, and alpha-1-antitrypsin (hAAT). It is known in the art that the nucleotide sequences of such promoters can be modified to increase or decrease the efficiency of mRNA transcription. See, for example, Gao et al. (2018) Mol. Ther.: Nucleic Acids 12:135-145 (modifying TATA box of 7SK, U6 and H1 promoters to abolish RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription). Synthetic derived promoters can be used for ubiquitous or tissue-specific expression. Additionally, viral derived promoters, some of which are described above, such as CMV, HIV, adenovirus, and AAV promoters, can be useful in the methods disclosed herein. In some embodiments, the promoter is used in conjunction with at least one enhancer to increase transcription efficiency. Non-limiting examples of enhancers include the interstitial retinoid binding protein (IRBP) enhancer, the RSV enhancer, or the CMV enhancer.

[0097] In some embodiments, the promoter sequence is selected from the group consisting of a Rous sarcoma virus (RSV) LTR promoter sequence (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter sequence, an SV40 promoter sequence, a dihydrofolate reductase promoter sequence, a β-actin promoter sequence, a phosphoglycerol kinase (PGK) promoter sequence, a U6 promoter sequence, an H1 promoter sequence, a ubiquitous chicken β-actin hybrid (CBh) promoter sequence, a small nuclear RNA (U1a or U1b) promoter sequence, a MeCP2 promoter sequence, a MeP418 promoter sequence, a MeP426 promoter sequence, a MeP428 promoter sequence, a MeP429 promoter sequence, a MeP430 promoter sequence, a MeP440 promoter sequence, a MeP450 promoter sequence, a MeP460 promoter sequence, a MeP470 promoter sequence, a MeP480 promoter sequence, a MeP490 promoter sequence, a MeP491 promoter sequence, a MeP492 promoter sequence, a MeP493 promoter sequence, a MeP494 promoter sequence, a MeP495 promoter sequence, a MeP496 promoter sequence, a MeP497 promoter sequence, a MeP498 promoter sequence, a MeP499 promoter sequence, a MeP499 promoter sequence, a MeP491 promoter sequence, a MeP492 ...3 promoter sequence, a MeP494 promoter sequence, a MeP495 promoter sequence, a MeP496 promoter sequence, a MeP497 promoter sequence, a MeP498 promoter sequence, a MeP499 promoter sequence, a MeP499 promoter sequence, a MeP499 promoter sequence The promoter sequence may comprise, consist essentially of, or consist of a minimal MeCP2 promoter sequence, a VMD2 promoter sequence, an mRho promoter sequence, an EFI promoter sequence, an EF1 promoter sequence, a Ubc promoter sequence, a human β-actin promoter sequence, a CAG promoter sequence, a TRE sequence, an Ac5 promoter sequence, a polyhedrin promoter sequence, a CaMKIIa promoter sequence, a Gal1 promoter sequence, a TEF1 promoter sequence, a GDS promoter sequence, an ADH1 promoter sequence, a Ubi promoter sequence, or an alpha-1-antitrypsin (hAAT) promoter sequence.

[0098] In some embodiments, the promoter sequence can be a cardiac specific promoter. In some embodiments, the cardiac specific promoter is a TNNI2 promoter. In some embodiments, the cardiac specific promoter is a NKX2.5 promoter. In some embodiments, the cardiac specific promoter is a cardiac troponin T (cTnT) promoter. In some embodiments, the cardiac troponin T promoter drives cardiac specific expression. In some embodiments, the cardiac troponin T promoter drives cardiomyocyte specific expression.

[0099] Enhancers are regulatory elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence capable of providing both promoter and enhancer functions. For example, retroviral long terminal repeats contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous," or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that is placed in juxtaposition to a gene by means of genetic engineering (i.e., molecular biological techniques) or by synthetic techniques such that transcription of that gene is directed by the linked enhancer / promoter. Non-limiting examples of linked enhancers / promoters for use in the methods, compositions, and constructs provided herein include PDE promoter + IRBP enhancer, or CMV enhancer + U1a promoter. It is understood in the art that enhancers can act from a distance and regardless of their orientation relative to the location of the endogenous or heterologous promoter. Thus, an enhancer that acts at a distance from a promoter is therefore further understood to be "operably linked" to that promoter, regardless of its location within the vector or its orientation relative to the location of the promoter.

[0100] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene (i.e., a transgene) under the control of a promoter to which it is spatially connected. A promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. A promoter may be located 5' (upstream) of the gene under its control. The distance between a promoter and a gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. Variations in the distance between a promoter and a gene may be accommodated without loss of promoter function.

[0101] In some embodiments, the promoter sequence may comprise, consist essentially of, or consist of a cardiac troponin T (cTnT) promoter sequence. The cTnT promoter sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:2.

[0102] In some aspects, a bacterial plasmid of the present disclosure may contain a prokaryotic promoter.

[0103] Transgene Nucleic Acid Molecules In some embodiments, the transgene nucleic acid molecule may comprise a nucleic acid sequence encoding a PKP2 polypeptide, or at least a fragment thereof. In some embodiments, the transgene nucleic acid molecule may comprise a nucleic acid sequence encoding a biological equivalent of a PKP2 polypeptide, or at least a fragment thereof.

[0104] In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:13, or a fragment thereof. In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to at least a portion of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:13, or a fragment thereof.

[0105] In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the amino acid sequence set forth in SEQ ID NO: 1, or a fragment thereof. In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to at least a portion of the amino acid sequence set forth in SEQ ID NO: 1, or a fragment thereof.

[0106] In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the amino acid sequence set forth in SEQ ID NO: 13, or a fragment thereof. In some embodiments, the PKP2 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to at least a portion of the amino acid sequence set forth in SEQ ID NO: 13, or a fragment thereof.

[0107] In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to any one of the nucleic acid sequences set forth in SEQ ID NO: 4 or SEQ ID NO: 14. In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14. A nucleic acid sequence encoding a PKP2 polypeptide may be referred to as a PKP2 sequence.

[0108] In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to any one of the nucleic acid sequences set forth in SEQ ID NO: 4. In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 4.

[0109] In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to any one of the nucleic acid sequences set forth in SEQ ID NO: 14. In some embodiments, a nucleic acid sequence encoding a PKP2 polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the nucleic acid sequence set forth in SEQ ID NO: 14.

[0110] In some aspects, the transgene nucleic acid molecule may comprise, consist essentially of, or consist of a nucleic acid sequence encoding a reporter protein. As used herein, a reporter protein is a detectable protein operably linked to a promoter to assay the expression (e.g., tissue specificity and / or strength) of the promoter. In aspects, the reporter protein may be operably linked to a polypeptide. In aspects, the reporter protein may be used in DNA delivery methods, functional identification and characterization of promoter and enhancer elements, translational and transcriptional regulation, mRNA processing, and monitoring protein:protein interactions. Non-limiting examples of reporter proteins are β-galactosidase, fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP), luciferase, glutathione-S-transferase, and maltose binding protein.

[0111] In some aspects, the transgene nucleic acid molecule may further comprise a nucleic acid sequence encoding a signal peptide.

[0112] In some embodiments, a transgene nucleic acid molecule present in a rAAV vector can be under the transcriptional control of a promoter sequence that is also present in the same rAAV vector.

[0113] Post-transcriptional regulatory elements For example, various post-transcriptional regulatory elements can be used in viral vectors to increase the expression level of a protein of interest in a host cell. In some embodiments, the post-transcriptional regulatory element can be a viral post-transcriptional regulatory element. Non-limiting examples of viral post-transcriptional regulatory elements include Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE), Hepatitis B Virus Post-transcriptional Regulatory Element (HBVPRE), RNA Transport Element (RTE), and any variants thereof. In some aspects, the post-transcriptional regulatory element can be an optimized post-transcriptional regulatory element (oPRE). An oPRE can comprise a nucleic acid sequence that comprises, consists essentially of, or consists of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the nucleic acid sequence set forth in SEQ ID NO:5, SEQ ID NO:27, or SEQ ID NO:28.

[0114] PolyA sequence In some embodiments, the polyadenylation (polyA) sequence can include any polyA sequence known in the art. Non-limiting examples of polyA sequences include, but are not limited to, MeCP2 polyA sequence, retinol dehydrogenase 1 (RDH1) polyA sequence, bovine growth hormone (BGH) polyA sequence, SV40 polyA sequence, SPA49 polyA sequence, sNRP-TK65 polyA sequence, sNRP polyA sequence, rabbit beta-globin polyA sequence, or TK65 polyA sequence.

[0115] Thus, the polyA sequence may comprise, consist essentially of, or consist of a MeCP2 polyA sequence, a retinol dehydrogenase 1 (RDH1) polyA sequence, a bovine growth hormone (BGH) polyA sequence, an SV40 polyA sequence, an SPA49 polyA sequence, a sNRP-TK65 polyA sequence, a sNRP polyA sequence, or a TK65 polyA sequence.

[0116] In some embodiments, the polyA sequence may comprise, consist essentially of, or consist of a rabbit beta-globin polyA sequence. In some embodiments, the rabbit beta-globin polyA sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the sequence set forth in SEQ ID NO:6.

