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US20220168447A1 - Plakophilin-2 (pkp2) gene therapy using aav vector - Google Patents

Plakophilin-2 (pkp2) gene therapy using aav vector Download PDF

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US20220168447A1
US20220168447A1 US17/670,390 US202217670390A US2022168447A1 US 20220168447 A1 US20220168447 A1 US 20220168447A1 US 202217670390 A US202217670390 A US 202217670390A US 2022168447 A1 US2022168447 A1 US 2022168447A1
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vector
promoter
pkp2
seq
aav
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US17/670,390
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Christopher Dean HERZOG
Chester Bittencort SACRAMENTO
Raj Prabhakar
David RICKS
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Spacecraft Seven LLC
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Spacecraft Seven LLC
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Priority to US17/670,390 priority Critical patent/US20220168447A1/en
Assigned to SPACECRAFT SEVEN, LLC reassignment SPACECRAFT SEVEN, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RICKS, David, SACRAMENTO, Chester Bittencort, HERZOG, Christopher Dean, PRABHAKAR, Raj
Publication of US20220168447A1 publication Critical patent/US20220168447A1/en
Priority to US18/399,438 priority patent/US20240390519A1/en
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • Arrhythmogenic right ventricular cardiomyopathy is a form of adult-onset heart disease, which impacts an estimated 1 in 1,000 to 1 in 1,250 people. It manifests as breakdown of the muscular wall of the heart (the myocardium) over time, which leads to increased risk of abnormal heartbeat (arrhythmia) and an increased risk of sudden death when an affected individual exercises strenuously. Individuals may also experience a sensation of fluttering or pounding in the chest (palpitations), light-headedness, fainting (syncope), shortness of breath, and abnormal swelling in the legs or abdomen. Over time, ARVC can lead to heart failure.
  • At least 13 genes are implicated in ARVC, many of which are involved in the biogenesis of desmosomes, which are intracellular junctions that provide strong adhesion between cells.
  • desmosomes When desmosomes fail to form properly, myocardial cells may detach from one another and die. The right ventricle in particular may develop weakness, while fatty deposits and scar tissue may replace the damaged myocardium, leading to distension of the right ventricle.
  • PGP2 Autosomal dominant plakophilin-2
  • cardiomyopathy is an inherited ARVC in which mutations affecting PKP2 are detected.
  • compositions and methods disclosed herein address this need.
  • the present invention relates generally to gene therapy for a disease or disorder, e.g., a cardiac disease or disorder, using a vector expressing PKP2 or a functional variant thereof.
  • the disclosure provides polynucleotide, comprising an expression cassette and optionally flanking adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein the polynucleotide comprises a polynucleotide sequence encoding a Plakophilin-2 (PKP2) or a functional variant thereof, operatively linked to a promoter.
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the promoter is a cardiac-specific promoter.
  • the promoter is a muscle-specific promoter.
  • the promoter is a cardiomyocyte-specific promoter.
  • the promoter is a Myosin Heavy-chain Creatine Kinase 7 (MHCK7) promoter.
  • the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 31.
  • the promoter is a cardiac troponin T (hTNNT2) promoter.
  • the hTNNT2 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 32.
  • the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene, wherein optionally the hTNNT2 promoter and exon 1 together share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 32.
  • the promoter is a ubiquitous promoter, optionally a CMV promoter or a CAG promoter.
  • the expression cassette comprises a polyA signal.
  • the polyA signal is a human growth hormone (hGH) polyA.
  • the expression cassette comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally a WPRE(x).
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the Plakophilin-2 (PKP2) or functional variant thereof is a PKP2.
  • the PKP2 is a functional PKP2.
  • the PKP2 is a human PKP2.
  • the PKP2 is PKP2 isoform A.
  • the PKP2 isoform A shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 1.
  • the PKP2 is PKP2 isoform B.
  • the PKP2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2.
  • the polynucleotide sequence encoding PKP2 is a human PKP2 polynucleotide.
  • the polynucleotide sequence encoding PKP2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 3.
  • the polynucleotide sequence encoding PKP2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 4.
  • the polynucleotide comprises at least about 4.0 kb, at least about 4.1 kb, at least about 4.2 kb, at least about 4.3 kb, at least about 4.4 kb, or at least about 4.5 kb.
  • the polynucleotide comprises at most about 4.1 kb, at most about 4.2 kb, at most about 4.3 kb, at most about 4.4 kb, at most about 4.5 kb, or at most about 4.6 kb.
  • the polynucleotide comprises 4.0 kb to 4.6 kb, 4.0 kb to 4.5 kb, or 4.0 kb to 4.4 kb or wherein the polynucleotide comprises 4.0 kb to 4.3 kb, 4.0 kb to 4.2 kb, or 4.0 kb to 4.1 kb.
  • the PKP2 or functional variant thereof comprises at least 800 or at least 830 amino acids.
  • the polynucleotide shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 8-15.
  • the expression cassette is flanked by 5′ and 3′ inverted terminal repeats (ITRs)
  • the ITRs are AAV2 ITRs and/or the ITRs share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NO: 20-26.
  • the disclosure provides a gene therapy vector, comprising the polynucleotide of any one of the preceding embodiments.
  • the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.
  • rAAV recombinant adeno-associated virus
  • the rAAV vector is an AAV9 or a functional variant thereof.
  • the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 77.
  • the rAAV vector is an AAVrh10 or a functional variant thereof.
  • the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 79.
  • the rAAV vector is an AAV6 or a functional variant thereof.
  • the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 78.
  • the rAAV vector is an AAVrh74 or a functional variant thereof.
  • the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 80.
  • the disclosure provides a method of treating and/or preventing a disease or disorder in a subject in need thereof, comprising administering the vector of any one of the preceding embodiments to the subject.
  • the disease or disorder is a cardiac disorder.
  • the disease or disorder is cardiomyopathy.
  • the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC).
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • the cardiomyopathy is hypertrophic cardiomyopathy or dilated cardiomyopathy.
  • the disease or disorder is characterized by fibrofatty infiltration of myocardium.
  • the disease or disorder is heart failure.
  • the subject is a mammal.
  • the subject is a primate.
  • the subject is a human.
  • the subject has a mutation in a PKP2 gene.
  • the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and/or cardiac catheterization.
  • the administration increases PKP2 expression by at least about 5%.
  • the administration increases PKP2 expression by at least about 30%.
  • the administration increases PKP2 expression by at least about 70%.
  • the administration increases PKP2 expression by about 5% to about 10%.
  • the administration increases PKP2 expression by about 30% to about 50%.
  • the administration increases PKP2 expression by about 50% to about 70%.
  • the administration increases PKP2 expression by about 70% to about 100%.
  • the method treats and/or prevents the disease or disorder.
  • the method comprises administering an effective amount of the vector.
  • the disease or disorder is related to or caused by loss of function in PKP2 in the subject.
  • the disease or disorder is related to or caused by gain of function in PKP2 in the subject.
  • the subject has a mutation that causes an amino acid substitution selected from p.Arg490Trp, Asp26Asn, Thr50 Va151SerfsX60, Arg79X, Tyr86X, Gln133X, Va1406SerfsX3, Tyr616X, Trp676X, Cys796Arg, Cys796E, Tyr807X, Glu62Lys, S688P, Trp848X, Y86X, V406X, Y616X, W848X, and Y807X, relative to a human PKP2 gene encoding a human PKP2 having the sequence of SEQ ID NO: 2.
  • the method comprises administering a pharmaceutical composition comprising an effective amount of the vector.
  • the method comprises administering between about 1 ⁇ 10 11 vector genomes and about 1 ⁇ 10 13 vector genomes of the vector to the subject, administering between about 1 ⁇ 10 12 vector genomes and about 1 ⁇ 10 14 vector genomes of the vector to the subject, or administering between about 1 ⁇ 10 13 vector genomes and about 1 ⁇ 10 15 vector genomes of the vector to the subject.
  • the disclosure provides a pharmaceutical composition comprising the vector of any one of the preceding embodiments.
  • the disclosure provides a kit comprising the vector of any one of the preceding embodiments or the pharmaceutical composition of the preceding embodiment and optionally instructions for use.
  • the disclosure provides a use of the vector of any one of the preceding embodiments in treating a disease or disorder, optionally according to the method of any one of the preceding embodiments.
  • the disclosure provides a vector according to any one of the preceding embodiments for use in treating a disease or disorder, optionally according to the method of any one of the preceding embodiments.
  • the disclosure provides a polynucleotide, comprising a polynucleotide sequences that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 12-15 and 89-92 or to any one of SEQ ID NOs: 8-11 and 93-96.
  • the promoter is a MHCK7 promoter.
  • the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 31.
  • the PKP2 is a human PKP2.
  • the PKP2 is PKP2 isoform A.
  • the PKP2 isoform A shares at least 80%, 90%, 95%, 99% or 100% identity with SEQ ID NO: 1.
  • the disclosure provides a gene therapy vector, comprising the polynucleotide of any one of the preceding embodiments.
  • the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.
  • rAAV recombinant adeno-associated virus
  • the rAAV vector is an AAV9 vector.
  • the rAAV vector is an AAVrh74 vector.
  • the disclosure provides a method of treating and/or preventing a cardiac disorder in a subject identified as having a mutation in the PRP2 gene, comprising administering the vector of any one of the preceding embodiments to the subject.
  • the cardiac disorder is cardiomyopathy, optionally arrhythmogenic right ventricular cardiomyopathy (ARVC), hypertrophic cardiomyopathy, or dilated cardiomyopathy.
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • the cardiac disorder is heart failure.
  • the subject is a mammal.
  • the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and/or cardiac catheterization.
  • the method prevents or reduces a decrease in left ventricle ejection fraction percentage (LVEF %), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject identified as having a mutation in the PRP2 gene.
  • LVEF % left ventricle ejection fraction percentage
  • the method prevents or reduces a decrease in left ventricle fractional shortening percentage (FS %), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject identified as having a mutation in the PRP2 gene.
  • FS % left ventricle fractional shortening percentage
  • the method prevents or reduces an increase in right ventricle area in millimeters squared (RV Area (mm2), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject identified as having a mutation in the PRP2 gene.
  • RV Area millimeters squared
  • the method prevents or reduces a decrease in right ventricle velocity time integral in millimeters per second (RV VTI (mm/sec), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject identified as having a mutation in the PRP2 gene.
  • RV VTI right ventricle velocity time integral in millimeters per second
  • the method prevents or reduces an increase in left ventricle or right ventricle fibrosis, optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject identified as having a mutation in the PKP2 gene.
  • FIG. 1 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 12.
  • the expression cassette is SEQ ID NO: 8.
  • the MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG. 2 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 13.
  • the expression cassette is SEQ ID NO: 9.
  • FIG. 3 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 14.
  • the expression cassette is SEQ ID NO: 10.
  • the MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG. 4 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 15.
  • the expression cassette is SEQ ID NO: 11.
  • FIGS. 5A-5B show PKP2 protein expression in transduced differentiated AC16 cells.
  • FIG. 5A shows Western Blots (WB) of PKP2 (top panel) or loading control, GAPDH (bottom panel).
  • FIG. 5B show a bar graph of the Western Blot.
  • the AAV vector serotype (AAV9 or rh74) and the promoter (MHCK7 or hTnT) are noted.
  • FIGS. 6A-6D show left ventricle ejection fraction percentage (LVEF %) for normal mice (left bars), untreated PKP2 knockout mice (middle bars, cKO PKP2), or treated mice (right bars).
  • FIG. 6A shows results with the AAV9 vector and MHCK7 promoter (AAV9-MHCK7).
  • FIG. 6B shows results with the AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7).
  • FIG. 6C shows results with the AAV9 vector and hTnT promoter (AAV9-hTnT).
  • FIG. 6D shows results with the AAVrh.74 vector and hTnT promoter (AAVrh.74-hTnT).
  • FIGS. 7A-7D show left ventricle fractional shortening percentage (FS %) for normal mice (left bars), untreated PKP2 knockout mice (middle bars, cKO PKP2), or treated mice (right bars).
  • FIG. 7A shows results with the AAV9 vector and MHCK7 promoter (AAV9-MHCK7).
  • FIG. 7B shows results with the AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7).
  • FIG. 7C shows results with the AAV9 vector and hTnT promoter (AAV9-hTnT).
  • FIG. 7D shows results with the AAVrh.74 vector and the hTnT promoter (AAVrh.74-hTnT).
  • FIGS. 8A-8D show right ventricle area in millimeters squared (RV Area (mm 2 )) for normal mice (left bars), untreated PKP2 knockout mice (middle bars, cKO PKP2), or treated mice (right bars).
  • FIG. 8A shows results with the AAV9 vector and MHCK7 promoter (AAV9-MHCK7).
  • FIG. 8B shows results with the AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7).
  • FIG. 8C shows results with the AAV9 vector and hTnT promoter (AAV9-hTnT).
  • FIG. 8D shows results with the AAVrh.74 vector and hTnT promoter (AAVrh.74-hTnT).
  • FIGS. 9A-9D show right ventricle velocity time integral in millimeters per second (RV VTI (mm/sec)) for normal mice (left bars), untreated PKP2 knockout mice (middle bars, cKO PKP2), or treated mice (right bars).
  • FIG. 9A shows results with the AAV9 vector and MHCK7 promoter (AAV9-MHCK7).
  • FIG. 9B shows results with the AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7).
  • FIG. 9C shows results with the AAV9 vector and hTnT promoter (AAV9-hTnT).
  • FIG. 9D shows results with the AAVrh.74 vector and hTnT promoter (AAVrh.74-hTnT).
  • FIGS. 10A-10B illustrate the degree of fibrosis in left and right ventricles based on quantitation of the Percent Collagen following trichrome histological staining of the heart.