[0117] In some embodiments, the polyA sequence may comprise, consist essentially of, or consist of a BGH polyA sequence. In some embodiments, the BGH polyA sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the sequence set forth in SEQ ID NO:17.

[0118] Bacterial plasmids In some aspects, the rAAV vector of the present disclosure can be contained in a bacterial plasmid to allow the rAAV vector to propagate in vitro.Thus, the present disclosure provides a bacterial plasmid that includes any of the rAAV vectors described herein.The bacterial plasmid can further include an origin of replication sequence.The bacterial plasmid can further include an antibiotic resistance gene.The bacterial plasmid can further include a prokaryotic promoter.

[0119] In a non-limiting example, an rAAV vector in a bacterial plasmid includes, from 5' to 3', a 5' ITR, a cTnT promoter sequence, a transgene nucleic acid molecule encoding a PKP2 polypeptide, an oPRE sequence, a BGH polyA sequence, and a 3' ITR.

[0120] In some embodiments, the bacterial plasmid of the present disclosure may comprise, consist essentially of, or consist of the nucleic acid sequence set forth in SEQ ID NO:10.

[0121] Replication origin sequence In some aspects, the origin of replication sequence can comprise, consist essentially of, or consist of any origin of replication sequence known in the art. The origin of replication sequence can be a bacterial origin of replication sequence, thereby allowing rAAV vectors containing the bacterial origin of replication sequence to be produced, propagated, and maintained in bacteria using methods standard in the art.

[0122] In some embodiments, the origin of replication sequence may comprise, consist essentially of, or consist of a pUC origin of replication sequence. The pUC19 origin of replication sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:11.

[0123] Antibiotic resistance genes In some aspects, the rAAV vectors and / or rAAV viral vectors of the present disclosure may include antibiotic resistance genes.

[0124] In some aspects, the antibiotic resistance gene may comprise, consist essentially of, or consist of any antibiotic resistance gene known in the art, examples of which include, but are not limited to, the kanamycin resistance gene, the spectinomycin resistance gene, the streptomycin resistance gene, the ampicillin resistance gene, the carbenicillin resistance gene, the bleomycin resistance gene, the erythromycin resistance gene, the polymyxin B resistance gene, the tetracycline resistance gene, and the chloramphenicol resistance gene.

[0125] In some embodiments, the antibiotic resistance gene may comprise, consist essentially of, or consist of an ampicillin antibiotic resistance gene. The ampicillin antibiotic resistance gene may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO: 12.

[0126] AAV viral vectors A "viral vector" is defined as a recombinantly produced virus or virus particle that contains a polynucleotide that is delivered to a host cell either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, for example, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.

[0127] "AAV virion" or "AAV viral particle" or "AAV viral vector" or "rAAV viral vector" or "AAV vector particle" or "AAV particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector. Thus, the production of a rAAV viral vector necessarily includes the production of a rAAV vector, since such a vector is contained within the rAAV vector.

[0128] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a viral particle. The capsid functions to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidated" means enclosed within the viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins: VP1, VP2, and VP3. The mixture of VP1, VP2, and VP3 contains 60 monomers arranged in a T=1 dodecahedron in a ratio of 1:1:10 (VP1:VP2:VP3) or 1:1:20 (VP1:VP2:VP3), as described in Sonntag F et al., (June 2010) "A viral assembly factor promotes AAV2 capsid formation in the nucleolus". Proceedings of the National Academy of Sciences of the United States of America. 107(22):10220-5, and Rabinowitz JE, Samulski RJ (December 2000) "Building a better vector: the manipulation of AAV virions". Virology. 278(2):301-8, each of which is incorporated herein by reference in its entirety.

[0129] The present disclosure provides an rAAV viral vector comprising: a) any of the rAAV vectors described herein; and b) an AAV capsid protein.

[0130] The AAV capsid protein can be any AAV capsid protein known in the art. The AAV capsid protein can be AAV1 capsid protein, AAV2 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAV13 capsid protein, AAVPHP.B capsid protein, AAVrh74 capsid protein, or AAVrh10 capsid protein. In some embodiments, the capsid protein can be AAV9 capsid protein. In some embodiments, the capsid protein can be AAVrh10 capsid protein.

[0131] Compositions and pharmaceutical compositions The present disclosure provides a composition comprising any of the isolated polynucleotides, rAAV vectors, and / or rAAV viral vectors described herein.In some aspects, the composition can be a pharmaceutical composition.Thus, the present disclosure provides a pharmaceutical composition comprising any of the isolated polynucleotides, rAAV vectors, and / or rAAV viral vectors described herein.

[0132] The pharmaceutical compositions described herein may be formulated by any method known or developed in the art of pharmacology, including, but not limited to, contacting the active ingredient (e.g., viral particle or recombinant vector) with excipients and / or additives and / or other accessory ingredients and dividing or packaging the product into dosage units. The viral particles of the present disclosure may be formulated to have desirable characteristics, such as increased stability, increased cell transfection, sustained or delayed release, biodistribution or disposition, regulated or enhanced translation of the encoded protein in vivo, and release profile of the encoded protein in vivo.

[0133] Thus, the pharmaceutical composition may further comprise saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for implantation into a subject), nanoparticle mimics, or combinations thereof. In some embodiments, the pharmaceutical composition is formulated as a nanoparticle. In some embodiments, the nanoparticle is a self-assembled nucleic acid nanoparticle.

[0134] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage. The formulations of the present invention may include one or more excipients and / or additives in amounts that together increase the stability of the viral vector, increase cell transfection or transduction by the viral vector, increase the expression of the viral vector encoding a protein, and / or modify the release profile of the viral vector encoding a protein. In some embodiments, the pharmaceutical composition includes an excipient and / or additive. Non-limiting examples of excipients and / or additives include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, or combinations thereof.

[0135] In some embodiments, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent capable of reducing or eliminating damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0136] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the Federal or State government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, in addition to other formulations that are safe for use in animals, and more specifically, humans and / or non-human mammals.

[0137] As used herein, the term "pharmaceutical acceptable carrier" includes standard pharmaceutical carriers such as phosphate buffered saline, water, and emulsions such as oil / water or water / oil emulsions, as well as any of various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm.Sci., 15th Ed. (Mack Publ.Co., Easton).

[0138] In some aspects, pharmaceutical compositions of the present disclosure may include tris(hydroxymethyl)aminomethane (Tris), magnesium chloride, sodium chloride, poloxamer, sucrose, or any combination thereof.

[0139] In some embodiments, the pharmaceutical composition may include sodium chloride, wherein the sodium chloride is present at a concentration of about 100 mM to about 500 mM, or about 200 mM to about 400 mM, or about 300 mM to about 400 mM. In some embodiments, the sodium chloride may be present at a concentration of about 200 mM.

[0140] In some embodiments, the pharmaceutical composition may include Tris, wherein Tris is present at a concentration of about 10 mM to about 100 mM, or about 10 mM to about 50 mM, or about 15 mM to about 25 mM, hi some embodiments, Tris may be present at a concentration of about 20 mM.

[0141] In some embodiments, the pharmaceutical composition may include magnesium chloride, wherein the magnesium chloride is present at a concentration of about 0.1 mM to about 50 mM, or about 0.1 mM to about 5 mM, or about 0.5 mM to about 2.5 mM. In some embodiments, the magnesium chloride may be present at a concentration of about 1 mM.

[0142] In some embodiments, the pharmaceutical composition may include poloxamer 188, wherein the poloxamer 188 is present at a concentration of about 0.001% to about 0.1%, or about 0.005% to about 0.05%. In some embodiments, the poloxamer 188 may be present at a concentration of about 0.01%.

[0143] In some embodiments, the pharmaceutical composition may include sucrose, wherein the sucrose is present at a concentration of about 0.1% to about 10%, or about 0.5% to about 5%. In some embodiments, the sucrose may be present at a concentration of about 1%.

[0144] In some embodiments, the pharmaceutical composition may be formulated at a pH of about 6.5 to about 8.5, or about 7.0 to about 8.0, or about 7.4 to about 7.8, In some embodiments, the pharmaceutical composition may be formulated at a pH of about 7.6.

[0145] Methods of Using the Disclosed Compositions The present disclosure provides for the use of the disclosed compositions and pharmaceutical compositions for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or described herein, for example, by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal, or subject. In one aspect, the subject is a mammal. Preferably, the subject is a human. In this specification, the terms "subject" and "patient" are used interchangeably. For example, the terms "subject" and "patient" can refer to a mammalian subject, including primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc.

[0146] The present disclosure provides a method of preventing or treating a disorder comprising, consisting essentially of, or consisting of administering to a subject a therapeutically effective amount of any one of the rAAV vectors, rAAV viral vectors, compositions, and / or pharmaceutical compositions disclosed herein.

[0147] In some aspects, the disclosure provides a method for preventing or treating cardiac arrhythmia. In an embodiment, the present invention provides a method for preventing or treating arrhythmogenic right ventricular cardiomyopathy (ARVC). In some aspects, the disease can be a genetic disorder involving the PKP2 gene. As will be understood by those skilled in the art, ARVC or PKP2-related genetic disorders can cause one or more symptoms in a subject, including, but not limited to, cardiac arrhythmia, fainting, cardiac palpitations, dizziness, shortness of breath, chest pain, fatigue, persistent cough, ventricular premature contractions, ventricular tachycardia (VT), heart failure, cardiac fibrosis, and / or cardiac arrest. In some aspects, ARVC or PKP2-related genetic disorders are associated with left ventricular dysfunction and / or fibrofatty degeneration of myocardium, which leads to ventricular arrhythmia and sudden cardiac death.

[0148] In some embodiments, ARVC is characterized by defects in cardiac desmosomes.As used herein, the term "desmosome" refers to the cell structure specialized for cell-cell adhesion.Desmosomes are a type of junction complex that are randomly arranged localized spot-like adhesions on the lateral side of plasma membrane.Desmosomes found in cardiac tissue are called cardiac desmosomes.

[0149] In some embodiments, the disease can be characterized by the loss of function of at least one copy of the PKP2 gene in the genome of the subject.In some embodiments, the disease can be characterized by the reduced function of at least one copy of the PKP2 gene in the genome of the subject.In some embodiments, the disease can be characterized by at least one mutation in at least one mutation in at least one copy of the PKP2 gene in the genome of the subject.

[0150] The mutation of the PKP2 gene can be any type of mutation known in the art.Non-limiting examples of mutations include somatic mutations, single nucleotide variants (SNVs), nonsense mutations, insertions, deletions, duplications, frameshift mutations, repeat expansions, short insertions and deletions (INDELs), long INDELs, alternative splicing, products of alternative splicing, altered translation initiation, products of altered translation initiation, proteomic truncation, products of proteomic truncation.

[0151] In some embodiments, the disease can be a disease characterized by decreased expression of the PKP2 gene in a subject compared to a control subject without the disease. In some embodiments, the decreased expression can be at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 100%.

[0152] In some embodiments, the disease can be characterized by a decrease in the amount of PKP2 in a subject compared to a control subject without the disease. In some embodiments, the decrease in the amount of PKP2 can be at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 100%.

[0153] In some embodiments, the disease can be characterized by decreased activity of PKP2 in a subject compared to a control subject without the disease. In some embodiments, the decrease in PKP2 activity can be at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 100%.

[0154] In some embodiments, the rAAV vector or rAAV viral vector comprising nucleic acid sequence encoding PKP2 can stabilize cardiac desmosomes in a subject. In some embodiments, the rAAV vector or rAAV viral vector comprising nucleic acid sequence encoding PKP2 can rescue the loss of cardiac intercellular junction proteins in a subject. In some embodiments, the rAAV vector or rAAV viral vector comprising nucleic acid sequence encoding PKP2 can reconstitute intercellular junction proteins in a subject. In some embodiments, the desmosomal proteins include PKP2, desmoplakin (DSP), desmoglein-2 (DSG2), plakoglobin (JUP). In some embodiments, the intercellular junction proteins include connexin 43 (CX43).

[0155] In some embodiments, rAAV vectors or rAAV viral vectors comprising nucleic acid sequences encoding PKP2 can improve the electrical and structural integrity associated with ARVC in a subject. In some embodiments, rAAV vectors or rAAV viral vectors comprising nucleic acid sequences encoding PKP2 can preserve electrical and structural integrity in a subject to prevent ARVC.

[0156] A subject to be treated using the disclosed methods, compositions, pharmaceutical compositions, rAAV vectors, or rAAV viral vectors may have any of the diseases and / or conditions described herein.

[0157] In some embodiments, the subject may be less than 0.5 years old, or less than 1 year old, or less than 1.5 years old, or less than 2 years old, or less than 2.5 years old, or less than 3 years old, or less than 3.5 years old, or less than 3.5 years old, or less than 4 years old, or less than 4.5 years old, or less than 5 years old, or less than 5.5 years old, or less than 6 years old, or less than 6.5 years old, or less than 7 years old, or less than 7.5 years old, or less than 8 years old, or less than 8.5 years old, or less than 9 years old, or less than 9.5 years old, or less than 10 years old. In some embodiments, the subject may be less than 0.5 years old. In some embodiments, the subject may be under the age of 4. In some embodiments, the subject may be under the age of 10. In some embodiments, the subject may be 18 or older.

[0158] The therapeutic and preventative methods disclosed herein may be combined with appropriate diagnostic techniques to identify and select patients for therapy or prevention.

[0159] The present disclosure provides a method for increasing the level of a protein in a host cell, comprising contacting a host cell with any one of the rAAV viral vectors disclosed herein, wherein the rAAV viral vector comprises any one of the rAAV vectors disclosed herein, the rAAV viral vector comprising a transgene nucleic acid molecule encoding a protein. In some embodiments, the protein is a therapeutic protein. In some embodiments, the host cell is in vitro, in vivo, or ex vivo. In some embodiments, the host cell is derived from a subject. In some embodiments, the subject is suffering from a disorder that results in a reduction in the level and / or functionality of the protein compared to the level and / or functionality of the protein in a normal subject.

[0160] In some embodiments, the level of PKP2 protein is increased to a level equal to or greater than endogenous PKP2 expression. In some embodiments, the level of PKP2 protein is increased by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 105%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to the PKP2 expression level before treatment.

[0161] The present disclosure provides a method for introducing a gene of interest into a cell of a subject, the method comprising contacting the cell with an effective amount of any one of the rAAV viral vectors disclosed herein, wherein the rAAV viral vector contains any one of the rAAV vectors disclosed herein that includes the gene of interest.

[0162] In some aspects of the methods of the present disclosure, in addition to administering the rAAV vector or rAAV viral vector of the present disclosure to the subject, the subject may also be administered a prophylactic immunosuppressant treatment regimen. In some aspects, the immunosuppressant treatment regimen may include administering at least one immunosuppressant therapeutic agent. Non-limiting examples of immunosuppressant therapeutic agents include, but are not limited to, sirolimus (rapamycin), acetaminophen, diphenhydramine, IV methylprednisolone, prednisone, or any combination thereof. The immunosuppressant therapeutic agent may be administered the day before administration of the rAAV vector and / or rAAV viral vector, the same day as administration of the rAAV vector and / or rAAV viral vector, or any day after administration of the rAAV vector and / or rAAV viral vector.

[0163] A "subject" of diagnosis or treatment may be a cell, or an animal, such as a mammal, or a human. Subjects are not limited to a particular species and include non-human animals that are the subject of diagnosis or treatment, and those that are subject to infection, or animal models, including, but not limited to, monkeys, mice, rats, dogs, or Leporidae species, as well as other farm animals, sport animals, or pets. In some embodiments, the subject is a human.

[0164] As used herein, "treating" or "treatment" of a disease in a subject refers to (1) preventing a symptom or disease from occurring in a subject who is predisposed to the disease or who does not yet exhibit symptoms thereof, (2) inhibiting or arresting the development of the disease, or (3) ameliorating or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results may include, but are not limited to, one or more of the following: alleviation or amelioration of one or more symptoms, whether detectable or undetectable, reduction in the extent of a condition (including a disease), a stable (i.e., non-worsening) stage of a condition (including a disease), delay or slowing of a condition (including a disease), progression, amelioration, or alleviation, stage, and remission (partial or complete) of a condition (including a disease).

[0165] As used herein, and unless otherwise specified, the terms "prevent", "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or one or more symptoms thereof. In certain embodiments, these terms refer to treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agents, prior to the onset of symptoms, particularly to subjects at risk of a disease or disorder provided herein. These terms encompass the inhibition or reduction of symptoms of a particular disease. In certain embodiments, subjects with a family history of a disease are potential candidates for a preventive regimen. In certain embodiments, subjects with a medical history of recurrent symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be used interchangeably with the term "prophylactic treatment".

[0166] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder or prevent its recurrence. A prophylactically effective amount of a compound refers to an amount of a therapeutic agent that alone, or in combination with one or more other agents, provides a prophylactic benefit in the prevention of a disease. The term "prophylactically effective amount" can include an amount that improves overall prophylaxis or enhances the prophylactic effectiveness of another prophylactic agent.

[0167] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic use, the effective amount will depend on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, the effective amount may be an amount sufficient to cause partial or complete restoration of function of a gene that is deficient in the subject. In some embodiments, an effective amount of a rAAV viral vector is an amount sufficient to cause expression of a gene in a subject such that PKP2 is produced. In some embodiments, an effective amount is an amount necessary to increase galactose metabolism in a subject in need thereof. Those skilled in the art will be able to determine the appropriate amount depending on these and other factors.

[0168] In some aspects, the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the target subject and the method of use. Those skilled in the art will be able to determine the effective amount based on these and other considerations. The effective amount may comprise, consist essentially of, or consist of one or more administrations of the composition, depending on the embodiment.

[0169] As used herein, the term "administer" or "administration" is intended to mean the delivery of a substance to a subject, such as an animal or human. Administration can be in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosages of administration are known to those skilled in the art and will vary with the composition used in the therapy, the purpose of the therapy, and the age, health, or sex of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern selected by the treating physician, or, in the case of pets and other animals, by the treating veterinarian.