  • Control animals without the conditional PKP2 gene knock-out (Cre Neg group) were found to have very little collagen, while control PKP2 knock-out animals receiving Formulation Buffer (CKO FB group) were found to have substantially greater proportion of collagen in both left and right ventricles.
  • FIG. 10A is a bar graph of percent collagen in the left ventricle.
  • FIG. 10B is a bar graph of percent collagen in the right ventricle.
  • FIG. 11 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 89.
  • the expression cassette is SEQ ID NO: 93.
  • the MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG. 12 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 90.
  • the expression cassette is SEQ ID NO: 94.
  • FIG. 13 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 91.
  • the expression cassette is SEQ ID NO: 95.
  • the MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG. 14 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 92.
  • the expression cassette is SEQ ID NO: 96.
  • the present disclosure provided gene therapy vectors for PKP2 that deliver a polynucleotide encoding a PKP2 polypeptide or a functional variant thereof, along with methods of use, and other compositions and methods.
  • the disclosure relates to a gene therapy vector comprising a promoter sequence operatively linked to a polynucleotide encoding a PKP2 polypeptide or a functional variant thereof.
  • the promoter is a Myosin Heavy-chain Creatine Kinase 7 (MHCK7) promoter.
  • the AAV vector is an AAV9 vector.
  • the promoter is an MHCK7 promoter and the AAV vector is an AAV9 vector.
  • the promoter is a hTNNT2 promoter. In some embodiment, the promoter is an hTNNT2 promoter and the AAV vector is an AAV9 vector. In some embodiments, the PKP2 is human PKP2a. In some embodiments, the PKP2 is human PKP2b. In some embodiment, the AAV vector is an rh74 vector. In some embodiments, the promoter is an MHCK7 promoter and the AAV vector is an rh74 vector. In some embodiments, the promoter is a hTNNT2 promoter. In some embodiment, the promoter is an hTNNT2 promoter and the AAV vector is an rh74 vector. In some embodiments, the PKP2 is human PKP2a. In some embodiments, the PKP2 is human PKP2b.
  • This disclosure further provides methods of treating a disorder or disorder in a subject by administering a gene therapy vector of the disclosure to the subject.
  • the disorder or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC).
  • the subject being treated is an ARVC patient having one or more mutation in a PKP2 gene. More than half of ARVC patients carry mutations in the desmosomal gene PKP2 encoding the protein Plakophilin-2 (PKP2). PKP2 is also associated with Brugada syndrome (BrS) and idiopathic ventricular fibrillation. It is a member of the armadillo repeat and plakophilin protein family. The protein contains nine central, conserved armadillo repeat domains flanked by N-terminal and C-terminal domains. It functions to link cadherins to intermediate filaments in the cytoskeleton
  • Plakophilin 2 localizes to cell desmosomes and nuclei and binds plakoglobin, desmoplakin, and the desmosomal cadherins via an N-terminal head domain.
  • PKP2 provides a lateral stabilizing force with the desmosomal-intermediate filament assembly facilitating cell-to-cell contact. It may also serve roles in intracellular signaling regulation, electrophysiologic and trafficking regulation, and control of transcription processes.
  • a polynucleotide encoding a PKP2 or functional variant thereof, wherein the PKP2 or functional variant thereof comprises at least 800 or at least 830 amino acids (e.g., no C terminal truncation at Arg-735), may be employed in generating a gene therapy vector.
  • the resulting vector may be employed in treating diseases or disorders, e.g. a PKP2-related disease or disorder, e.g. ARVC, Brugada syndrome (BrS), idiopathic ventricular fibrillation, dilated cardiomyopathy (DCM), and others.
  • diseases or disorders e.g. a PKP2-related disease or disorder, e.g. ARVC, Brugada syndrome (BrS), idiopathic ventricular fibrillation, dilated cardiomyopathy (DCM), and others.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • the term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%.
  • the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated.
  • the use of the alternative e.g., “or” should be understood to mean either one, both, or any combination thereof of the alternatives.
  • the term “and/or” should be understood to mean either one, or both of the alternatives.
  • the terms “include” and “comprise” are used synonymously.
  • identity refers, with respect to a polypeptide or polynucleotide sequence, to the percentage of exact matching residues in an alignment of that “query” sequence to a “subject” sequence, such as an alignment generated by the BLAST algorithm. Identity is calculated, unless specified otherwise, across the full length of the subject sequence.
  • a query sequence “shares at least x % identity to” a subject sequence if, when the query sequence is aligned to the subject sequence, at least x % (rounded down) of the residues in the subject sequence are aligned as an exact match to a corresponding residue in the query sequence.
  • residues denoted X residues denoted X
  • Sequence alignments may be performed using the NCBI Blast service (BLAST+ version 2.12.0).
  • operatively linked refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter sequence is operatively linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operatively linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • an “AAV vector” or “rAAV vector” refers to a recombinant vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV inverted terminal repeat sequences (ITRs).
  • AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a plasmid encoding and expressing rep and cap gene products.
  • AAV vectors can be packaged into infectious particles using a host cell that has been stably engineered to express rep and cap genes.
  • an “AAV virion” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector.
  • the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector.”
  • production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle.
  • promoter refers to a polynucleotide sequence capable of promoting initiation of RNA transcription from a polynucleotide in a eukaryotic cell.
  • vector genome refers to the polynucleotide sequence packaged by the vector (e.g., an rAAV virion), including flanking sequences (in AAV, inverted terminal repeats).
  • expression cassette and “polynucleotide cassette” refer to the portion of the vector genome between the flanking ITR sequences. “Expression cassette” implies that the vector genome comprises at least one gene encoding a gene product operatively linked to an element that drives expression (e.g., a promoter).
  • the term “patient in need” or “subject in need” refers to a patient or subject at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a recombinant gene therapy vector or gene editing system disclosed herein.
  • a patient or subject in need may, for instance, be a patient or subject diagnosed with a disorder associated with heart.
  • a subject may have a mutation in an PKP2 gene or deletion of all or a part of PKP2 gene, or of gene regulatory sequences, that causes aberrant expression of the PKP2 protein.
  • Subject and “patient” are used interchangeably herein.
  • the subject treated by the methods described herein may be an adult or a child. Subjects may range in age.
  • variant refers to a protein that has one or more amino-acid substitution, insertion, or deletion as compared to a parental protein.
  • functional variant refers to a protein that has one or more amino-acid substitution, insertion, or deletion as compared to a parental protein, and which retains one or more desired activities of the parental protein.
  • treating refers to ameliorating one or more symptoms of a disease or disorder.
  • preventing refers to delaying or interrupting the onset of one or more symptoms of a disease or disorder or slowing the progression of PKP2-related disease or disorder, e.g., arrhythmogenic right ventricular cardiomyopathy (ARVC).
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • Adeno-associated virus is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including two ⁇ 145-nucleotide inverted terminal repeat (ITRs).
  • ITRs inverted terminal repeat
  • serotypes when classified by antigenic epitopes.
  • the nucleotide sequences of the genomes of the AAV serotypes are known.
  • 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 Srivastava et al., J.
  • the sequence of the AAVrh.74 genome is provided in U.S. Pat. No. 9,434,928, incorporated herein by reference.
  • Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs.
  • Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes.
  • the two rep promoters (p5 and p19), coupled with the differential splicing of the 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.
  • Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.
  • the cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3.
  • Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins.
  • a single consensus polyadenylation 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).
  • AAV possesses unique features 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 noncytopathic, and natural infection of humans and other animals is silent and asymptomatic.
  • AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo.
  • AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).
  • the AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible.
  • the 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 replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA.
  • the rep and cap proteins may be provided in trans.
  • Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65° C. for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.
  • Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology.
  • viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins, which mediate cell transduction.
  • Such recombinant viruses may be produced by techniques known in the art, e.g., by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • Typical examples of virus packaging cells include but are not limited to HeLa cells, SF9 cells (optionally with a baculovirus helper vector), 293 cells, etc.
  • a Herpesvirus-based system can be used to produce AAV vectors, as described in US20170218395A1.
  • Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95/14785, WO96/22378, U.S. Pat. Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056 and WO94/19478, the complete contents of each of which is hereby incorporated by reference.
  • the present disclosure contemplates compositions and methods of use related to Plakophilin-2 (PKP2) proteins or polypeptides.
  • PPKP2 Plakophilin-2
  • Various mutations in PKP2 are known to be associated with cardiomyopathy and heart failure, including diseases like those described in van Tintelen et al. Circulation 113:1650-58 (2006); Novelli Front. Cardiovasc. Med . (2008); and in other sources.
  • Viral vector-mediated delivery of the PKP2 gene may therefore serve as a viable therapeutic for PKP2-related human diseases such as cardiomyopathy and heart failure.
  • PKP2 gene More than 230 mutations in the PKP2 gene have been identified in people with arrhythmogenic right ventricular cardiomyopathy (ARVC). (See “PKP2 gene,” MedlinePlus). This condition most commonly affects the myocardium surrounding the right ventricle, one of the two lower chambers of the heart. ARVC increases the risk of an abnormal heartbeat (arrhythmia) and sudden death.
  • Some PKP2 gene mutations lead to the production of an abnormally short version of plakophilin 2.
  • Other mutations alter the structure of plakophilin 2 by adding, deleting, or changing one or more of its protein building blocks (amino acids). Studies suggest that the altered protein impairs the formation and function of desmosomes.
  • PKP2-related disease may be found in the following references: Bonne et al. Genomics 51:452-454 (1998) [PubMed: 9721216]; Bonne et al. Cytogenet. Cell Genet.
  • PKP2a-837 amino acids (SEQ ID NO: 1) 1 MAAPGAPAEY GYIRTVLGQQ ILGQLDSSSL ALPSEAKLKL 41 AGSSGRGGQT VKSLRIQEQV QQTLARKGRS SVGNGNLHRT 81 SSVPEYVYNL HLVENDFVGG RSPVPKTYDM LKAGTTATYE 121 GRWGRGTAQY SSQKSVEERS LRHPLRRLEI SPDSSPERAH 161 YTHSDYQYSQ RSQAGHTLHH QESRRAALLV PPRYARSEIV 201 GVSRAGTTSR QRHFDTYHRQ YQHGSVSDTV FDSIPANPAL 241 LTYPRPGTSR SMGNLLEKEN YLTAGLTVGQ VRPLVPLQPV 281 TQNRASRSSW HQSSFHSTRT LREAGPSVAV DSSGRRAHLT 321 VGQAAAGGSG NLLTERSTFT DSQLGNADME MTLERAVSML
  • R735X is numbered according to the PKP2b isoform.
  • the R735X mutant of PKP2a is 690 amino acids in length, due to C-terminal truncation at Arg-690, (Arg-735 relative to PKP2b, SEQ ID NO: 2).
  • the PKP2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the PKP2 protein is a wild-type or native PKP2 protein, e.g. human PKP2a or human PKP2b.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) virion, comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an PKP2 or a functional variant thereof, operatively linked to a promoter.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) virion, comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an PKP2, operatively linked to a promoter.
  • the polynucleotide encoding the PKP2a may comprise a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3.
  • the polynucleotide encoding the PKP2b may comprise a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
  • the polynucleotide sequence encoding the vector genome may comprise a Kozak sequence, including but not limited to GCCACCATGG (SEQ ID NO: 5).
  • Kozak sequence may overlap the polynucleotide sequence encoding an PKP2a protein or a functional variant thereof.
  • the vector genome may comprise a polynucleotide sequence (with first ten nucleotides constituting the Kozak sequence) at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6.
  • Kozak sequence may overlap the polynucleotide sequence encoding an PKP2b protein or a functional variant thereof.
  • the vector genome may comprise a polynucleotide sequence (with first ten nucleotides constituting the Kozak sequence) at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • the Kozak sequence is an alternative Kozak sequence comprising or consisting of any one of:
  • the vector genome comprises no Kozak sequence.
  • the polynucleotide sequence may be codon-optimized.
  • the vector genome may comprise a polynucleotide sequence encoding a PKP2a that shares at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 87.
  • the vector genome may comprise a polynucleotide sequence encoding a PKP2b that shares at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 88.
  • the AAV virions of the disclosure comprise a vector genome.
  • the vector genome may comprise an expression cassette (or a polynucleotide cassette for gene-editing applications not requiring expression of the polynucleotide sequence).
  • Any suitable inverted terminal repeats (ITRs) may be used.
  • the ITRs may be AAV ITRs from the same serotype as the capsid present in the AAV virion, or a different serotype from the capsid (e.g., AAV2 ITRs may be used with an AAV virion having an AAV9 capsid or an AAVrh74 capsid). In each case, the serotype of the capsid determines the name applied to the virion.
  • the ITR are generally the most 5′ and most 3′ elements of the vector genome.
  • the vector genome will also generally contain, in 5′ to 3′ order, a promoter, a transgene, 3′ untranslated region (UTR) sequences (e.g., a WPRE element), and a polyadenylation sequence.
  • the vector genome includes an enhancer element (generally 5′ to the promoter) and/or an exon (generally 3′ to the promoter).
  • the vector genomes of the disclosure encode a partial or complete transgene sequence used as a repair template in a gene editing system.
  • the vector genome may comprise an exogenous promoter, or the gene editing system may insert the transgene into a locus in the genome having an endogenous promoter, such as a cardiac- or myocyte-specific promoter.
  • the 5′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20.
  • the 5′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21.
  • the 5′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22)
  • the 5′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23.
  • the 3′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24.
  • the 3′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25.
  • the 3′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26.
  • the vector genome comprises one or more filler sequences, e.g., at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.
  • the polynucleotide sequence encoding an PKP2 protein or functional variant thereof is operatively linked to a promoter.
  • the promoter is an MHCK7 promoter.
  • the promoter is an TNNT2 promoter.
  • Promoters useful in embodiments of the present disclosure include, without limitation, a cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, or a promoter sequence comprised of the CMV enhancer and portions of the chicken beta-actin promoter and the rabbit beta-globin gene (CAG).