[0170] Methods for determining the most effective means and dosage of administration are known to those skilled in the art and will vary with the composition used for therapy, the purpose of the therapy, and the subject being treated. Single or multiple administrations can be carried out with the dosage level and pattern selected by the treating physician. It should be noted that dosage can be affected by the route of administration. Suitable dosage formulations and methods of administering drugs are known in the art. A non-limiting example of such a suitable dosage is 10 mg / kg per administration. 9 As low as vector genome ~10 17 It can be as high as the vector genome.

[0171] In some embodiments of the methods described herein, the number of viral particles (e.g., rAAV viral vectors) administered to a subject is about 10 9 ~about 10 17 In some embodiments, the range is about 10 10 ~about 10 12 , about 10 11 ~about 10 13 , about 10 11 ~about 10 12 , about 10 11 ~about 10 14 , about 10 12 ~about 10 16 , about 10 13 ~about 10 16 , about 10 14 ~about 10 15 , about 5×10 11 ~Approx. 5×10 12 , or about 10 12 ~about 10 13 The viral particles are administered to the subject.

[0172] In some embodiments of the methods described herein, the number of viral particles (e.g., rAAV viral vectors) administered to a subject is at least about 10 10 , or at least about 10 11 , or at least about 10 12 , or at least about 10 13 , or at least about 10 14 , or at least about 10 15 , or at least about 1016 , or at least about 10 17 Each virus particle is called a virion.

[0173] In some embodiments, the rAAV vectors, rAAV viral vectors, compositions, and / or pharmaceutical compositions disclosed herein are capable of delivering approximately 1.0×10 11 Vector genome (vg) / kg ~ approx. 1.0 × 10 15 In some embodiments, the dose is administered to the subject at a dose ranging from about 1.0×10 12 vg / kg ~ approx. 1.0×10 14 In some embodiments, the dose is about 1.0×10 12 vg / kg ~ approx. 1.0×10 13 In some embodiments, the dose is administered in the range of about 1.0×10 12 vg / kg, approximately 1.5×10 12 vg / kg, approx. 2.0×10 12 vg / kg, approx. 2.5×10 12 vg / kg, approx. 3.0×10 12 vg / kg, approximately 4.0×10 12 vg / kg, approximately 4.5×10 12 vg / kg, approximately 5.0×10 12 vg / kg, approximately 5.5×10 12 vg / kg, approximately 6.0×10 12 vg / kg, approximately 6.5×10 12 vg / kg, approximately 7.0×10 12 vg / kg, approximately 7.5×10 12 vg / kg, approximately 8.0×10 12 vg / kg, approximately 8.5×10 12 vg / kg, approximately 9.0×10 12 vg / kg, approx. 9.5×10 12 vg / kg, approximately 1.0×10 13 vg / kg, approximately 1.5×10 13 vg / kg, approx. 2.0×10 13 vg / kg, approx. 2.5×10 13 vg / kg, approx. 3.0×10 13 vg / kg, approximately 4.0×10 13 vg / kg, approximately 4.5×10 13vg / kg, approximately 5.0×10 13 vg / kg, approximately 5.5×10 13 vg / kg, approximately 6.0×10 13 vg / kg, approximately 6.5×10 13 vg / kg, approximately 7.0×10 13 vg / kg, approximately 7.5×10 13 vg / kg, approximately 8.0×10 13 vg / kg, approximately 8.5×10 13 vg / kg, approximately 9.0×10 13 vg / kg, approx. 9.5×10 13 vg / kg, approximately 8.0×10 12 vg / kg, approximately 8.5×10 12 vg / kg, approximately 9.0×10 12 vg / kg, approx. 9.5×10 12 vg / kg, approximately 1.0×10 14 vg / kg, approximately 1.5×10 14 vg / kg, approx. 2.0×10 14 vg / kg, approx. 2.5×10 14 vg / kg, approx. 3.0×10 14 vg / kg, approximately 4.0×10 14 vg / kg, approximately 4.5×10 14 vg / kg, approximately 5.0×10 14 vg / kg, approximately 5.5×10 14 vg / kg, approximately 6.0×10 14 vg / kg, approximately 6.5×10 14 vg / kg, approximately 7.0×10 14 vg / kg, approximately 7.5×10 14 vg / kg, approximately 8.0×10 14 vg / kg, approximately 8.5×10 14 vg / kg, approximately 9.0×10 14 vg / kg, or approximately 9.5 × 10 14 vg / kg.

[0174] In some aspects, the amount of viral particles in a composition, pharmaceutical composition, or administered to a patient can be calculated based on the percentage of viral particles predicted to contain the viral genome.

[0175] In some aspects, the rAAV viral vectors of the present disclosure can be introduced into a subject intravenously, intrathecally, intracerebral, intraventricularly, intranasally, intratracheally, intraaurally, intraocularly or periocularly, orally, rectally, transmucosally, by inhalation, transdermal, parenterally, subcutaneously, intradermal, intramuscularly, intracisternally, intraneurally, intrathoracically, topically, intralymphatically, intracisternally, and such introduction can also be intraarterially, intracardiacly, intraventricularly, intravenous, intraretinal, intracerebral, intraretinal, intravitreal, intraarticularly, intraperitoneally, intrauterinely, intraneurally, or any combination thereof. In some aspects, the viral particles are delivered to a desired target tissue, such as, for example, cardiac tissue, as a non-limiting example. In some aspects, the delivery of the viral particles is systemic. The intracisternal administration route involves administering the drug directly into the cerebrospinal fluid of the ventricles of the brain. It can be performed by direct injection into the cisterna magna or via a permanently placed tube. In some embodiments, the rAAV viral vector of the present disclosure is administered parenterally. In some embodiments, the rAAV viral vector of the present disclosure is administered via intraperitoneal administration. In some embodiments, the rAAV viral vector of the present disclosure is administered intravenously.

[0176] In some embodiments, the rAAV viral vectors of the present disclosure repair a genetic defect in a subject. In some embodiments, the ratio of repaired target polynucleotides or polypeptides to unrepaired target polynucleotides or polypeptides in a successfully treated cell, tissue, organ, or subject is at least about 1.5:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 50:1, about 100:1, about 1000:1, about 10,000:1, about 100,000:1, or about 1,000,000:1. The amount or ratio of repaired target polynucleotides or polypeptides may be determined by any method known in the art, including, but not limited to, Western blot, Northern blot, Southern blot, PCR, sequencing, mass spectrometry, flow cytometry, immunohistochemistry, immunofluorescence, fluorescent in situ hybridization, next generation sequencing, immunoblot, and ELISA.

[0177] The administration of the rAAV vector, rAAV viral vector, composition, or pharmaceutical composition of the present disclosure can be performed in one dose, continuously, or intermittently throughout the course of treatment.In some embodiments, the rAAV vector, rAAV viral vector, composition, or pharmaceutical composition of the present disclosure is administered parenterally by injection, infusion, or implantation.In some embodiments, the rAAV vector, rAAV viral vector, composition, or pharmaceutical composition of the present disclosure is administered repeatedly.In some embodiments, the rAAV vector, rAAV viral vector, composition, or pharmaceutical composition of the present disclosure is administered in a single dose.

[0178] In some aspects, the rAAV viral vectors of the present disclosure exhibit enhanced tropism for cardiac tissue.

[0179] Manufacturing method The rAAV viral vector of the present disclosure can be produced using a variety of approaches. In some aspects, packaging is achieved by using a helper virus or helper plasmid and a cell line. The helper virus or helper plasmid contains elements and sequences that facilitate viral vector production. In another aspect, the helper plasmid is stably integrated into the genome of the packaging cell line, so that the packaging cell line does not require additional transfection with the helper plasmid.

[0180] In some aspects, the rAAV viral vectors of the present disclosure can be produced following baculovirus infection of insect cells.

[0181] In some embodiments, the cells are packaging or helper cell lines. In some embodiments, the helper cell lines are eukaryotic cells, such as HEK293 cells or 293T cells. In some embodiments, the helper cells are yeast cells or insect cells.

[0182] In some embodiments, the cell comprises a nucleic acid encoding a tetracycline activator protein and a promoter that regulates expression of the tetracycline activator protein. In some embodiments, the promoter that regulates expression of the tetracycline activator protein is a constitutive promoter. In some embodiments, the promoter is a phosphoglycerate kinase promoter (PGK) or a CMV promoter.

[0183] The helper plasmid can contain, for example, at least one viral helper DNA sequence derived from a replication-incompetent viral genome that encodes in trans all the virion proteins necessary to package replication-incompetent AAV without producing replication-incompetent AAV and to produce virion proteins capable of packaging replication-incompetent AAV at high titers.

[0184] Helper plasmids for packaging AAV are known in the art, see, for example, US Patent Publication No. 2004 / 0235174A1, which is incorporated herein by reference.As described herein, AAV helper plasmids can contain, as non-limiting examples, the Ad5 genes E2A, E4, and VA controlled by their respective native promoters or heterologous promoters as helper virus DNA sequences.In addition, AAV helper plasmids can contain expression cassettes for the expression of marker proteins, such as fluorescent proteins, to allow simple detection of transfection of desired target cells.

[0185] The present disclosure provides a method for producing a rAAV viral vector, comprising transfecting a packaging cell line with any one of the AAV helper plasmids disclosed herein and any one of the rAAV vectors disclosed herein. In some embodiments, the AAV helper plasmid and the rAAV vector are co-transfected into the packaging cell line. In some embodiments, the cell line is a mammalian cell line, for example, a human embryonic kidney (HEK) 293 cell line. The present disclosure provides a cell comprising any one of the rAAV vectors and / or rAAV viral vectors disclosed herein.

[0186] As used herein, the term "helper" in reference to a virus or plasmid refers to a virus or plasmid used to provide additional components necessary for replication and packaging of any one of the rAAV vectors disclosed herein. The components encoded by the helper virus can include any gene required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus or plasmid can encode an enzyme required for replication of the viral genome. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus). In some aspects, the pHELP plasmid can be a pHELPK plasmid in which the ampicillin expression cassette has been replaced with a kanamycin expression cassette.

[0187] As used herein, packaging cells (or helper cells) are cells used to produce viral vectors. To produce recombinant AAV viral vectors, Rep and Cap proteins provided in trans, as well as gene sequences from adenovirus that help AAV replication, are required. In some embodiments, the packaging / helper cells containing the plasmid are stably integrated into the genome of the cell. In other embodiments, the packaging cells can be transiently transfected. Typically, the packaging cells are eukaryotic cells, such as mammalian cells or insect cells.

[0188] kit The isolated polynucleotides, rAAV vectors, rAAV viral vectors, compositions, and / or pharmaceutical compositions described herein may be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. In some aspects, the kits of the present disclosure include any one of the isolated polynucleotides, rAAV vectors, rAAV viral vectors, compositions, pharmaceutical compositions, host cells, isolated tissues described herein.

[0189] In some embodiments, the kit further comprises instructions for use. Specifically, such kits may include one or more of the agents described herein, together with instructions describing the intended use and appropriate use of these agents. In some embodiments, the kit may include instructions for mixing one or more components of the kit, and / or for isolating and mixing samples and applying to a subject. In some embodiments, the agents of the kit are pharmaceutical formulations and dosages suitable for a particular use and method of administration of the agents. Kits for research purposes may contain components in appropriate concentrations or amounts for carrying out various experiments.

[0190] The kits may be designed to facilitate the use of the methods described herein and may take many forms. Each of the compositions of the kit may be provided in liquid form (e.g., solution) or solid form (e.g., dry powder), as applicable. In certain cases, some of the compositions may be configurable or processable (e.g., into an active form), for example, by the addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. In some aspects, the compositions may be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor® (Stem Cell Technologies, Vancouver, Canada). In some aspects, the preservation solution contains an amount of a metalloprotease inhibitor.

[0191] In some embodiments, the kit contains any one or more of the components described herein in one or more containers. Thus, in some embodiments, the kit may include a container-containing agent described herein. The drug may be in liquid, gel, or solid (powder) form. The drug may be sterilely prepared, packaged in a syringe, and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may have another drug that is sterilely prepared. Alternatively, the kit may include an active agent that is premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the components necessary to administer the drug to a subject, such as a syringe, topical application device, or IV needle tube and bag.

[0192] Further definitions It is specifically contemplated that the various features of the invention described herein can be used in any combination unless the context indicates otherwise. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein can be excluded or omitted. For illustrative purposes, if the present specification states that a complex comprises components A, B, and C, it is specifically contemplated that any of A, B, or C, or combinations thereof, alone or in any combination, can be omitted and eliminated.

[0193] Unless expressly stated otherwise, all specific aspects, embodiments, features, and terms are intended to include both the recited aspect, embodiment, feature, or term and biological equivalents thereof.

[0194] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Current Protocols In Molecular Biology (FMA Usubel, et al. eds., (1987)), the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)).

[0195] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) is to be interpreted as including the recited materials or steps and those that do not materially affect the basic and novel characteristics of the recited embodiment. Thus, the term "consisting essentially of" as used herein should not be interpreted as equivalent to "comprising." "Consisting of" is intended to mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. The aspects defined by each of these transitional terms are within the scope of the present disclosure. In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with any of the other two terms while maintaining their ordinary meaning.

[0196] When introducing elements of the present invention or preferred embodiments thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0197] It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Also, herein, values ​​or ranges may be expressed as "about," "about" from one particular value, and / or "about" to another particular value. When such values ​​or ranges are expressed, other embodiments disclosed include the particular values ​​recited, from the one particular value, and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It is further understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. In embodiments, "about" can be used to mean, for example, within 10% of the recited value, within 5% of the recited value, or within 2% of the recited value.

[0198] All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, or alternatively by a variation of + / - 15%, 10%, 5%, 2%, as appropriate. Although not always explicitly stated, all numerical designations are understood to be preceded by the term "about". Also, although not always explicitly stated, it is understood that the reagents described herein are merely exemplary and that equivalents of such are known in the art. As used herein, the term "about", when referring to a measurable value such as an amount or concentration, is meant to encompass a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0199] When used to describe the selection of any component, range, dosage form, etc. disclosed herein, the terms "acceptable," "effective," or "sufficient" intend that the component, range, dosage form, etc. is suitable for the purposes of the disclosure.

[0200] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0201] The term "combination" refers to either a fixed combination in one dosage unit form or a kit of parts for combined administration, where one or more active compounds and combination partners (e.g., another drug described below, also referred to as a "therapeutic agent" or "adjunct agent") can be administered simultaneously or separately within a time interval. In some situations, combination partners exhibit a cooperative effect, e.g., a synergistic effect. As used herein, terms such as "co-administration" or "combined administration" are meant to encompass administration of selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include therapeutic regimens in which agents are not necessarily administered by the same route of administration or at the same time.

[0202] As used herein, the term "pharmaceutical combination" refers to a product obtained by mixing or combining two or more active ingredients, including fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that both active ingredients, e.g., both compounds and combination partners, are administered to a patient at the same time in the form of a single entity or dose. The term "non-fixed combination" means that both active ingredients, e.g., both compounds and combination partners, are administered to a patient simultaneously, concurrently, or sequentially as separate entities without specific time constraints, where such administration provides the two compounds in a therapeutically effective concentration in the patient's body. The latter also applies to cocktail therapy, e.g., administration of three or more active ingredients.

[0203] Unless specifically recited, the term "host cell" includes eukaryotic host cells, including, for example, fungal cells, yeast cells, higher plant cells, insect cells, and mammalian cells. Non-limiting examples of eukaryotic host cells include monkey, bovine, porcine, murine, rat, avian, reptilian, and human, e.g., HEK293 cells and 293T cells.

[0204] As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free of other materials.

[0205] The "sequence" of a nucleic acid refers to the order and identity of nucleotides in a nucleic acid. The sequence is typically read in the 5' to 3' direction. The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of amino acid residues or nucleotides that are the same when compared and aligned to the closest match, e.g., using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) "Basic local alignment search tool" J. Mol. Biol. 215:403-410, Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet. 3:266-272, Madden et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141, Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and Annotation” Genome Res. 7:649-656. Many other optimal alignment algorithms are also known in the art and are optionally utilized to determine percent sequence identity.

[0206] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers that comprise, consist essentially of, or consist of purine and pyrimidine bases, or other naturally occurring, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

[0207] "Nucleic acid" or "nucleic acid molecule" refers to a polymeric compound containing two or more covalently linked nucleosides or nucleoside analogs with nitrogen-containing heterocyclic bases or base analogs, where the nucleosides are linked together by phosphodiester or other bonds to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and analogs thereof. The nucleic acid backbone can be composed of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages, phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of the nucleic acid can be ribose, deoxyribose, or similar compounds with known substituents (e.g., 2'-methoxy and 2'-halide substituents). The nitrogen-containing bases can be conventional bases (A, G, C, T, U), or their analogs (e.g., inosine, 5-methylisocytosine, isoguanine). Nucleic acids may contain only conventional sugars, bases, and linkages found in RNA and DNA, or may contain conventional building blocks and substituents (e.g., conventional bases linked by a 2'-methoxy backbone, or nucleic acids containing a mixture of conventional bases and one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNAs), in which one or more nucleotide monomers have a bicyclic furanose unit locked in RNA that mimics the sugar conformation, enhancing hybridization affinity to complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA). Nucleic acids may contain modified bases to alter the function or behavior of the nucleic acid (e.g., addition of a 3'-terminal dideoxynucleotide to block additional nucleotides from being added to the nucleic acid). Synthetic methods for making nucleic acids in vitro are well known in the art, but nucleic acids may be purified from natural sources using routine techniques. Nucleic acids may be single-stranded or double-stranded.