  • CMV cytomegalovirus
  • PGK phosphoglycerate kinase
  • CAG rabbit beta-globin gene
  • the promoter may be a synthetic promoter.
  • Exemplary synthetic promoters are provided by Schlabach et al. PNAS USA. 107(6):2538-43 (2010).
  • the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30.
  • a polynucleotide sequence encoding an PKP2 protein or functional variant thereof is operatively linked to an inducible promoter.
  • An inducible promoter may be configured to cause the polynucleotide sequence to be transcriptionally expressed or not transcriptionally expressed in response to addition or accumulation of an agent or in response to removal, degradation, or dilution of an agent.
  • the agent may be a drug.
  • the agent may be tetracycline or one of its derivatives, including, without limitation, doxycycline.
  • the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically-regulated promoter, a physically-regulated promoter (i.e., a promoter that responds to presence or absence of light or to low or high temperature).
  • Inducible promoters include heavy metal ion inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and the promoters from T7 phage which are active in the presence of T7 RNA polymerase. This list of inducible promoters is non-limiting.
  • the promoter is a tissue-specific promoter, such as a promoter capable of driving expression in a cardiac cell to a greater extent than in a non-cardiac cell.
  • tissue-specific promoter is a selected from any various cardiac cell-specific promoters including but not limited to, desmin (Des), alpha-myosin heavy chain ( ⁇ -MHC), myosin light chain 2 (MLC-2), cardiac troponin C (cTnC), cardiac troponin T (hTNNT2), muscle creatine kinase (CK) and combinations of promoter/enhancer regions thereof, such as MHCK7.
  • the promoter is a ubiquitous promoter.
  • a “ubiquitous promoter” refers to a promoter that is not tissue-specific under experimental or clinical conditions.
  • the ubiquitous promoter is any one of Cytomegalovirus (CMV), Cytomegalovirus early enhancer element chicken beta-Actin gene intron with the splice acceptor of the rabbit beta-Globin gene (CAG), ubiquitin C (UBC), Phosphoglycerate Kinase (PGK), Eukaryotic translation elongation factor 1 alpha 1 (EF1-alpha), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), simian virus 40 (SV40), Hepatitis B virus (HBV), chicken beta-actin, and human beta-actin promoters.
  • CMV Cytomegalovirus
  • CAG Cytomegalovirus early enhancer element chicken beta-Actin gene intron with the splice acceptor of the rabbit beta-Globin gene
  • UBC ubiquitin C
  • PGK Phos
  • the promoter sequence is selected from Table 3.
  • the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS: 31-51.
  • the promoter comprises a fragment of a polynucleotide sequence of any one of SEQ ID NOs: 31-51, e.g., a fragment comprising at least 25%, at least 50%, at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of SEQ ID NOs: 31-51.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33.
  • promoters are the SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements.
  • HSV tk Herpes Simplex Virus thymidine kinase
  • CMV cytomegalovirus
  • LTR elements various retroviral promoters including LTR elements.
  • a large variety of other promoters are known and generally available in the art, and the sequences of many such promoters are available in sequence databases such as the GenBank database.
  • vectors of the present disclosure further comprise one or more regulatory elements selected from the group consisting of an enhancer, an intron, a poly-A signal, a 2A peptide encoding sequence, a WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element), and a HPRE (Hepatitis B posttranscriptional regulatory element).
  • regulatory elements selected from the group consisting of an enhancer, an intron, a poly-A signal, a 2A peptide encoding sequence, a WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element), and a HPRE (Hepatitis B posttranscriptional regulatory element).
  • the vector comprises a CMV enhancer.
  • the vectors comprise one or more enhancers.
  • the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a (3-actin enhancer sequence, or an EF1- ⁇ enhancer sequence. Sequences of the foregoing are known in the art.
  • the sequence of the CMV immediate early (IE) enhancer is SEQ ID NO: 50.
  • the vectors comprise one or more introns.
  • the intron is a rabbit globin intron sequence, a chicken ⁇ -actin intron sequence, a synthetic intron sequence, an SV40 intron, or an EF1- ⁇ intron sequence.
  • the vectors comprise a polyA sequence.
  • the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence.
  • the poly-A signal may be a bovine growth hormone polyadenylation signal (bGHpA).
  • the vectors comprise one or more transcript stabilizing element.
  • the transcript stabilizing element is a WPRE sequence, a HPRE sequence, a scaffold-attachment region, a 3′ UTR, or a 5′ UTR.
  • the vectors comprise both a 5′ UTR and a 3′ UTR.
  • the vector comprises a 5′ untranslated region (UTR) selected from Table 4.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 51-61.
  • the vector comprises a 3′ untranslated region selected from Table 5.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 62-70.
  • the vector comprises a polyadenylation (polyA) signal selected from Table 6.
  • the polyA signal comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 71-75.
  • Illustrative vector genomes are depicted in FIGS. 1-4 and 11-14 ; and provided as SEQ ID NOs: 12-15 and 89-92.
  • the expression cassette of each sequence is SEQ ID NOs: 8-11 or 93-96.
  • the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 12-15 and 89-92, optionally with or without the ITR sequences.
  • the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 8-11 and 93-96.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; a PKPa transgene; an WPRE(x) element; an pAGH-HS sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; any one of SEQ ID NOs: 3, 6, and 87; SEQ ID NO: 63; and SEQ ID NO: 75; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; a PKPa transgene; an WPRE(x) element; an pAGH-HS sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 3, 6, and 87; SEQ ID NO: 63; and SEQ ID NO: 75; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; a PKPb transgene; an WPRE(x) element; an pAGH-HS sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; any one of SEQ ID NOs: 4, 7, and 88; SEQ ID NO: 63; and SEQ ID NO: 75; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; a PKPb transgene; an WPRE(x) element; an pAGH-HS sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 4, 7, and 88; SEQ ID NO: 63; and SEQ ID NO: 75; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; a PKPa transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; any one of SEQ ID NOs: 3, 6, and 87; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; a PKPa transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 3, 6, and 87; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; a PKPb transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; any one of SEQ ID NOs: 4, 7, and 88; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; a PKPb transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 4, 7, and 88; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; SV40 intron; a PKPa transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 3, 6, and 87; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; SV40 intron; a PKPa transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 3, 6, and 87; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; SV40 intron; a PKPb transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 4, 7, and 88; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; SV40 intron; a PKPb transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR.
  • the vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 4, 7, and 88; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing.
  • this vector genome is packaged in an AAV9 or AAVrh74 vector.
  • the PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • WPRE element may be present or absent.
  • AAV vectors useful in the practice of the present invention can be packaged into AAV virions (viral particles) using various systems including adenovirus-based and helper-free systems.
  • Standard methods in AAV biology include those described in Kwon and Schaffer. Pharm Res . (2008) 25(3):489-99; Wu et al. Mol. Ther . (2006) 14(3):316-27. Burger et al. Mol. Ther . (2004) 10(2):302-17; Grimm et al. Curr Gene Ther . (2003) 3(4):281-304; Deyle D R, Russell D W. Curr Opin Mol Ther. (2009) 11(4):442-447; McCarty et al. Gene Ther .
  • AAV DNA in the rAAV genomes may be from any AAV variant or serotype for which a recombinant virus can be derived including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAVrh10.
  • Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692.
  • Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. 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.
  • the rAAV comprises a self-complementary genome.
  • an rAAV comprising a “self-complementary” or “double stranded” genome refers to an rAAV which has been engineered such that the coding region of the rAAV is configured to form an intra-molecular double-stranded DNA template, as described in McCarty et al.
  • Self-complementary recombinant adeno-associated virus (scAAV) vectors promoter efficient transduction independently of DNA synthesis. Gene Therapy. 8 (16): 1248-54 (2001).
  • the present disclosure contemplates the use, in some cases, of an rAAV comprising a self-complementary genome because upon infection (such transduction), rather than waiting for cell mediated synthesis of the second strand of the rAAV genome, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription.
  • dsDNA double stranded DNA
  • the rAAV vector comprises a single stranded genome.
  • a “single standard” genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAVs have singled stranded DNA genomes. There have been some indications that rAAVs should be scAAVs to achieve efficient transduction of cells. The present disclosure contemplates, however, rAAV vectors that maybe have singled stranded genomes, rather than self-complementary genomes, with the understanding that other genetic modifications of the rAAV vector may be beneficial to obtain optimal gene transcription in target cells.
  • the present disclosure relates to single-stranded rAAV vectors capable of achieving efficient gene transfer to anterior segment in the mouse eye. See Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE 12(8): e0182473 (2017).
  • the rAAV vector is of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74.
  • Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692.
  • Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).
  • the rAAV vector is of the serotype AAV9.
  • said rAAV vector is of serotype AAV9 and comprises a single stranded genome. In some embodiments, said rAAV vector is of serotype AAV9 and comprises a self-complementary genome. In some embodiments, a rAAV vector comprises the inverted terminal repeat (ITR) sequences of AAV2. In some embodiments, the rAAV vector comprises an AAV2 genome, such that the rAAV vector is an AAV-2/9 vector, an AAV-2/6 vector, or an AAV-2/8 vector.
  • ITR inverted terminal repeat
  • AAV vectors may comprise wild-type AAV sequence or they may comprise one or more modifications to a wild-type AAV sequence.
  • an AAV vector comprises one or more amino acid modifications, optionally substitutions, deletions, or insertions, within a capsid protein, optionally VP1, VP2 and/or VP3.
  • the modification provides for reduced immunogenicity when the AAV vector is provided to a subject.
  • Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as endothelial cells or more particularly endothelial tip cells.
  • the rAAV is directly injected into the intracerebroventricular space of the subject.
  • the rAAV virion is an AAV2 rAAV virion.
  • the capsid many be an AAV2 capsid or functional variant thereof.
  • the AAV2 capsid shares at least 98%, 99%, or 100% identity to a reference AAV2 capsid, e.g., SEQ ID NO: 76.
  • the rAAV virion is an AAV9 rAAV virion.
  • the capsid many be an AAV9 capsid or functional variant thereof.
  • the AAV9 capsid shares at least 98%, 99%, or 100% identity to a reference AAV9 capsid, e.g., SEQ ID NO: 77.
  • the rAAV virion is an AAV6 rAAV virion.
  • the capsid many be an AAV9 capsid or functional variant thereof.
  • the AAV6 capsid shares at least 98%, 99%, or 100% identity to a reference AAV6 capsid, e.g., SEQ ID NO: 78.
  • the rAAV virion is an AAVrh.10 rAAV virion.
  • the capsid many be an AAV9 capsid or functional variant thereof.
  • the AAVrh.10 capsid shares at least 98%, 99%, or 100% identity to a reference AAVrh.10 capsid, e.g., SEQ ID NO: 79.
  • the capsid protein is encoded by a polynucleotide supplied on a plasmid in trans to the transfer plasmid.
  • the polynucleotide sequence of wild-type AAVrh74 cap is provided as SEQ ID NO: 80.
  • the disclosure further provides protein sequences for AAVrh74 VP1, VP2, and VP3, including SEQ ID NOs: 81-83, and homologs or functional variants thereof.
  • the AAVrh74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 81.
  • the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh74 VP1 which is set forth in SEQ ID NO: 81.
  • the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh74 VP2 which is set forth in SEQ ID NO: 82.
  • the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh74 VP3 which is set forth in SEQ ID NO: 83.
  • the rAAV virion is an AAV-PHP.B rAAV virion or a neutrotrophic variant thereof, such as, without limitation, those disclosed in Int'l Pat. Pub. Nos. WO 2015/038958 A1 and WO 2017/100671 A1.
  • the AAV capsid may comprise at least 4 contiguous amino acids from the sequence TLAVPFK (SEQ ID NO:85) or KFPVALT (SEQ ID NO:86), e.g., inserted between a sequence encoding for amino acids 588 and 589 of AAV9.
  • the capsid many be an AAV-PHP.B capsid or functional variant thereof.
  • the AAV-PHP.B capsid shares at least 98%, 99%, or 100% identity to a reference AAV-PHP.B capsid, e.g., SEQ ID NO: 84.
  • AAV capsids used in the rAAV virions of the disclosure include those disclosed in Pat. Pub. Nos. WO 2009/012176 A2 and WO 2015/168666 A2.
  • an AAV9 vector, AAVrh.74, or an AAVrh.10 vector will confer desirable cardiac tropism on the vector.
  • the present inventors have further determined that an AAV9 vector, AAVrh.74, or an AAVrh.10 vector may provide desired specificity to cardiac cells.
  • the disclosure provides pharmaceutical compositions comprising the rAAV virion of the disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • aqueous solutions For purposes of administration, optionally by injection, various solutions can be employed, such as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose.
  • Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as Poloxamer 188, e.g., at 0.001% or 0.01%.
  • a dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
  • the pharmaceutical forms suitable for injectable use include but are not limited to sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form is sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the certain methods of preparation are vacuum drying and the freeze-drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • the disclosure comprises a kit comprising an rAAV virion of the disclosure and instructions for use.
  • the disclosure provides a method of increasing PKP2 activity in a cell, comprising contacting the cell with an rAAV of the disclosure. In another aspect, the disclosure provides a method of increasing PKP2 activity in a subject, comprising administering to the subject an rAAV of the disclosure.
  • the cell and/or subject is deficient in PKP2 messenger RNA or PKP2 protein expression levels and/or activity and/or comprises a loss-of-function mutation in PKP2.
  • the cell may be a cardiac cell, e.g. a cardiomyocyte cell.
  • the subject is a mammal, e.g., a human.
  • the method promotes survival of cardiac cell, e.g. a cardiomyocyte cell, in cell culture and/or in vivo. In some embodiments, the method promotes and/or restores function of the heart.
  • cardiac cell e.g. a cardiomyocyte cell
  • the method promotes and/or restores function of the heart.
  • the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of an rAAV virion of the disclosure.
  • the disease or disorder is a cardiac disease or disorder.
  • Illustrative cardiac disorders include heart failure, arrhythmogenic right ventricular cardiomyopathy (ARVC), Brugada syndrome (BrS) and idiopathic ventricular fibrillation.
  • the subject suffers from or is at risk for arrhythmogenic right ventricular cardiomyopathy (ARVC).
  • the subject is a mammal, e.g., a human, having a loss-of-function mutation in a PKP2 gene.
  • treatment with the rAAV virion results in expression of the PKP2 protein encoded by the rAAV virion in the subject, e.g., in the subject's heart or cardiac tissue.
  • treatment with the rAAV virion results in at least two-fold, at least five-fold, at least ten-fold, or more PKP2 protein levels detectable in the subject's heart.
  • the AAV-mediated delivery of PKP2 protein to the heart may increase life span, prevent or attenuate cardiac cell degeneration, heart failure, scarring, reduced ejection fraction, arrythmia, angina, exercise intolerance, angina (chest pain), sudden cardiac death, exertional myalgias and cramps.
  • the AAV-mediated delivery of PKP2 protein to the heart may show improvement from, or prevent normal disease course detected by use of pathological electrocardiogram, cardiac MRI, heart biopsy, decrease in paroxysmal ventricular arrhythmias, decrease in sudden cardiac death, and/or decrease in or lack of further development of fibro-fatty deposits in right ventricular myocardium.
  • the methods of the disclosure may prevent a decrease in, restore, and/or increase right ventricular ejection fraction (RVEF).
  • the methods disclosed herein may provide efficient biodistribution in the heart. They may result in sustained in expression in all, or a substantial fraction of, cardiac cells, e.g., cardiomyocytes. Notably, the methods disclosed herein may provide long-lasting expression of PKP2 protein throughout the life of the subject following AAV vector administration. In some embodiments, PKP2 protein expression in response to treatment lasts at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 years.
  • Combination therapies are also contemplated by the invention. Combinations of methods of the invention with standard medical treatments (e.g., corticosteroids or topical pressure reducing medications) are specifically contemplated, as are combinations with novel therapies.
  • a subject may be treated with a steroid and/or combination of immune suppressing agents to prevent or to reduce an immune response to administration of a rAAV described herein.
  • the AAV vector is administered at a dose of between about 1 ⁇ 10 12 and 5 ⁇ 10 14 vector genomes (vg) or between about 1 ⁇ 10 12 and 6 ⁇ 10 14 vg of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered at a dose of between about 1 ⁇ 10 13 and 5 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered at a dose of between about 5 ⁇ 10 13 and 3 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered at a dose of between about 5 ⁇ 10 13 and 1 ⁇ 10 14 vg/kg.
  • the AAV vector is administered at a dose of less than about 1 ⁇ 10 12 vg/kg, less than about 3 ⁇ 10 12 vg/kg, less than about 5 ⁇ 10 12 vg/kg, less than about 7 ⁇ 10 12 vg/kg, less than about 1 ⁇ 10 13 vg/kg, less than about 3 ⁇ 10 13 vg/kg, less than about 5 ⁇ 10 13 vg/kg, less than about 7 ⁇ 10 13 vg/kg, less than about 1 ⁇ 10 13 vg/kg, less than about 3 ⁇ 10 14 vg/kg, less than about 5 ⁇ 10 14 vg/kg, less than about 7 ⁇ 10 14 vg/kg, less than about 1 ⁇ 10 15 vg/kg, less than about 3 ⁇ 10 15 vg/kg, less than about 5 ⁇ 10 15 vg/kg, or less than about 7 ⁇ 10 15 vg/kg.
  • the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74
  • the AAV vector is administered at a dose of about 1 ⁇ 10 12 vg/kg, about 3 ⁇ 10 12 vg/kg, about 5 ⁇ 10 12 vg/kg, about 7 ⁇ 10 12 vg/kg, about 1 ⁇ 10 13 vg/kg, about 3 ⁇ 10 13 vg/kg, about 5 ⁇ 10 13 vg/kg, about 7 ⁇ 10 13 vg/kg, about 1 ⁇ 10 14 vg/kg, about 3 ⁇ 10 14 vg/kg, about 5 ⁇ 10 14 vg/kg, about 7 ⁇ 10 14 vg/kg, about 1 ⁇ 10 15 vg/kg, about 3 ⁇ 10 15 vg/kg, about 5 ⁇ 10 15 vg/kg, or about 7 ⁇ 10 15 vg/kg.
  • the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • the AAV vector is administered at a dose of 1 ⁇ 10 12 vg/kg, 3 ⁇ 10 12 vg/kg, 5 ⁇ 10 12 vg/kg, 7 ⁇ 10 12 vg/kg, 1 ⁇ 10 13 vg/kg, 3 ⁇ 10 13 vg/kg, 5 ⁇ 10 13 vg/kg, 7 ⁇ 10 13 vg/kg, 1 ⁇ 10 14 vg/kg, 3 ⁇ 10 14 vg/kg, 5 ⁇ 10 14 vg/kg, 7 ⁇ 10 14 vg/kg, 1 ⁇ 10 15 vg/kg, 3 ⁇ 10 15 vg/kg, 5 ⁇ 10 15 vg/kg, or 7 ⁇ 10 15 vg/kg.
  • the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • the AAV vector is administered systemically at a dose of between about 1 ⁇ 10 12 and 5 ⁇ 10 14 vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered systemically at a dose of between about 1 ⁇ 10 13 and 5 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of between about 5 ⁇ 10 13 and 3 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of between about 5 ⁇ 10 13 and 1 ⁇ 10 14 vg/kg.
  • the AAV vector is administered systemically at a dose of less than about 1 ⁇ 10 12 vg/kg, less than about 3 ⁇ 10 12 vg/kg, less than about 5 ⁇ 10 12 vg/kg, less than about 7 ⁇ 10 12 vg/kg, less than about 1 ⁇ 10 13 vg/kg, less than about 3 ⁇ 10 13 vg/kg, less than about 5 ⁇ 10 13 vg/kg, less than about 7 ⁇ 10 13 vg/kg, less than about 1 ⁇ 10 14 vg/kg, less than about 3 ⁇ 10 14 vg/kg, less than about 5 ⁇ 10 14 vg/kg, less than about 7 ⁇ 10 14 vg/kg, less than about 1 ⁇ 10 15 vg/kg, less than about 3 ⁇ 10 15 vg/kg, less than about 5 ⁇ 10 15 vg/kg, or less than about 7 ⁇ 10 15 vg/kg.
  • the AAV vector delivered at any of these doses is an AAV9 vector or an AAV r
  • the AAV vector is administered systemically at a dose of about 1 ⁇ 10 12 vg/kg, about 3 ⁇ 10 12 vg/kg, about 5 ⁇ 10 12 vg/kg, about 7 ⁇ 10 12 vg/kg, about 1 ⁇ 10 13 vg/kg, about 3 ⁇ 10 13 vg/kg, about 5 ⁇ 10 13 vg/kg, about 7 ⁇ 10 13 vg/kg, about 1 ⁇ 10 14 vg/kg, about 3 ⁇ 10 14 vg/kg, about 5 ⁇ 10 14 vg/kg, about 7 ⁇ 10 14 vg/kg, about 1 ⁇ 10 15 vg/kg, about 3 ⁇ 10 15 vg/kg, about 5 ⁇ 10 15 vg/kg, or about 7 ⁇ 10 15 vg/kg.
  • the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • the AAV vector is administered systemically at a dose of 1 ⁇ 10 12 vg/kg, 3 ⁇ 10 12 vg/kg, 5 ⁇ 10 12 vg/kg, 7 ⁇ 10 12 vg/kg, 1 ⁇ 10 13 vg/kg, 3 ⁇ 10 13 vg/kg, 5 ⁇ 10 13 vg/kg, 7 ⁇ 10 13 vg/kg, 1 ⁇ 10 14 vg/kg, 3 ⁇ 10 14 vg/kg, 5 ⁇ 10 14 vg/kg, 7 ⁇ 10 14 vg/kg, 1 ⁇ 10 15 vg/kg, 3 ⁇ 10 15 vg/kg, 5 ⁇ 10 15 vg/kg, or 7 ⁇ 10 15 vg/kg.
  • the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • the AAV vector is administered intravenously at a dose of between about 1 ⁇ 10 12 and 5 ⁇ 10 14 vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered intravenously at a dose of between about 1 ⁇ 10 13 and 5 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of between about 5 ⁇ 10 13 and 3 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of between about 5 ⁇ 10 13 and 1 ⁇ 10 14 vg/kg.
  • the AAV vector is administered intravenously at a dose of less than about 1 ⁇ 10 12 vg/kg, less than about 3 ⁇ 10 12 vg/kg, less than about 5 ⁇ 10 12 vg/kg, less than about 7 ⁇ 10 12 vg/kg, less than about 1 ⁇ 10 13 vg/kg, less than about 3 ⁇ 10 13 vg/kg, less than about 5 ⁇ 10 13 vg/kg, less than about 7 ⁇ 10 13 vg/kg, less than about 1 ⁇ 10 14 vg/kg, less than about 3 ⁇ 10 14 vg/kg, less than about 5 ⁇ 10 14 vg/kg, less than about 7 ⁇ 10 14 vg/kg, less than about 1 ⁇ 10 15 vg/kg, less than about 3 ⁇ 10 15 vg/kg, less than about 5 ⁇ 10 15 vg/kg, or less than about 7 ⁇ 10 15 vg/kg.
  • the AAV vector delivered at any of these doses is an AAV9 vector or an AAV
  • the AAV vector is administered intravenously at a dose of about 1 ⁇ 10 12 vg/kg, about 3 ⁇ 10 12 vg/kg, about 5 ⁇ 10 12 vg/kg, about 7 ⁇ 10 12 vg/kg, about 1 ⁇ 10 13 vg/kg, about 3 ⁇ 10 13 vg/kg, about 5 ⁇ 10 13 vg/kg, about 7 ⁇ 10 13 vg/kg, about 1 ⁇ 10 14 vg/kg, about 3 ⁇ 10 14 vg/kg, about 5 ⁇ 10 14 vg/kg, about 7 ⁇ 10 14 vg/kg, about 1 ⁇ 10 15 vg/kg, about 3 ⁇ 10 15 vg/kg, about 5 ⁇ 10 15 vg/kg, or about 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered intravenously at a dose of 1 ⁇ 10 12 vg/kg, 3 ⁇ 10 12 vg/kg, 5 ⁇ 10 12 vg/kg, 7 ⁇ 10 12 vg/kg, 1 ⁇ 10 13 vg/kg, 3 ⁇ 10 13 vg/kg, 5 ⁇ 10 13 vg/kg, 7 ⁇ 10 13 vg/kg, 1 ⁇ 10 14 vg/kg, 3 ⁇ 10 14 vg/kg, 5 ⁇ 10 14 vg/kg, 7 ⁇ 10 14 vg/kg, 1 ⁇ 10 15 vg/kg, 3 ⁇ 10 15 vg/kg, 5 ⁇ 10 15 vg/kg, or 7 ⁇ 10 15 vg/kg.
  • the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • NHA Class New York Heart Association functional classification
  • pathological electrocardiogram cardiac MRI
  • heart biopsy decrease in paroxysmal ventricular arrhythmias
  • decrease in sudden cardiac death and/or decrease in or lack of further development of fibro-fatty deposits in right ventricular myocardium.
  • Benefit may be observed in electrocardiographic features normally associated with arrhythmogenic right ventricular cardiomyopathy such as T wave inversion, prolonged S-wave upstroke, localized QRS widening, and/or paroxysmal episodes of ventricular tachycardia.
  • the method prevents or reduces a decrease in left ventricle ejection fraction percentage (LVEF %), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • LVEF % left ventricle ejection fraction percentage
  • the method prevents or reduces a decrease in left ventricle fractional shortening percentage (FS %), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • FS % left ventricle fractional shortening percentage
  • the method prevents or reduces an increase in right ventricle area in millimeters squared (RV Area (mm2), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • RV Area millimeters squared
  • the method prevents or reduces a decrease in right ventricle velocity time integral in millimeters per second (RV VTI (mm/sec), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • RV VTI millimeters per second
  • the method prevents or reduces an increase in left ventricle or right ventricle fibrosis, optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, systemic, local, direct injection, intravenous, intracardiac administration. In some cases, administration comprises systemic, local, direct injection, intravenous, intracardiac injection. Administration may be performed by cardiac catheterization.
  • systemic administration may be administration into the circulatory system so that the entire body is affected.
  • Systemic administration includes parental administration through injection, infusion or implantation.
  • Routes of administration for the compositions disclosed herein include intravenous (“IV”) administration, intraperitoneal (“IP”) administration, intramuscular (“IM”) administration, intralesional administration, or subcutaneous (“SC”) administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, a depot formulation, etc.
  • the methods of the disclosure comprise administering an AAV vector of the disclosure, or pharmaceutical composition thereof by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic or intracranial administration.
  • administration of rAAV of the present invention may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal. Administration includes, but is not limited to, injection into the heart.
  • the methods of the disclosure comprise intracardiac delivery.
  • Infusion may be performed using specialized cannula, catheter, syringe/needle using an infusion pump.
  • Administration may comprise delivery of an effective amount of the rAAV virion, or a pharmaceutical composition comprising the rAAV virion, to the heart. These may be achieved, e.g., via intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic or intracranial administration.
  • the compositions of the disclosure may further be administered intravenously.
  • the method of treatment disclosed herein may reduce and/or prevent one or more symptoms including but not limited to ventricular hypertrophy, ventricular tachycardia, exercise intolerance, angina, and reduced RVEF.