[0208] "Gene" refers to a polynucleotide that contains at least one open reading frame (ORF) capable of encoding a particular polypeptide or protein. "Gene product" or alternatively "gene expression product" refers to the amino acid sequence (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0209] Nucleic acids are typically single- or double-stranded and will generally contain phosphodiester bonds, although in some cases, as outlined herein, may contain, for example and without limitation, phosphoramide bonds (see Beaucage et al. (1993) Tetrahedron 49(10):1925 and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. Acids Res. 14:3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) Chemica Scripta 26:1419, phosphorothioates (Mag et al. (1991) Nucleic Acids Res. 19:1437 and U.S. Pat. No. 5,644,048, both of which are incorporated by reference), phosphorodithioates (Briu et al. (1989) J. Am. Chem. Soc. 111:2321, both of which are incorporated by reference), O-methyl phosphoramidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press (1992), each of which is incorporated by reference), and peptide nucleic acid backbones and linkages (Egholm (1992) J. Am. Chem. Soc. 114:1895, Meier et al. (1992) J. Am. Chem. Soc. 114:1895, each of which is incorporated by reference). al. (1992) Chem. Int. Ed. Engl. 31:1008; Nielsen (1993) Nature 365:566; and Carlsson et al. (1996) Nature 380:207.Other similar nucleic acids include those with positively charged backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92:6097, which are incorporated by reference), non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863, each of which is incorporated by reference; Angew (1991) Chem. Intl. Ed. English 30:423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597, Chapters 2 and 3, ASC Symposium Series, 1999). 580, "Carbohydrate Modifications in Antisense Research", Ed. YS Sanghvi and P. Dan Cook, Mesmaeker et al. (1994) Bioorganic & Medicinal Chem:Lett. 4:395, Jeffs et al. (1994) J. Biomolecular NMR 34:17, and Tetrahedron Lett. 37:743 (1996)), and those with non-ribose backbones including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. YS Sanghvi and P. Dan Cook, each of which references are incorporated by reference. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995) Chem. Soc. Rev. pp 169-176, incorporated by reference). Also, some nucleic acid analogs are described, for example, in Rawls, C & E News Jun. 2, 1997 page 35, incorporated by reference.These modifications of the ribose-phosphate backbone may be made to facilitate the addition of additional moieties, such as labels, or to alter the stability and half-life of such molecules in physiological environments.

[0210] In addition to the naturally occurring heterocyclic bases typically found in nucleic acids (e.g., adenine, guanine, thymine, cytosine, and uracil), nucleic acid analogs also include those with non-naturally occurring heterocyclic bases or modified bases, many of which are described or referenced herein. In particular, many non-naturally occurring bases are further described, for example, in Seela et al. (1991) Helv. Chim. Acta 74:1790, Grein et al. (1994) Bioorg. Med. Chem. Lett. 4:971-976, and Seela et al. (1999) Helv. Chim. Acta 82:1640, each of which is incorporated by reference. To further illustrate, certain bases used in nucleotides that act as melting temperatures (TO) modifiers are optionally included. For example, some of these include 7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.), pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC, etc.), etc. See, for example, "SYNTHESIS OF 7-DEAZA-2'-DEOXYGUANOSINE," published Nov. 23, 1999 to Seela, which is incorporated by reference. No. 5,990,303 entitled "Nucleotide-Based NUCLEOTIDES." Other representative heterocyclic bases include, for example, hypoxanthine, inosine, xanthine; 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine, and the 8-aza derivatives of xanthine; adenine, guanine, 2-aminopurine, 2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine, and These include 7-deaza-8-aza derivatives of xanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine; 5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil; 5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil; 5-ethynyluracil; 5-propynyluracil, and the like.

[0211] Examples of modified bases and nucleotides can also be found in, for example, "OLIGONUCLEOTIDES CONTAINING 5-PROPYLENE GLYCOL ET AL.," published Jan. 16, 1996 by Froehler et al., each of which is incorporated herein by reference.

[0212] No. 5,484,908 entitled "ENHANCED TRIPLE-HELIX AND DOUBLE-HELIX FORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES" issued on July 8, 1997 to Froehler et al., U.S. Patent No. 5,645,985 entitled "ENHANCED TRIPLE-HELIX AND DOUBLE-HELIX FORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES" issued on November 3, 1998 to Froehler et al., U.S. Patent No. 5,830,653 entitled "METHODS OF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES" issued on October 28, 2003 to Kochkine et al., U.S. Patent No. 6,639,059 entitled "SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES" issued on October 16, 2001 to Skouv, U.S. Patent No. 6,639,059 entitled "ONE STEP No. 6,303,315, entitled "SAMPLE PREPARATION AND DETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES," and U.S. Patent Application Publication No. 2003 / 0092905, published May 15, 2003, by Kochkine et al., entitled "SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES."

[0213] As used herein, "expression" refers to the two-step process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0214] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on being operably linked to elements that contribute to or facilitate the initiation of transcription. By "operably linked" it is intended that the polynucleotides are positioned in a manner that allows them to function within the cell. In one aspect, a promoter may be operably linked to a downstream sequence.

[0215] The term "encoding" as applied to a polynucleotide and / or nucleic acid sequence refers to a polynucleotide and / or a nucleic acid sequence that is said to "encode" a polynucleotide that, in its natural state or when manipulated by methods well known to those of skill in the art, can be transcribed to produce an mRNA of a polypeptide and / or fragment thereof. The antisense strand is the complementary strand of such a nucleic acid, and a coding sequence can be deduced therefrom.

[0216] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise, consist essentially of, or consist of a protein or peptide sequence. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0217] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is usually present at the N-terminus of a newly synthesized secretory or membrane polypeptide or protein. It acts to direct the polypeptide to a specific cellular location, for example, through the cell membrane, to the cell membrane, or to the nucleus. In some embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381, 5,958,736, and 8,795,965. In some embodiments, the signal peptide can be an IDUA signal peptide.

[0218] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecule, biological material, or cellular material and are intended to have minimal homology while still maintaining a desired structure or functionality. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, or at least about 99% identity to a reference polypeptide (e.g., a wild-type polypeptide), or polypeptides encoded by a polynucleotide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, at least about 97% sequence identity, or at least about 99% sequence identity to a reference polynucleotide (e.g., a wild-type polynucleotide).

[0219] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. The percent identity can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. If a position in the compared sequence is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences correlates to the number of matching positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 25% identity with one of the sequences of the present disclosure. Alignment and percent sequence identity for nucleic acid or amino acid sequences provided herein can be determined by importing the nucleic acid or amino acid sequence into and using ClustalW (available at https: / / genome.jp / tools-bin / clustalw / ). For example, the ClustalW parameters used to perform the protein sequence alignments found herein were generated using the Gonnet (for proteins) weight matrix. In some embodiments, the ClustalW parameters used to perform nucleic acid sequence alignments using the nucleic acid sequences found herein are generated using the ClustalW (for DNA) weight matrix.

[0220] As used herein, an amino acid modification can be an amino acid substitution, an amino acid deletion, or an amino acid insertion. An amino acid substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution. A conservative amino acid substitution (also called a conservative mutation, conservative substitution, or conservative variation) is an amino acid replacement in a protein that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, or size). As used herein, a "conservative variation" refers to the replacement of an amino acid residue with another biologically similar residue. Examples of conservative variations include the replacement of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another, or the replacement of one charged or polar residue with another, such as the replacement of arginine with lysine, glutamic acid with aspartic acid, and glutamine with asparagine. Other illustrative examples of conservative substitutions include changing alanine to serine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glycine to proline, histidine to asparagine or glutamine, lysine to arginine, glutamine, or glutamic acid, phenylalanine to tyrosine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and the like.

[0221] The polynucleotides disclosed herein may be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery," "gene transfer," "transduction," and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the transfer. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery, and various other techniques used for the transfer of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell, or is integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. As known in the art and described herein, several vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0222] A "plasmid" is a DNA molecule that is typically separated from chromosomal DNA and capable of replicating independently of chromosomal DNA. It is often circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and typically provide a selective advantage under a given environmental condition. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in competitive environmental niches, or alternatively, the proteins produced may act as toxins under similar circumstances. Although plasmid vectors often exist as extrachromosomal circular DNA molecules, it is known in the art that plasmid vectors can also be designed to stably integrate into host chromosomes either randomly or in a targeted manner, and such integration can be achieved using either circular plasmids or plasmids that are linearized prior to being introduced into the host cell.

[0223] "Plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids for such use are commercially available. The gene to be replicated is inserted into a copy of the plasmid, which contains a gene that makes the cell resistant to a particular antibiotic, and a multiple cloning site (MCS, or polylinker), a short region that contains several commonly used restriction sites, allowing easy insertion of DNA fragments at this position. Another major use of plasmids is to make large amounts of proteins. In this case, researchers can grow bacteria or eukaryotic cells that contain a plasmid with the gene of interest and induce the bacteria or eukaryotic cells to produce large amounts of the protein from the inserted gene.

[0224] In aspects in which gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide that comprises, consists essentially of, or consists of the viral genome or a portion thereof and the transgene.

[0225] The term "tissue" is used herein to refer to tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, diseased, and / or genetically altered. Biological tissues can include any single tissue (e.g., a collection of cells that may be interconnected), or a group of tissues that make up an organ, or a body part or region of an organism. Tissues can include, consist essentially of, or consist of homogenous cellular material, or can be composite structures such as those found in regions of the body including the chest, which can include, for example, lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, those derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary tree, duodenum, abdominal aorta, iliac vein, heart, and intestine, including any combination thereof. EXAMPLES

[0226] Example 1 Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primarily genetically based cardiac disease characterized by right and, more recently, left ventricular dysfunction and fibrofatty replacement of the myocardium leading to ventricular arrhythmias and sudden cardiac death in young people and athletes (1). ARVC accounts for 10% of sudden cardiac deaths in people ≤65 years of age and 24% in people ≤30 years of age (2,3). ARVC is thought to occur in 1 in 1,000–5,000 individuals, although the prevalence may be higher as some patients are undiagnosed or misdiagnosed due to poor diagnostic markers (4,5). Increasing evidence reveals earlier onset as the pediatric population, ranging from infants to teenagers, is also particularly vulnerable to ARVC (6–10), highlighting the critical need to identify and treat patients at an early stage of the disease.

[0227] Over the past decade, ARVC has been recognized as a disease of the cardiac desmosome (specialized intercellular junctions) since 40–50% of cases are associated with mutations / defects in multiple genes associated with the desmosome (desmoglein-2, desmocollin-2, plakoglobin, plakophilin-2, desmoplakin) and therefore is never considered a monogenic disease (11). Critical to the disease, mutations / defects in one component of the desmosome complex have devastating cascading effects on other members of the desmosome complex as well as other parts of the cardiac intercellular junctions (attachment fascial junctions associated with the contractile mechanism, and gap junctions associated with electrical coupling), driving the cardiac structural and electrical defects that underlie ARVC. The hierarchical dissolution of the desmosome complex along with functionally important adjacent cell junction components highlights the need for strategies that target restorative effects on the entire cardiac intercellular junction complex, not just the desmosome itself. The present disclosure provides studies demonstrating a single gene strategy (plakophilin-2) to reconstitute cardiac intercellular junction complexes and prevent ARVC disease development.

[0228] The present invention is a targeted gene therapy that delivers PKP2 cDNA and ultimately increases PKP2 protein levels in the heart (Figure 1A-C). This gene therapy utilizes cardioactive AAV serotype 9 (AAV9) or AAVrhlO and cardiac specific promoter cardiac troponin T to drive cardiomyocyte specific expression (Figure 1B-C, Figure 8A). PKP2 expression can effectively remodel cardiac desmosomes (Figure 2, Figure 3A-B, Figure 6F, Figure 7B, Figure 8A, 8E), which serves as a molecular scaffold to circumvent cardiac cell-cell junction defects underlying ARVC. PKP2 stabilizes desmosomes as well as other cell-cell junction complexes (gap junctions and adherens fascia junctions), which are downstream cascade defects underlying disease progression (Figure 2, Figure 3A-B, Figure 6F, Figure 7B, Figure 8A, 8E). AAV9 PKP2 gene therapy may function as a prevention by early delivery before disease onset, or to halt disease progression in patients with existing disease (Figures 3C, 4, 5, 6A-F, 7D, 7E, 8B-D).

[0229] The present disclosure provides studies showing that treatment of PKP2-containing AAV vectors improved cell-cell junction protein levels (PKP2, DSP, DSG2, JUP, CX43) in PKP2 mutant neonatal cardiomyocytes (Figure 1A, Figure 2). The present disclosure provides AAV9 or AAVrh10 vectors containing sequences encoding PKP2 that can normally express PKP2 under the control of the cardiac troponin T promoter in the heart in vivo (Figures 1B-C, Figure 8A). PKP2 mutant mice were injected with 5 x 10 of AAV9 PKP2 on postnatal day 2. 11A single intraperitoneal injection of 100 viral particles was performed (Figure 3). Four weeks after injection, we analyzed cardiac lysates via Western blot and found that AAV9 PKP2 administration could improve the levels of cell-cell junction proteins (PKP2, DSP, DSG2, JUP, CX43) (Figure 3B). Early AAV9 PKP2 injection could also prevent the development of ARVC disease at 4 weeks of age (Figure 3C), because there was preserved cardiac mechanical and electrical function, significantly less fibrosis in the myocardium, and prolonged survival (Figures 4, 5, 6A). Cell-cell junction protein levels, cardiac mechanical function, and cardiac electrical function were still preserved 6 months after AAV9 PKP2 injection (Figures 6B-6F), highlighting the sustained impact of PKP2 gene therapy on ARVC disease development. We also treated PKP2 mutant mice with AAV9 PKP2 at the time when all disease features were present (4 weeks of age) and showed improved cardiac intercellular junction proteins (PKP2, DSP, DSG2, JUP, N-Cad) and cardiac mechanical function (approximately 15% improvement in LV / RV ejection fraction) 2 weeks after AAV9 PKP2 injection into PKP2 mutant mice compared to PKP2 mutant mice that received AAV9 GFP (Figure 7).

[0230] Studies were also performed with AAVrhlO-encapsidated PKP2 rAAV vectors. These studies demonstrate that the AAV vectors of the present disclosure, including AAV9 and AAVrhlO vector serotypes, can stably express human PKP2 protein in adult mouse hearts and avoid ARVC disease outcomes in PKP2 Hom mice. Figure 8A shows Western blot analysis that hPKP2 can be expressed as early as 10 days in the hearts of adult wild-type control mice using AAV9 at a dose of 5e13vg / kg compared to uninjected wild-type controls (the higher migration size band corresponds to hPKP2, whereas the lower endogenous migration band corresponds to mouse PKP2). Western blot analysis further shows that expression of hPKP2 becomes more robust 21 days after infection in the hearts of adult PKP2 heterozygous (HET) mutant mice using AAVrhlO at a dose of 5e13vg / kg. Expression of hPKP2 could also be detected in the liver, albeit at a low level. Furthermore, AAVrh10-hPKP2 has an "on-target" effect as it stabilizes desmoplakin (DSP) levels, an immediate binding partner to PKP2, in adult PKP2 Het hearts. DSP levels are restored to control levels compared to wild-type control uninjected mice. GAPDH and beta-actin served as loading controls. These data indicate that AAV9 and AAVrh10-PKp2 constructs are expressed in adult mouse hearts and that AAVrh10-PKP2 functionally rescues defects in cardiac desmosomes in adult PKP2 Het mouse hearts.

[0231] Figure 8B shows 4-week survival curves after early administration (postnatal day 2 (P2)) of formulations and hPKP2 (via AAV9 and AAVrh10) in PKP2 Hom mice. Note that AAV9-hPKP2 and AAVrh10-hPKP2 are sufficient to prevent early death in PKP Hom mice, which is observed in formulation-treated PKP2 Hom mice. n=5 AAVrh10 hPKP2, n=5 Hom-AAV9 PKP2, n=5 formulations.

[0232] FIG. 8C shows surface ECG analysis of formulations and hPKP2 (via AAV9 and AAVrh10) and bar graph analysis of ectopic beats / premature ventricular contractions (PVCs) in PKP2 Hom mice after early administration (P2). Note that AAV9-hPKP2 (n=2) and AAVrh10-hPKP2 (n=4) are sufficient to prevent premature ventricular contractions in PKP Hom mice, as no PVCs were observed in these groups by 4 weeks of age. In contrast, early death was observed in two of the three formulation-treated PKP2 Hom mice at 4 weeks of age: n=4 AAVrh10 hPKP2, n=2 Hom-AAV9 PKP2, n=3 formulations.

[0233] FIG. 8D shows representative cardiac short-axis views of magnetic resonance images at end diastole from wild-type control untreated mice, formulation-treated PKP2 Hom, AAV9-hPKP2-treated PKP2 Hom, and AAVrh10-hPKP2-treated PKP2 Hom. The right ventricular (RV) and left ventricular (LV) dimensions of each group at end diastole are outlined in color. Note the significant reduction in RV and LV dimensions in AAVrh10-hPKP2-treated PKP2 Hom compared to formulation-treated mice. Note the significant reduction in LV dimensions in AAVrh10-hPKP2-treated PKP2 Hom compared to formulation-treated mice.

[0234] FIG. 8E shows Western blot analysis of PKP2 and cell-cell junction proteins (desmoplakin (DSP), desmoglein-2 (DSG2), connexin 43 (Cx43), N-cadherin (NCAD), plakoglobin (JUP)) in hearts from wild-type control untreated mice, formulation-treated PKP2 Hom, AAV9-hPKP2-treated PKP2 Hom, and AAVrh10-hPKP2-treated PKP2 Hom. Figure 8E shows Western blot analysis of PKP2 and cell-cell junction proteins (desmoplakin (DSP), desmoglein-2 (DSG2), connexin 43 (Cx43), N-cadherin (NCAD), plakoglobin (JUP)) in hearts from wild-type control untreated mice, formulation-treated PKP2 Hom, AAV9-hPKP2-treated PKP2 Hom, and AAVrh10-hPKP2-treated PKP2 Hom, highlighting prevention of cardiac cell-cell junction lysis in hPKP2-treated PKP2 Hom hearts. Note the significant upregulation in cardiac protein expression of DSP, DSG2, Cx43 (compared to its degraded form found in formulation-treated mice), JUP in Hom. GAPDH is used as a loading control. Note that formulation-treated mice that died are indicated by an asterisk.