  • FIGS. 1-4 Vectors illustrated in FIGS. 1-4 are tested.
  • AAV vectors or respective expression cassettes are tested in vitro using cultured cardiomyocytes (e.g., induced pluripotent stem cell cardiomyocytes, iPSC-CMs) or other cells amenable to transfection or transduction with these constructs.
  • iPSC-CMs induced pluripotent stem cell cardiomyocytes
  • PKP2 is assessed by immunofluorescence and Western blot.
  • Cell-based studies employing patient iPSC-derived cardiomyocytes will reveal benefit of overexpression of PKP2 transgene (either following AAV vector transduction and/or transfection with vector plasmids) by a decreased adipogenic potential (e.g. less lipid accumulation), decreased upregulation or abnormal peroxisome proliferator-activated receptor gamma activation, associated with increased density of PKP2.
  • PDP2-cKO mutant mouse models of cardiomyopathy
  • Evidence of benefit of AAV mediated overexpression of PKP2 may be revealed using a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line, referred to as “PKP2-cKO”.
  • This mouse model allows control of the onset of PKP2 loss of expression, limits loss of PKP2 to adult myocytes, and initiates a progression of molecular and functional events leading to an arrhythmogenic cardiomyopathy, with right ventricular predominance in this mouse.
  • Additional mouse models that result in similar course of pathology may also be utilized to reveal benefit of AAV-mediated overexpression of PKP2 in cardiomyocytes.
  • Benefit of AAV-mediated PKP2 overexpression would be evidenced by increase in survival, mitigation of the normal progression of cardiomyopathy observed on echocardiograms from left and/or right ventricle (e.g. greater left ventricular ejection fraction, greater left ventricle fractional shortening, and greater right ventricle velocity time interval, compared to PKP cKO formulation buffer control animals).
  • Electrophysiological evidence of functional benefit of AAV-mediated delivery of PKP2 protein is demonstrated by mitigation of disease-related disrupted calcium dynamics in affected cardiomyocytes, most notably on measures of L-type calcium current, sarcoplasmic reticulum calcium leak, diastolic calcium leak, as well as standard measures of calcium transients in affected (e.g., PKP2-deficient) cardiomyocytes such as time to peak amplitude and relaxation time constants. Histological analyses will reveal benefit of AAV-mediated PKP2 overexpression by diminished appearance of disease-related collagen deposition (e.g., via trichrome stain) in various regions of the heart including ventricles, compared to cKO formulation buffer injected controls.
  • cardiomyocyte ventricular proteins involved in calcium signaling pathways measured by increased (i.e., normalized) relative levels of Casq2, and/or Trdn, and/or Cav 1.2, and/or AnkB and/or RyR2, relative to non AAV-PKP2 treated, PKP2-cKO diseased controls.
  • AAV vectors are described herein (see FIGS. 11-12 ) were prepared and used to transduce differentiated AC16 cells, a human cardiomyocyte cell line. Expression levels of PKP2 (PKP2a isoform) were assessed by Western Blot ( FIGS. 5A-5B ). Surprisingly, the MHCK7 promoter causes robust expression of PKP2 in cardiomyocytes, whereas the hTnnT2 promoter (“hTnT”) generates marginal PKP2 levels above background under the current testing conditions. The AAVrh.74 serotype induced higher expression of PKP2 than the AAV9 serotype vector.
  • AAV9 vectors or AAVrh74 vectors can effectively be used to express PKP2 in cardiomyocytes, and that the MHCK7 promoter is superior to the hTnnT2 promoter when solely evaluating the relative levels of PKP2 expression.
  • PGP2-cKO mouse model of PKP2-deficiency, as described in Cerrone et al., Nat Comm., 2017 was obtained.
  • This cardiomyocyte-specific, tamoxifen-activated PKP2 knockout murine line (aMHC-Cre-ER(T2)/Pkp2 fl/fl; referred to as “PKP2-cKO”) was utilized to control the onset of PKP2 loss of expression (see Cerrone et al., Nat Comm, 2017).
  • PKP2 deficiency in adult ventricular myocytes is sufficient to cause an arrhythmogenic cardiomyopathy of RV predominance, which includes the ‘hallmark’ functional, molecular, and structural indices consistent with the disease phenotype of ARVC.
  • PKP2-Cko mice were injected with tamoxifen, causing myocyte-specific knockout of PKP2.
  • Mice were injected with AAV vectors (as described below) at 3 ⁇ 10 13 vg/kg by intravenous (tail vein) injection.
  • myocyte-specific knockout of PKP2 was induced by treatment of the mice with tamoxifen.
  • the vector genomes used were:
  • Each vector genome was tested in a AAV9 serotype or AAVrh74 serotype vector.
  • mice were evaluated for various physiology parameters, essentially as described in Cerrone et al., Nat Comm, 2017 or using standard methodologies known in the art. Efficacy in treating disease was assessed by left ventricle ejection fraction percentage (LVEF %) ( FIGS. 6A-6D ), left ventricle fractional shortening percentage (FS %) ( FIGS. 7A-7D ), right ventricle area in millimeters squared (RV Area (mm 2 )) ( FIGS. 8A-8D ), right ventricle velocity time integral in millimeters per second (RV VTI (mm/sec)) ( FIGS.
  • LVEF % left ventricle ejection fraction percentage
  • FS left ventricle fractional shortening percentage
  • RV Area mm 2
  • FIGS. 8A-8D right ventricle velocity time integral in millimeters per second
  • FIGS. 10A-10B degree of fibrosis
  • AAV9-mediated PKP expression evidence for mitigation of the disease phenotype was observed following both AAV9- and AAVrh.74-mediated PKP expression, to varying degrees.
  • AAV9 surprisingly produced the most robust effects on all parameters.
  • AAVrh.74 given the cardiotropism of AAVrh74 and given that biological effects were observed with AAVrh.74-mediated overexpression of PKP2 in this model (e.g. LVEF %, FS %, and right ventricular area), optimization of the dose of AAVrh.74 in combination with the appropriate promoter (i.e., either MHCK7 or hTnnT2) could enable robust therapeutic potential for this vector.
  • the appropriate promoter i.e., either MHCK7 or hTnnT2
  • AAV9 and AAVrh.74 can be used to treat PKP2-related diseases, such as Arrhythmogenic right ventricular cardiomyopathy (ARVC) [also known as Arrhythmogenic Right Ventricular Dysplasia (ARVD) or Arrhythmogenic Cardiomyopathy (ACM)] for which the PKP-cKO mouse is considered an appropriate model.
  • ARVC Arrhythmogenic right ventricular cardiomyopathy
  • ARVD Arrhythmogenic Right Ventricular Dysplasia
  • ACM Arrhythmogenic Cardiomyopathy

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Abstract

Provided herein is a gene therapy for PKP2 (Plakophilin-2), e.g. using an adeno-associated virus (AAV) vector. The promoter of the vector may be a MHCK7 promoter or a cardiac troponin T (HTNNT2) promoter. The capsid may be an AAV9 or AAVrh74 capsid or a functional variant thereof. Other promoters or capsids may be used. Further provided are methods of treatment, such as by intravenous, intracoronary, intracarotid or intracardiac administration of the rAAV vector, and other compositions and methods.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of International Application No. PCT/US2021/045220, filed Aug. 9, 2021, which claims the benefit of U.S. Provisional Application No. 63/063,032, filed Aug. 7, 2020, which is incorporated by reference herein in its entirety.
    • STATEMENT REGARDING THE SEQUENCE LISTING
  • The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is ROPA_021_01WO_ST25.txt. The text file is about 212 KB, created on Aug. 8, 2021, and is being submitted electronically via EFS-Web.
    • BACKGROUND
  • Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a form of adult-onset heart disease, which impacts an estimated 1 in 1,000 to 1 in 1,250 people. It manifests as breakdown of the muscular wall of the heart (the myocardium) over time, which leads to increased risk of abnormal heartbeat (arrhythmia) and an increased risk of sudden death when an affected individual exercises strenuously. Individuals may also experience a sensation of fluttering or pounding in the chest (palpitations), light-headedness, fainting (syncope), shortness of breath, and abnormal swelling in the legs or abdomen. Over time, ARVC can lead to heart failure.
  • At least 13 genes are implicated in ARVC, many of which are involved in the biogenesis of desmosomes, which are intracellular junctions that provide strong adhesion between cells. When desmosomes fail to form properly, myocardial cells may detach from one another and die. The right ventricle in particular may develop weakness, while fatty deposits and scar tissue may replace the damaged myocardium, leading to distension of the right ventricle. These alterations ultimately prevent effective heart pumping and disrupt the electrical signals that control the heartbeat, leading to arrhythmia. Autosomal dominant plakophilin-2 (PKP2) cardiomyopathy is an inherited ARVC in which mutations affecting PKP2 are detected.
  • There remains, therefore, an unmet need in the art for treatments for PKP2-related diseases and disorders, including ARVC. The compositions and methods disclosed herein address this need.
    • SUMMARY
  • The present invention relates generally to gene therapy for a disease or disorder, e.g., a cardiac disease or disorder, using a vector expressing PKP2 or a functional variant thereof.
  • In one aspect, the disclosure provides polynucleotide, comprising an expression cassette and optionally flanking adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein the polynucleotide comprises a polynucleotide sequence encoding a Plakophilin-2 (PKP2) or a functional variant thereof, operatively linked to a promoter.
  • In some embodiments, the promoter is a cardiac-specific promoter.
  • In some embodiments, the promoter is a muscle-specific promoter.
  • In some embodiments, the promoter is a cardiomyocyte-specific promoter.
  • In some embodiments, the promoter is a Myosin Heavy-chain Creatine Kinase 7 (MHCK7) promoter.
  • In some embodiments, the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 31.
  • In some embodiments, the promoter is a cardiac troponin T (hTNNT2) promoter.
  • In some embodiments, the hTNNT2 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 32.
  • In some embodiments, the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene, wherein optionally the hTNNT2 promoter and exon 1 together share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 32.
  • In some embodiments, the promoter is a ubiquitous promoter, optionally a CMV promoter or a CAG promoter.
  • In some embodiments, the expression cassette comprises a polyA signal.
  • In some embodiments, the polyA signal is a human growth hormone (hGH) polyA.
  • In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), optionally a WPRE(x).
  • In some embodiments, the Plakophilin-2 (PKP2) or functional variant thereof is a PKP2.
  • In some embodiments, the PKP2 is a functional PKP2.
  • In some embodiments, the PKP2 is a human PKP2.
  • In some embodiments, the PKP2 is PKP2 isoform A.
  • In some embodiments, the PKP2 isoform A shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 1.
  • In some embodiments, the PKP2 is PKP2 isoform B.
  • In some embodiments, the PKP2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2.
  • In some embodiments, the polynucleotide sequence encoding PKP2 is a human PKP2 polynucleotide.
  • In some embodiments, the polynucleotide sequence encoding PKP2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 3.
  • In some embodiments, the polynucleotide sequence encoding PKP2 shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 4.
  • In some embodiments, the polynucleotide comprises at least about 4.0 kb, at least about 4.1 kb, at least about 4.2 kb, at least about 4.3 kb, at least about 4.4 kb, or at least about 4.5 kb.
  • In some embodiments, the polynucleotide comprises at most about 4.1 kb, at most about 4.2 kb, at most about 4.3 kb, at most about 4.4 kb, at most about 4.5 kb, or at most about 4.6 kb.
  • In some embodiments, the polynucleotide comprises 4.0 kb to 4.6 kb, 4.0 kb to 4.5 kb, or 4.0 kb to 4.4 kb or wherein the polynucleotide comprises 4.0 kb to 4.3 kb, 4.0 kb to 4.2 kb, or 4.0 kb to 4.1 kb.
  • In some embodiments, the PKP2 or functional variant thereof comprises at least 800 or at least 830 amino acids.
  • In some embodiments, the polynucleotide shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NOs: 8-15.
  • In some embodiments, the expression cassette is flanked by 5′ and 3′ inverted terminal repeats (ITRs)
  • In some embodiments, the ITRs are AAV2 ITRs and/or the ITRs share at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with any one of SEQ ID NO: 20-26.
  • In another aspect, the disclosure provides a gene therapy vector, comprising the polynucleotide of any one of the preceding embodiments.
  • In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.
  • In some embodiments, the rAAV vector is an AAV9 or a functional variant thereof.
  • In some embodiments, the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 77.
  • In some embodiments, the rAAV vector is an AAVrh10 or a functional variant thereof.
  • In some embodiments, the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 79.
  • In some embodiments, the rAAV vector is an AAV6 or a functional variant thereof.
  • In some embodiments, the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 78.
  • In some embodiments, the rAAV vector is an AAVrh74 or a functional variant thereof.
  • In some embodiments, the rAAV vector comprises a capsid protein that shares 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 80.
  • In another aspect, the disclosure provides a method of treating and/or preventing a disease or disorder in a subject in need thereof, comprising administering the vector of any one of the preceding embodiments to the subject.
  • In some embodiments, the disease or disorder is a cardiac disorder.
  • In some embodiments, the disease or disorder is cardiomyopathy.
  • In some embodiments, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy (ARVC).
  • In some embodiments, the cardiomyopathy is hypertrophic cardiomyopathy or dilated cardiomyopathy.
  • In some embodiments, the disease or disorder is characterized by fibrofatty infiltration of myocardium.
  • In some embodiments, the disease or disorder is heart failure.
  • In some embodiments, the subject is a mammal.
  • In some embodiments, the subject is a primate.
  • In some embodiments, the subject is a human.
  • In some embodiments, the subject has a mutation in a PKP2 gene.
  • In some embodiments, the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and/or cardiac catheterization.
  • In some embodiments, the administration increases PKP2 expression by at least about 5%.
  • In some embodiments, the administration increases PKP2 expression by at least about 30%.
  • In some embodiments, the administration increases PKP2 expression by at least about 70%.
  • In some embodiments, the administration increases PKP2 expression by about 5% to about 10%.
  • In some embodiments, the administration increases PKP2 expression by about 30% to about 50%.
  • In some embodiments, the administration increases PKP2 expression by about 50% to about 70%.
  • In some embodiments, the administration increases PKP2 expression by about 70% to about 100%.
  • In some embodiments, the method treats and/or prevents the disease or disorder.
  • In some embodiments, the method comprises administering an effective amount of the vector.
  • In some embodiments, the disease or disorder is related to or caused by loss of function in PKP2 in the subject.
  • In some embodiments, the disease or disorder is related to or caused by gain of function in PKP2 in the subject.
  • In some embodiments, the subject has a mutation that causes an amino acid substitution selected from p.Arg490Trp, Asp26Asn, Thr50 Va151SerfsX60, Arg79X, Tyr86X, Gln133X, Va1406SerfsX3, Tyr616X, Trp676X, Cys796Arg, Cys796E, Tyr807X, Glu62Lys, S688P, Trp848X, Y86X, V406X, Y616X, W848X, and Y807X, relative to a human PKP2 gene encoding a human PKP2 having the sequence of SEQ ID NO: 2.
  • In some embodiments, the method comprises administering a pharmaceutical composition comprising an effective amount of the vector.
  • In some embodiments, the method comprises administering between about 1×1011 vector genomes and about 1×1013 vector genomes of the vector to the subject, administering between about 1×1012 vector genomes and about 1×1014 vector genomes of the vector to the subject, or administering between about 1×1013 vector genomes and about 1×1015 vector genomes of the vector to the subject.
  • In another aspect, the disclosure provides a pharmaceutical composition comprising the vector of any one of the preceding embodiments.
  • In another aspect, the disclosure provides a kit comprising the vector of any one of the preceding embodiments or the pharmaceutical composition of the preceding embodiment and optionally instructions for use.
  • In another aspect, the disclosure provides a use of the vector of any one of the preceding embodiments in treating a disease or disorder, optionally according to the method of any one of the preceding embodiments.
  • In another aspect, the disclosure provides a vector according to any one of the preceding embodiments for use in treating a disease or disorder, optionally according to the method of any one of the preceding embodiments.
  • In another aspect, the disclosure provides a polynucleotide, comprising a polynucleotide sequences that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 12-15 and 89-92 or to any one of SEQ ID NOs: 8-11 and 93-96.
  • In some embodiments, the promoter is a MHCK7 promoter.
  • In some embodiments, the MHCK7 promoter shares at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 31.
  • In some embodiments, the PKP2 is a human PKP2.
  • In some embodiments, the PKP2 is PKP2 isoform A.
  • In some embodiments, the PKP2 isoform A shares at least 80%, 90%, 95%, 99% or 100% identity with SEQ ID NO: 1.
  • In another aspect, the disclosure provides a gene therapy vector, comprising the polynucleotide of any one of the preceding embodiments.
  • In some embodiments, the gene therapy vector is a recombinant adeno-associated virus (rAAV) vector.
  • In some embodiments, the rAAV vector is an AAV9 vector.
  • In some embodiments, the rAAV vector is an AAVrh74 vector.
  • In another aspect, the disclosure provides a method of treating and/or preventing a cardiac disorder in a subject identified as having a mutation in the PRP2 gene, comprising administering the vector of any one of the preceding embodiments to the subject.
  • In some embodiments, the cardiac disorder is cardiomyopathy, optionally arrhythmogenic right ventricular cardiomyopathy (ARVC), hypertrophic cardiomyopathy, or dilated cardiomyopathy.
  • In some embodiments, the cardiac disorder is heart failure.
  • In some embodiments, the subject is a mammal.
  • In some embodiments, the vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, and/or cardiac catheterization.
  • In some embodiments, the method prevents or reduces a decrease in left ventricle ejection fraction percentage (LVEF %), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject identified as having a mutation in the PRP2 gene.
  • In some embodiments, the method prevents or reduces a decrease in left ventricle fractional shortening percentage (FS %), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject identified as having a mutation in the PRP2 gene.
  • In some embodiments, the method prevents or reduces an increase in right ventricle area in millimeters squared (RV Area (mm2), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject identified as having a mutation in the PRP2 gene.
  • In some embodiments, the method prevents or reduces a decrease in right ventricle velocity time integral in millimeters per second (RV VTI (mm/sec), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject identified as having a mutation in the PRP2 gene.
  • In some embodiments, the method prevents or reduces an increase in left ventricle or right ventricle fibrosis, optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject identified as having a mutation in the PKP2 gene.
  • Various other aspects and embodiments are disclosed in the detailed description that follows. The invention is limited solely by the appended claims.
    • BRIEF DESCRIPTION OF FIGURES
  • FIG. 1 shows a diagram illustrating a non-limiting example of a vector genome. The full polynucleotide sequence of the vector genome is SEQ ID NO: 12. The expression cassette is SEQ ID NO: 8. The MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG. 2 shows a diagram illustrating a non-limiting example of a vector genome. The full polynucleotide sequence of the vector genome is SEQ ID NO: 13. The expression cassette is SEQ ID NO: 9.
  • FIG. 3 shows a diagram illustrating a non-limiting example of a vector genome. The full polynucleotide sequence of the vector genome is SEQ ID NO: 14. The expression cassette is SEQ ID NO: 10. The MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG. 4 shows a diagram illustrating a non-limiting example of a vector genome. The full polynucleotide sequence of the vector genome is SEQ ID NO: 15. The expression cassette is SEQ ID NO: 11.
  • FIGS. 5A-5B show PKP2 protein expression in transduced differentiated AC16 cells. FIG. 5A shows Western Blots (WB) of PKP2 (top panel) or loading control, GAPDH (bottom panel). FIG. 5B show a bar graph of the Western Blot. The AAV vector serotype (AAV9 or rh74) and the promoter (MHCK7 or hTnT) are noted. Controls included a GFP vector (CON-GFP) and no transduction (No Tdxn).
  • FIGS. 6A-6D show left ventricle ejection fraction percentage (LVEF %) for normal mice (left bars), untreated PKP2 knockout mice (middle bars, cKO PKP2), or treated mice (right bars). FIG. 6A shows results with the AAV9 vector and MHCK7 promoter (AAV9-MHCK7). FIG. 6B shows results with the AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7). FIG. 6C shows results with the AAV9 vector and hTnT promoter (AAV9-hTnT). FIG. 6D shows results with the AAVrh.74 vector and hTnT promoter (AAVrh.74-hTnT).
  • FIGS. 7A-7D show left ventricle fractional shortening percentage (FS %) for normal mice (left bars), untreated PKP2 knockout mice (middle bars, cKO PKP2), or treated mice (right bars). FIG. 7A shows results with the AAV9 vector and MHCK7 promoter (AAV9-MHCK7). FIG. 7B shows results with the AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7). FIG. 7C shows results with the AAV9 vector and hTnT promoter (AAV9-hTnT). FIG. 7D shows results with the AAVrh.74 vector and the hTnT promoter (AAVrh.74-hTnT).
  • FIGS. 8A-8D show right ventricle area in millimeters squared (RV Area (mm2)) for normal mice (left bars), untreated PKP2 knockout mice (middle bars, cKO PKP2), or treated mice (right bars). FIG. 8A shows results with the AAV9 vector and MHCK7 promoter (AAV9-MHCK7). FIG. 8B shows results with the AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7). FIG. 8C shows results with the AAV9 vector and hTnT promoter (AAV9-hTnT). FIG. 8D shows results with the AAVrh.74 vector and hTnT promoter (AAVrh.74-hTnT).
  • FIGS. 9A-9D show right ventricle velocity time integral in millimeters per second (RV VTI (mm/sec)) for normal mice (left bars), untreated PKP2 knockout mice (middle bars, cKO PKP2), or treated mice (right bars). FIG. 9A shows results with the AAV9 vector and MHCK7 promoter (AAV9-MHCK7). FIG. 9B shows results with the AAVrh.74 vector and MHCK7 promoter (AAVrh.74-MHCK7). FIG. 9C shows results with the AAV9 vector and hTnT promoter (AAV9-hTnT). FIG. 9D shows results with the AAVrh.74 vector and hTnT promoter (AAVrh.74-hTnT).
  • FIGS. 10A-10B illustrate the degree of fibrosis in left and right ventricles based on quantitation of the Percent Collagen following trichrome histological staining of the heart. Control animals without the conditional PKP2 gene knock-out (Cre Neg group) were found to have very little collagen, while control PKP2 knock-out animals receiving Formulation Buffer (CKO FB group) were found to have substantially greater proportion of collagen in both left and right ventricles. AAV-mediated overexpression of PKP2 resulted in robust attenuation of collagen, to varying degrees, in all AAV-injected groups [n=4 for all groups; p-values reflect results from One-way ANOVA with Bonferroni post-hoc analyses]. FIG. 10A is a bar graph of percent collagen in the left ventricle. FIG. 10B is a bar graph of percent collagen in the right ventricle.
  • FIG. 11 shows a diagram illustrating a non-limiting example of a vector genome. The full polynucleotide sequence of the vector genome is SEQ ID NO: 89. The expression cassette is SEQ ID NO: 93. The MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG. 12 shows a diagram illustrating a non-limiting example of a vector genome. The full polynucleotide sequence of the vector genome is SEQ ID NO: 90. The expression cassette is SEQ ID NO: 94.
  • FIG. 13 shows a diagram illustrating a non-limiting example of a vector genome. The full polynucleotide sequence of the vector genome is SEQ ID NO: 91. The expression cassette is SEQ ID NO: 95. The MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG. 14 shows a diagram illustrating a non-limiting example of a vector genome. The full polynucleotide sequence of the vector genome is SEQ ID NO: 92. The expression cassette is SEQ ID NO: 96.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • The present disclosure provided gene therapy vectors for PKP2 that deliver a polynucleotide encoding a PKP2 polypeptide or a functional variant thereof, along with methods of use, and other compositions and methods. In particular embodiments, the disclosure relates to a gene therapy vector comprising a promoter sequence operatively linked to a polynucleotide encoding a PKP2 polypeptide or a functional variant thereof. In some embodiments, the promoter is a Myosin Heavy-chain Creatine Kinase 7 (MHCK7) promoter. In some embodiment, the AAV vector is an AAV9 vector. In some embodiments, the promoter is an MHCK7 promoter and the AAV vector is an AAV9 vector. In some embodiments, the promoter is a hTNNT2 promoter. In some embodiment, the promoter is an hTNNT2 promoter and the AAV vector is an AAV9 vector. In some embodiments, the PKP2 is human PKP2a. In some embodiments, the PKP2 is human PKP2b. In some embodiment, the AAV vector is an rh74 vector. In some embodiments, the promoter is an MHCK7 promoter and the AAV vector is an rh74 vector. In some embodiments, the promoter is a hTNNT2 promoter. In some embodiment, the promoter is an hTNNT2 promoter and the AAV vector is an rh74 vector. In some embodiments, the PKP2 is human PKP2a. In some embodiments, the PKP2 is human PKP2b.
  • This disclosure further provides methods of treating a disorder or disorder in a subject by administering a gene therapy vector of the disclosure to the subject. In a certain embodiment, the disorder or disorder is arrhythmogenic right ventricular cardiomyopathy (ARVC).
  • In certain embodiments, the subject being treated is an ARVC patient having one or more mutation in a PKP2 gene. More than half of ARVC patients carry mutations in the desmosomal gene PKP2 encoding the protein Plakophilin-2 (PKP2). PKP2 is also associated with Brugada syndrome (BrS) and idiopathic ventricular fibrillation. It is a member of the armadillo repeat and plakophilin protein family. The protein contains nine central, conserved armadillo repeat domains flanked by N-terminal and C-terminal domains. It functions to link cadherins to intermediate filaments in the cytoskeleton
  • Plakophilin 2 localizes to cell desmosomes and nuclei and binds plakoglobin, desmoplakin, and the desmosomal cadherins via an N-terminal head domain. PKP2 provides a lateral stabilizing force with the desmosomal-intermediate filament assembly facilitating cell-to-cell contact. It may also serve roles in intracellular signaling regulation, electrophysiologic and trafficking regulation, and control of transcription processes.
  • Intravenous injection of an AAV9 vector encoding a C-terminal deletion mutant of PKP2a (R735X) in the heart of wild-type mice accelerates the appearance of ARVC when treated mice are subjected to exercise training. Cruz et al. J Am Coll Cardiol. 65(14):1438-50 (2015). Mutant PKP2a causes a disease phenotype; and control AAV9 vector expressing the non-mutant PKP2a causes no phenotypic change in wild type mice. Heterologous expression of wild-type human PKP2a does not induce disease or altered function. These studies demonstrate that mutant PKP2a can cause a disease phenotype. They fail to demonstrate a curative role for PKP2, because heterologous expression of non-mutant PKP2 resulted in no phenotypic change in the wild-type mouse.
  • In accordance with the present invention, a polynucleotide encoding a PKP2 or functional variant thereof, wherein the PKP2 or functional variant thereof comprises at least 800 or at least 830 amino acids (e.g., no C terminal truncation at Arg-735), may be employed in generating a gene therapy vector. The resulting vector may be employed in treating diseases or disorders, e.g. a PKP2-related disease or disorder, e.g. ARVC, Brugada syndrome (BrS), idiopathic ventricular fibrillation, dilated cardiomyopathy (DCM), and others.
  • Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In cases of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.
  • All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.
  • In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and/or” should be understood to mean either one, or both of the alternatives. As used herein, the terms “include” and “comprise” are used synonymously.