[0235] Therapeutic drugs for ARVC: 1) PKP2 can be targeted to therapy for ARVC by generating adeno-associated viral vectors containing PKP2 cDNA as a means to restore PKP2 levels in ARVC patients. 2) PKP2 can be targeted for therapy in ARVC by developing novel direct pharmacological activators of PKP2 as a means to restore PKP2 function in ARVC patients. 3) PKP2 can be targeted for therapy of ARVC by developing or utilizing drugs that target pathways downstream of PKP2 function to restore cell-cell junction complex reorganization in ARVC patients.

[0236] References 1.Idris,A.,S.R.Shah,and K.Park,Right ventricular dysplasia:management and treatment in light of current evidence.J Community Hosp Intern Med Perspect,2018.8(3):p.101-106. 2.Elias Neto,J.,et al.,Arrhythmogenic Right Ventricular Cardiomyopathy / Dysplasia (ARVC / D)-What We Have Learned after 40 Years of the Diagnosis of This Clinical Entity.Arq Bras Cardiol,2019.112(1):p.91-103. 3.Wang,W.,C.A.James,and H.Calkins,Diagnostic and therapeutic strategies for arrhythmogenic right ventricular dysplasia / cardiomyopathy patient.Europace,2019.21(1):p.9-21. 4.Sen-Chowdhry,S.,et al.,Arrhythmogenic cardiomyopathy:etiology,diagnosis,and treatment.Annu Rev Med,2010.61:p.233-53 5.Tabib,A.,et al.,Undetected cardiac lesions cause unexpected sudden cardiac death during occasional sport activity.A report of 80 cases.Eur Heart J,1999.20(12):p.900-3. 6.Tabib,A.,et al.,Circumstances of death and gross and microscopic observations in a series of 200 cases of sudden death associated with arrhythmogenic right ventricular cardiomyopathy and / or dysplasia.Circulation,2003.108(24):p.3000-5. 7.Thiene,G.,D.Corrado,and C.Basso,Arrhythmogenic right ventricular cardiomyopathy / dysplasia.Orphanet J Rare Dis,2007.2:p.45. 8.Peters,S.,M.Trummel,and W.Meyners,Prevalence of right ventricular dysplasia-cardiomyopathy in a non-referral hospital.Int J Cardiol,2004.97(3):p.499-501. 9.Nishikawa,T.,et al.,Programmed cell death in the myocardium of arrhythmogenic right ventricular cardiomyopathy in children and adults.Cardiovasc Pathol,1999.8(4):p.185-9. 10.Wong,A.R.,et al.,Arrhythmogenic right ventricular cardiomyopathy in an 11-year-old girl and typical echocardiographic features.Pediatr Cardiol,2008.29(2):p.427-30. 11.Rey,C.,et al.,Life-saving automated external defibrillation in a teenager:a case report.J Med Case Rep,2007.1:p.76. 12.Daliento,L.,et al.,Arrhythmogenic right ventricular cardiomyopathy in young versus adult patients:similarities and differences.J Am Coll Cardiol,1995.25(3):p.655-64. 13.Garcia-Gras,E.,et al.,Suppression of canonical Wnt / beta-catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy.J Clin Invest,2006.116(7):p.2012-21. 14.Marcus,F.I.,S.Edson,and J.A.Towbin,Genetics of arrhythmogenic right ventricular cardiomyopathy:a practical guide for physicians.J Am Coll Cardiol,2013.61(19):p.1945-8. 15.Sen-Chowdhry,S.,et al.,Arrhythmogenic cardiomyopathy:etiology,diagnosis,and treatment.Annu Rev Med,2010.61:p.233-53. 16.Elliott,PM.,et al.,Definition and treatment of arrhythmogenic cardiomyopathy:an updated expert panel report.Eur J Heart Fail,2019.8:p.955-964. 17.Elias Neto,J.,et al.,Arrhythmogenic Right Ventricular Cardiomyopathy / Dysplasia(ARVC / D)-What We Have Learned after 40 Years of the Diagnosis of This Clinical Entity.Arq Bras Cardiol,2019.112(1):p.91-103.

Claims

1. A recombinant adeno-associated virus (rAAV) vector comprising, in a 5' to 3' direction: a) a first AAV ITR sequence; and b) a promoter sequence; and c) a transgene nucleic acid molecule, said transgene nucleic acid molecule comprising a nucleic acid sequence encoding a plakophilin-2 (PKP2) polypeptide; and d) a post-transcriptional regulatory element; and e) a polyA sequence; and f) a second AAV ITR sequence.

2. 3. An rAAV vector comprising a nucleic acid sequence as set forth in SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:

30.

3. The rAAV vector of claim 1 , wherein the PKP2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:

13.

4. The rAAV vector of claim 1 , wherein the nucleic acid sequence encoding the PKP2 polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO:

14.

5. 2. The rAAV vector of claim 1, wherein the first AAV ITR sequence and / or the second AAV ITR sequence comprises a nucleic acid sequence set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:

25.

6. The promoter sequence is a cardiac-specific promoter sequence, and optionally The promoter sequence may be selected from the group consisting of a Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, an H1 promoter, a CAG promoter, a hybrid chicken β-actin promoter, a MeCP2 promoter, an EF1 promoter, a ubiquitous chicken β-actin hybrid (CBh) promoter, a U1a promoter, , U1b promoter, MeCP2 promoter, MeP418 promoter, MeP426 promoter, minimal MeCP2 promoter, VMD2 promoter, mRho promoter, EF1a promoter, Ubc promoter, human β-actin promoter, TRE promoter, Ac5 promoter, polyhedrin promoter, CaMKIIa promoter, Gal1 promoter, TEF1 promoter, GDS promoter, ADH1 promoter, Ubi promoter, or alpha-1-antitrypsin (hAAT) promoter, and optionally The promoter sequence comprises a cardiac troponin T (cTnT) promoter sequence, and optionally 2. The rAAV vector of claim 1 , wherein the cTnT promoter sequence comprises the nucleic acid sequence set forth in SEQ ID NO:

2.

7. the poly A sequence comprises a rabbit beta-globin poly A sequence, and optionally 2. The rAAV vector of claim 1, wherein the rabbit beta-globin poly A sequence comprises the nucleic acid sequence set forth in SEQ ID NO:

6.

8. the post-transcriptional regulatory element is an oPRE post-transcriptional regulatory element, and optionally The rAAV vector of claim 1, wherein the oPRE post-transcriptional regulatory element comprises a nucleic acid sequence set forth in SEQ ID NO:5, SEQ ID NO:27, or SEQ ID NO:

28.

9. In the 5' to 3' direction, a) a first AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:7; b) a promoter sequence comprising the nucleic acid sequence set forth in SEQ ID NO:2; c) a transgene nucleic acid molecule, said transgene nucleic acid molecule comprising a nucleic acid sequence encoding a PKP2 polypeptide, said nucleic acid sequence encoding the PKP2 polypeptide comprising the nucleic acid sequence set forth in SEQ ID NO: 4; d) a post-transcriptional regulatory element comprising the nucleic acid sequence set forth in SEQ ID NO:5; e) a polyA sequence comprising the nucleic acid sequence set forth in SEQ ID NO:6; f) a second AAV ITR sequence comprising the nucleic acid sequence set forth in SEQ ID NO:

8.

10. (i) AAV capsid proteins; and (ii) the rAAV vector of claim 1, optionally comprising: rAAV viral vector, wherein the AAV capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein, or an AAVrhlO capsid protein.

11. a) A pharmaceutical composition comprising the rAAV vector of claim 1 or the rAAV viral vector of claim 10, and at least one pharma- ceutically acceptable excipient and / or additive.

12. A method for treating a subject having a disease and / or disorder in which the PKP2 gene is involved, the method comprising administering to the subject at least one therapeutically effective amount of a rAAV vector described in claim 1, or a rAAV viral vector described in claim 10, or a pharmaceutical composition described in claim 11.

13. 13. The method of claim 12, wherein the disease and / or disorder in which the PKP2 gene is involved is a cardiovascular disease characterized by abnormal cardiac intercellular junction complexes, optionally comprising: The method, wherein the disease and / or disorder in which the PKP2 gene is involved is arrhythmogenic right ventricular cardiomyopathy (ARVC).

14. The effective amount is improving electrical and structural cardiac integrity in said subject; Rescuing and reconstituting intercellular junction proteins in the subject; improving cardiac function in said subject; and / or Maintaining electrical and structural integrity in the subject to prevent ARVC; The method of claim 12.

15. The rAAV viral vector or the pharmaceutical composition has a concentration of about 1.0×10 １２ vg / kg ~ approx. 2.5×10 １４ 13. The method of claim 12, wherein the subject is administered a dose in the range of 0.01 to 0.05 vg / kg, optionally including: The method, wherein said rAAV viral vector or said pharmaceutical composition is administered to said subject at a dose ranging from about 1.0×10 12 vg / kg to about 5.0×10 13 vg / kg.

16. 13. The method of claim 12, wherein the rAAV viral vector or pharmaceutical composition is administered to the subject intravenously, intrathecally, intracerebrally, intraventricularly, intranasally, intratracheally, intraaurally, intraocularly or periocularly, orally, rectally, transmucosally, by inhalation, transdermal, parenterally, subcutaneously, intradermally, intramuscularly, intracisternally, intraneurally, intrapleurally, topically, intralymphatically, intracisternally, or intraneurally.

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