  • As used herein, the terms “identity” and “identical” refer, with respect to a polypeptide or polynucleotide sequence, to the percentage of exact matching residues in an alignment of that “query” sequence to a “subject” sequence, such as an alignment generated by the BLAST algorithm. Identity is calculated, unless specified otherwise, across the full length of the subject sequence. Thus a query sequence “shares at least x % identity to” a subject sequence if, when the query sequence is aligned to the subject sequence, at least x % (rounded down) of the residues in the subject sequence are aligned as an exact match to a corresponding residue in the query sequence. Where the subject sequence has variable positions (e.g., residues denoted X), an alignment to any residue in the query sequence is counted as a match. Sequence alignments may be performed using the NCBI Blast service (BLAST+ version 2.12.0).
  • As used herein, the term “operatively linked” refers to a functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is operatively linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operatively linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
  • As used herein, an “AAV vector” or “rAAV vector” refers to a recombinant vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV inverted terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a plasmid encoding and expressing rep and cap gene products. Alternatively, AAV vectors can be packaged into infectious particles using a host cell that has been stably engineered to express rep and cap genes.
  • As used herein, an “AAV virion” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. As used herein, if the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector.” Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle.
  • As used herein, “promoter” refers to a polynucleotide sequence capable of promoting initiation of RNA transcription from a polynucleotide in a eukaryotic cell.
  • As used herein, “vector genome” refers to the polynucleotide sequence packaged by the vector (e.g., an rAAV virion), including flanking sequences (in AAV, inverted terminal repeats). The terms “expression cassette” and “polynucleotide cassette” refer to the portion of the vector genome between the flanking ITR sequences. “Expression cassette” implies that the vector genome comprises at least one gene encoding a gene product operatively linked to an element that drives expression (e.g., a promoter).
  • As used herein, the term “patient in need” or “subject in need” refers to a patient or subject at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a recombinant gene therapy vector or gene editing system disclosed herein. A patient or subject in need may, for instance, be a patient or subject diagnosed with a disorder associated with heart. A subject may have a mutation in an PKP2 gene or deletion of all or a part of PKP2 gene, or of gene regulatory sequences, that causes aberrant expression of the PKP2 protein. “Subject” and “patient” are used interchangeably herein. The subject treated by the methods described herein may be an adult or a child. Subjects may range in age.
  • As used herein, the term “variant” refers to a protein that has one or more amino-acid substitution, insertion, or deletion as compared to a parental protein. As used herein, the term “functional variant” refers to a protein that has one or more amino-acid substitution, insertion, or deletion as compared to a parental protein, and which retains one or more desired activities of the parental protein.
  • As used herein, “treating” refers to ameliorating one or more symptoms of a disease or disorder. The term “preventing” refers to delaying or interrupting the onset of one or more symptoms of a disease or disorder or slowing the progression of PKP2-related disease or disorder, e.g., arrhythmogenic right ventricular cardiomyopathy (ARVC).
  • Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including two ˜145-nucleotide inverted terminal repeat (ITRs). There are multiple known variants of AAV, also sometimes called serotypes when classified by antigenic epitopes. 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 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_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et 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 AAVrh.74 genome is provided in U.S. Pat. No. 9,434,928, incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the 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. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation 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).
  • AAV possesses unique features 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 noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible. Furthermore, because the 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 replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65° C. for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.
  • Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins, which mediate cell transduction. Such recombinant viruses may be produced by techniques known in the art, e.g., by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include but are not limited to HeLa cells, SF9 cells (optionally with a baculovirus helper vector), 293 cells, etc. A Herpesvirus-based system can be used to produce AAV vectors, as described in US20170218395A1. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO95/14785, WO96/22378, U.S. Pat. Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056 and WO94/19478, the complete contents of each of which is hereby incorporated by reference.
  • The present disclosure contemplates compositions and methods of use related to Plakophilin-2 (PKP2) proteins or polypeptides. Various mutations in PKP2 are known to be associated with cardiomyopathy and heart failure, including diseases like those described in van Tintelen et al. Circulation 113:1650-58 (2006); Novelli Front. Cardiovasc. Med. (2008); and in other sources. Viral vector-mediated delivery of the PKP2 gene may therefore serve as a viable therapeutic for PKP2-related human diseases such as cardiomyopathy and heart failure.
  • More than 230 mutations in the PKP2 gene have been identified in people with arrhythmogenic right ventricular cardiomyopathy (ARVC). (See “PKP2 gene,” MedlinePlus). This condition most commonly affects the myocardium surrounding the right ventricle, one of the two lower chambers of the heart. ARVC increases the risk of an abnormal heartbeat (arrhythmia) and sudden death. Some PKP2 gene mutations lead to the production of an abnormally short version of plakophilin 2. Other mutations alter the structure of plakophilin 2 by adding, deleting, or changing one or more of its protein building blocks (amino acids). Studies suggest that the altered protein impairs the formation and function of desmosomes.
  • Without normal desmosomes, cells of the myocardium detach from one another and die, particularly when the heart muscle is placed under stress (such as during vigorous exercise). The damaged myocardium is gradually replaced by fat and scar tissue. As this abnormal tissue builds up, the walls of the right ventricle become stretched out, preventing the heart from pumping blood effectively. These changes also disrupt the electrical signals that control the heartbeat, which can lead to arrhythmia. Description of PKP2-related disease may be found in the following references: Bonne et al. Genomics 51:452-454 (1998) [PubMed: 9721216]; Bonne et al. Cytogenet. Cell Genet. 88:286-287 (2000) [PubMed: 10828611]; Dalal et al., Circulation 113:1641-1649 (2006) [PubMed: 16549640]; Gerull et al. Nature Genet. 36:1162-1164 (2004 [PubMed: 15489853]; Grossmann et al. J. Cell Biol. 167:149-160 (2004) [PubMed: 15479741]; Marcus et al. Circulation 65:384-398 (1982) [PubMed: 7053899]; and Mertens et al. J. Cell Biol. 135:1009-1025 (1996) [PubMed: 8922383]. See also OMIM.org entry 602861 (“PLAKOPHILIN 2; PKP2”).
  • The native sequences of human PKP2a and its isoform PKP2b are shown below, with Arg-735 underlined:
  • PKP2a-837 amino acids
    (SEQ ID NO: 1)
    1 MAAPGAPAEY GYIRTVLGQQ ILGQLDSSSL ALPSEAKLKL
    41 AGSSGRGGQT VKSLRIQEQV QQTLARKGRS SVGNGNLHRT
    81 SSVPEYVYNL HLVENDFVGG RSPVPKTYDM LKAGTTATYE
    121 GRWGRGTAQY SSQKSVEERS LRHPLRRLEI SPDSSPERAH
    161 YTHSDYQYSQ RSQAGHTLHH QESRRAALLV PPRYARSEIV
    201 GVSRAGTTSR QRHFDTYHRQ YQHGSVSDTV FDSIPANPAL
    241 LTYPRPGTSR SMGNLLEKEN YLTAGLTVGQ VRPLVPLQPV
    281 TQNRASRSSW HQSSFHSTRT LREAGPSVAV DSSGRRAHLT
    321 VGQAAAGGSG NLLTERSTFT DSQLGNADME MTLERAVSML
    361 EADHMLPSRI SAAATFIQHE CFQKSEARKR VNQLRGILKL
    401 LQLLKVQNED VQRAVCGALR NLVFEDNDNK LEVAELNGVP
    441 RLLQVLKQTR DLETKKQITG LLWNLSSNDK LKNLMITEAL
    481 LTLTENIIIP FSGWPEGDYP KANGLLDFDI FYNVTGCLRN
    521 MSSAGADGRK AMRRCDGLID SLVHYVRGTI ADYQPDDKAT
    561 ENCVCILHNL SYQLEAELPE KYSQNIYIQN RNIQTDNNKS
    601 IGCFGSRSRK VKEQYQDVPM PEEKSNPKGV EWLWHSIVIR
    641 MYLSLIAKSV RNYTQEASLG ALQNLTAGSG PMPTSVAQTV
    681 VQKESGLQHT  R KMLHVGDPS VKKTAISLLR NLSRNLSLQN
    721 EIAKETLPDL 7SIIPDTVPS TDLLIETTAS ACYTLNNIIQ
    761 NSYQNARDLL NTGGIQKIMA ISAGDAYASN KASKAASVLL
    801 YSLWAHTELH HAYKKAQFKK TDFVNSRTAK AYHSLKD
    PKP2b-881 amino acids
    (SEQ ID NO: 2)
    1 MAAPGAPAEY GYIRTVLGQQ ILGQLDSSSL ALPSEAKLKL
    41 AGSSGRGGQT VKSLRIQEQV QQTLARKGRS SVGNGNLHRT
    81 SSVPEYVYNL HLVENDFVGG RSPVPKTYDM LKAGTTATYE
    121 GRWGRGTAQY SSQKSVEERS LRHPLRRLEI SPDSSPERAH
    161 YTHSDYQYSQ RSQAGHTLHH QESRRAALLV PPRYARSEIV
    201 GVSRAGTTSR QRHFDTYHRQ YQHGSVSDTV FDSIPANPAL
    241 LTYPRPGTSR SMGNLLEKEN YLTAGLTVGQ VRPLVPLQPV
    281 TQNRASRSSW HQSSFHSTRT LREAGPSVAV DSSGRRAHLT
    321 VGQAAAGGSG NLLTERSTFT DSQLGNADME MTLERAVSML
    361 EADHMLPSRI SAAATFIQHE CFQKSEARKR VNQLRGILKL
    401 LQLLKVQNED VQRAVCGALR NLVFEDNDNK LEVAELNGVP
    441 RLLQVLKQTR DLETKKQITD HTVNLRSRNG WPGAVAHACN
    481 PSTLGGQGGR ITRSGVRDQP DQHGLLWNLS SNDKLKNLMI
    521 TEALLTLTEN IIIPFSGWPE GDYPKANGLL DFDIFYNVTG
    561 CLRNMSSAGA DGRKAMRRCD GLIDSLVHYV RGTIADYQPD
    601 DKATENCVCI LHNLSYQLEA ELPEKYSQNI YIQNRNIQTD
    641 NNKSIGCFGS RSRKVKEQYQ DVPMPEEKSN PKGVEWLWHS
    681 IVIRMYLSLI AKSVRNYTQE ASLGALQNLT AGSGPMPTSV
    721 AQTVVQKESG LQHT R KMLHV GDPSVKKTAI SLLRNLSRNL
    761 SLQNEIAKET LPDLVSIIPD TVPSTDLLIE TTASACYTLN
    801 NIIQNSYQNA RDLLNTGGIQ KIMAISAGDA YASNKASKAA
    841 SVLLYSLWAH TELHHAYKKA QFKKTDFVNS RTAKAYHSLK
    881 D
  • One experimental model of PKP2-related disease is the R735X model, as described in Cruz et al. J Am Coll Cardiol 65:1438-50 (2015). R735X is numbered according to the PKP2b isoform. The R735X mutant of PKP2a is 690 amino acids in length, due to C-terminal truncation at Arg-690, (Arg-735 relative to PKP2b, SEQ ID NO: 2).
  • In some embodiments, the PKP2 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the PKP2 protein is a wild-type or native PKP2 protein, e.g. human PKP2a or human PKP2b.
  • In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) virion, comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an PKP2 or a functional variant thereof, operatively linked to a promoter. In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) virion, comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an PKP2, operatively linked to a promoter. The polynucleotide encoding the PKP2a may comprise a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3.
  • The polynucleotide encoding the PKP2b may comprise a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4.
  • Optionally, the polynucleotide sequence encoding the vector genome may comprise a Kozak sequence, including but not limited to GCCACCATGG (SEQ ID NO: 5). Kozak sequence may overlap the polynucleotide sequence encoding an PKP2a protein or a functional variant thereof. For example, the vector genome may comprise a polynucleotide sequence (with first ten nucleotides constituting the Kozak sequence) at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6.
  • Kozak sequence may overlap the polynucleotide sequence encoding an PKP2b protein or a functional variant thereof. For example, the vector genome may comprise a polynucleotide sequence (with first ten nucleotides constituting the Kozak sequence) at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7.
  • In some embodiments, the Kozak sequence is an alternative Kozak sequence comprising or consisting of any one of:
  • (SEQ ID NO: 16)
      (gcc)gccRccAUGG;
    AGNNAUGN;
    ANNAUGG;
    ANNAUGC;
    ACCAUGG;
    and
    (SEQ ID NO: 18)
    GACACCAUGG.
  • In some embodiments, the vector genome comprises no Kozak sequence.
  • The polynucleotide sequence may be codon-optimized. For example, the vector genome may comprise a polynucleotide sequence encoding a PKP2a that shares at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 87.
  • The vector genome may comprise a polynucleotide sequence encoding a PKP2b that shares at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 88.
  • The AAV virions of the disclosure comprise a vector genome. The vector genome may comprise an expression cassette (or a polynucleotide cassette for gene-editing applications not requiring expression of the polynucleotide sequence). Any suitable inverted terminal repeats (ITRs) may be used. The ITRs may be AAV ITRs from the same serotype as the capsid present in the AAV virion, or a different serotype from the capsid (e.g., AAV2 ITRs may be used with an AAV virion having an AAV9 capsid or an AAVrh74 capsid). In each case, the serotype of the capsid determines the name applied to the virion. The ITR are generally the most 5′ and most 3′ elements of the vector genome. The vector genome will also generally contain, in 5′ to 3′ order, a promoter, a transgene, 3′ untranslated region (UTR) sequences (e.g., a WPRE element), and a polyadenylation sequence. In variations, the vector genome includes an enhancer element (generally 5′ to the promoter) and/or an exon (generally 3′ to the promoter). In variations, the vector genomes of the disclosure encode a partial or complete transgene sequence used as a repair template in a gene editing system. In such variations, the vector genome may comprise an exogenous promoter, or the gene editing system may insert the transgene into a locus in the genome having an endogenous promoter, such as a cardiac- or myocyte-specific promoter.
  • In some embodiments, the 5′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20.
  • In some embodiments, the 5′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21.
  • In some embodiments, the 5′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22)
  • In some embodiments, the 5′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23.
  • In some embodiments, the 3′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24.
  • In some embodiments, the 3′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25.
  • In some embodiments, the 3′ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26.
  • In some embodiments the vector genome comprises one or more filler sequences, e.g., at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27; SEQ ID NO: 28; or SEQ ID NO: 29.
  • In some embodiments, the polynucleotide sequence encoding an PKP2 protein or functional variant thereof is operatively linked to a promoter. In certain embodiments, the promoter is an MHCK7 promoter. In certain embodiments, the promoter is an TNNT2 promoter.
  • The present disclosure contemplates use of various promoters. Promoters useful in embodiments of the present disclosure include, without limitation, a cytomegalovirus (CMV) promoter, phosphoglycerate kinase (PGK) promoter, or a promoter sequence comprised of the CMV enhancer and portions of the chicken beta-actin promoter and the rabbit beta-globin gene (CAG). In some cases, the promoter may be a synthetic promoter. Exemplary synthetic promoters are provided by Schlabach et al. PNAS USA. 107(6):2538-43 (2010). In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30.
  • In some embodiments, a polynucleotide sequence encoding an PKP2 protein or functional variant thereof is operatively linked to an inducible promoter. An inducible promoter may be configured to cause the polynucleotide sequence to be transcriptionally expressed or not transcriptionally expressed in response to addition or accumulation of an agent or in response to removal, degradation, or dilution of an agent. The agent may be a drug. The agent may be tetracycline or one of its derivatives, including, without limitation, doxycycline. In some cases, the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically-regulated promoter, a physically-regulated promoter (i.e., a promoter that responds to presence or absence of light or to low or high temperature). Inducible promoters include heavy metal ion inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and the promoters from T7 phage which are active in the presence of T7 RNA polymerase. This list of inducible promoters is non-limiting.
  • In some cases, the promoter is a tissue-specific promoter, such as a promoter capable of driving expression in a cardiac cell to a greater extent than in a non-cardiac cell. In some embodiments, tissue-specific promoter is a selected from any various cardiac cell-specific promoters including but not limited to, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2), cardiac troponin C (cTnC), cardiac troponin T (hTNNT2), muscle creatine kinase (CK) and combinations of promoter/enhancer regions thereof, such as MHCK7. In some cases, the promoter is a ubiquitous promoter. A “ubiquitous promoter” refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some cases, the ubiquitous promoter is any one of Cytomegalovirus (CMV), Cytomegalovirus early enhancer element chicken beta-Actin gene intron with the splice acceptor of the rabbit beta-Globin gene (CAG), ubiquitin C (UBC), Phosphoglycerate Kinase (PGK), Eukaryotic translation elongation factor 1 alpha 1 (EF1-alpha), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), simian virus 40 (SV40), Hepatitis B virus (HBV), chicken beta-actin, and human beta-actin promoters.
  • In some embodiments, the promoter sequence is selected from Table 3. In some embodiments, the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS: 31-51. In some embodiments, the promoter comprises a fragment of a polynucleotide sequence of any one of SEQ ID NOs: 31-51, e.g., a fragment comprising at least 25%, at least 50%, at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of SEQ ID NOs: 31-51.
  • TABLE 3
    SEQ ID
    PROMOTER SEQUENCE NO:
    MHCK7 ACCCTTCAGATTAAAAATAACTGAGGTAAGGGCCTGGGTAGG 31
    GGAGGTGGTGTGAGACGCTCCTGTCTCTCCTCTATCTGCCCA
    TCGGCCCTTTGGGGAGGAGGAATGTGCCCAAGGACTAAAAAA
    AGGCCATGGAGCCAGAGGGGCGAGGGCAACAGACCTTTCATG
    GGCAAACCTTGGGGCCCTGCTGTCTAGCATGCCCCACTACGG
    GTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCC
    GAGATGCCTGGTTATAATTAACCCAGACATGTGGCTGCCCCC
    CCCCCCCCAACACCTGCTGCCTCTAAAAATAACCCTGTCCCT
    GGTGGATCCCCTGCATGCGAAGATCTTCGAACAAGGCTGTGG
    GGGACTGAGGGCAGGCTGTAACAGGCTTGGGGGCCAGGGCTT
    ATACGTGCCTGGGACTCCCAAAGTATTACTGTTCCATGTTCC
    CGGCGAAGGGCCAGCTGTCCCCCGCCAGCTAGACTCAGCACT
    TAGTTTAGGAACCAGTGAGCAAGTCAGCCCTTGGGGCAGCCC
    ATACAAGGCCATGGGGCTGGGCAAGCTGCACGCCTGGGTCCG
    GGGTGGGCACGGTGCCCGGGCAACGAGCTGAAAGCTCATCTG
    CTCTCAGGGGCCCCTCCCTGGGGACAGCCCCTCCTGGCTAGT
    CACACCCTGTAGGCTCCTCTATATAACCCAGGGGCACAGGGG
    CTGCCCTCATTCTACCACCACCTCCACAGCACAGACAGACAC
    TCAGGAGCCAGCCAG
    Human cardiac CTCAGTCCATTAGGAGCCAGTAGCCTGGAAGATGTCTTTACC 33
    troponin T CCCAGCATCAGTTCAAGTGGAGCAGCACATAACTCTTGCCCT
    promoter CTGCCTTCCAAGATTCTGGTGCTGAGACTTATGGAGTGTCTT
    (without GGAGGTTGCCTTCTGCCCCCCAACCCTGCTCCCAGCTGGCCC
    exon 1) TCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTC
    hTnnT2/ AAGAGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGCAT
    HTNNT2 ATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGA
    CCACATGGGCTTATATGGCGTGGGGTACATGTTCCTGTAGCC
    TTGTCCCTGGCACCTGCCAAAATAGCAGCCAACACCCCCCAC
    CCCCACCGCCATCCCCCTGCCCCACCCGTCCCCTGTCGCACA
    TTCCTCCCTCCGCAGGGCTGGCTCACCAGGCCCCAGCCCACA
    TGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCACCCAGT
    Human cardiac CTCAGTCCATTAGGAGCCAGTAGCCTGGAAGATGTCTTTACC 32
    troponin T CCCAGCATCAGTTCAAGTGGAGCAGCACATAACTCTTGCCCT
    promoter (with CTGCCTTCCAAGATTCTGGTGCTGAGACTTATGGAGTGTCTT
    exon 1, GGAGGTTGCCTTCTGCCCCCCAACCCTGCTCCCAGCTGGCCC
    underlined) TCCCAGGCCTGGGTTGCTGGCCTCTGCTTTATCAGGATTCTC
    hTnnT2/ AAGAGGGACAGCTGGTTTATGTTGCATGACTGTTCCCTGCAT
    HTNNT2 ATCTGCTCTGGTTTTAAATAGCTTATCTGAGCAGCTGGAGGA
    CCACATGGGCTTATATGGCGTGGGGTACATGTTCCTGTAGCC
    TTGTCCCTGGCACCTGCCAAAATAGCAGCCAACACCCCCCAC
    CCCCACCGCCATCCCCCTGCCCCACCCGTCCCCTGTCGCACA
    TTCCTCCCTCCGCAGGGCTGGCTCACCAGGCCCCAGCCCACA
    TGCCTGCTTAAAGCCCTCTCCATCCTCTGCCTCACCCAGTCC
    CCGCTGAGACTGAGCAGACGCCTCCAGGATCTGTCGGCAG
    Mouse α- GGTACCGGATCCTGCAAGGTCACACAAGGGTCTCCACCCACC 34
    cardiac myosin AGGTGCCCTAGTCTCAATTTCAGTTTCCATGCCTTGTTCTCA
    heavy chain CAATGCTGGCCTCCCCAGAGCTAATTTGGACTTTGTTTTTAT
    promoter TTCAAAAGGGCCTGAATGAGGAGTAGATCTTGTGCTACCCAG
    (αMHC) CTCTAAGGGTGCCCGTGAAGCCCTCAGACCTGGAGCCTTTGC
    AACAGCCCTTTAGGTGGAAGCAGAATAAAGCAATTTTCCTTA
    AAGCCAAAATCCTGCCTCTAGACTCTTCTTCTCTGACCTCGG
    TCCCTGGGCTCTAGGGTGGGGAGGTGGGGCTTGGAAGAAGAA
    GGTGGGGAAGTGGCAAAAGCCGATCCCTAGGGCCCTGTGAAG
    TTCGGAGCCTTCCCTGTACAGCACTGGCTCATAGATCCTCCT
    CCAGCCAAACATAGCAAGAAGTGATACCTCCTTTGTGACTTC
    CCCAGGCCCAGTACCTGTCAGGTTGAAACAGGATTTAGAGAA
    GCCTCTGAACTCACCTGAACTCTGAAGCTCATCCACCAAGCA
    AGCACCTAGGTGCCACTGCTAGTTAGTATCCTACGCTGATAA
    TATGCAGAGCTGGGCCACAGAAGTCCTGGGGTGTAGGAACTG
    ACCAGTGACTTTTCAGTCGGCAAAGGTATGACCCCCTCAGCA
    GATGTAGTAATGTCCCCTTAGATCCCATCCCAGGCAGGTCTC
    TAAGAGGACATGGGATGAGAGATGTAGTCATGTGGCATTCCA
    AACACAGCTATCCACAGTGTCCCTTGCCCCTTCCACTTAGCC
    AGGAGGACAGTAACCTTAGCCTATCTTTCTTCCTCCCCATCC
    TCCCAGGACACACCCCCTGGTCTGCAGTATTCATTTCTTCCT
    TCACGTCCCCTCTGTGACTTCCATTTGCAAGGCTTTTGACCT
    CTGCAGCTGCTGGAAGATAGAGTTTGGCCCTAGGTGTGGCAA
    GCCATCTCAAGAGAAAGCAGACAACAGGGGGACCAGATTTTG
    GAAGGATCAGGAACTAAATCACTGGCGGGCCTGGGGGTAGAA
    AAAAGAGTGAGTGAGTCCGCTCCAGCTAAGCCAAGCTAGTCC
    CCGAGATACTCTGCCACAGCTGGGCTGCTCGGGGTAGCTTTA
    GGAATGTGGGTCTGAAAGACAATGGGATTGGAAGACATCTCT
    TTGAGTCTCCCCTCAACCCCACCTACAGACACACTCGTGTGT
    GGCCAGACTCCTGTTCAACAGCCCTCTGTGTTCTGACCACTG
    AGCTAGGCAACCAGAGCATGGGCCCTGTGCTGAGGATGAAGA
    GTTGGTTACCAATAGCAAAAACAGCAGGGGAGGGAGAACAGA
    GAACGAAATAAGGAAGGAAGAAGGAAAGGCCAGTCAATCAGA
    TGCAGTCAGAAGAGATGGGAAGCCAACACACAGCTTGAGCAG
    AGGAAACAGAAAAGGGAGAGATTCTGGGCATAAGGAGGCCAC
    AGAAAGAAGAGCCCAGGCCCCCCAAGTCTCCTCTTTATACCC
    TCATCCCGTCTCCCAATTAAGCCCACTCTTCTTCCTAGATCA
    GACCTGAGCTGCAGCGAAGAGACCCGTAGGGAGGATCACACT
    GGATGAAGGAGATGTGTGGAGAAGTCCAGGGAACCTAAGAGC
    CAGAGCCTAAAAGAGCAAGAGATAAAGGTGCTTCAAAGGTGG
    CCAGGCTGTGCACACAGAGGGTCGAGGACTGGTGGTAGAGCC
    TCAAGATAAGGATGATGCTCAGAATGGGCGGGGGGGGGGATT
    CTGGGGGGGGGAGAGAGAAGGTGAGAAGGAGCCTGGAACAGA
    GAATCTGGAAGCGCTGGAAACGATACCATAAAGGGAAGAACC
    CAGGCTACCTTTAGATGTAAATCATGAAAGACAGGGAGAAGG
    GAAGCTGGAGAGAGTAGAAGGACCCCGGGGCAAGACATTGAA
    GCAAGGACAAGCCAGGTTGAGCGCTCCGTGAAATCAGCCTGC
    TGAAGGCAGAGCCCTGGTATGAGCACCAGAACAGCAGAGGCT
    AGGGTTAATGTCGAGACAGGGAACAGAAGGTAGACACAGGAA
    CAGACAGAGACGGGGGAGCCAGGTAACAAAGGAATGGTCCTT
    CTCACCTGTGGCCAGAGCGTCCATCTGTGTCCACATACTCTA
    GAATGTTCATCAGACTGCAGGGCTGGCTTGGGAGGCAGCTGG
    AAAGAGTATGTGAGAGCCAGGGGAGACAAGGGGGCCTAGGAA
    AGGAAGAAGAGGGCAAACCAGGCCACACAAGAGGGCAGAGCC
    CAGAACTGAGTTAACTCCTTCCTTGTTGCATCTTCCATAGGA
    GGCAGTGGGAACTCTGTGACCACCATCCCCCATGAGCCCCCA
    CTACCCATACCAAGTTTGGCCTGAGTGGCATTCTAGGTTCCC
    TGAGGACAGAGCCTGGCCTTTGTCTCTTGGACCTGACCCAAG
    CTGACCCAATGTTCTCAGTACCTTATCATGCCCTCAAGAGCT
    TGAGAACCAGGCAGTGACATATTAGGCCATGGGCTAACCCTG
    GAGCTTGCACACAGGAGCCTCAAGTGACCTCCAGGGACACAG
    CTGCAGACAGGTGGCCTTTATCCCCAAAGAGCAACCATTTGG
    CATAGGTGGCTGCAAATGGGAATGCAAGGTTGAATCAGGTCC
    CTTCAAGAATACTGCATGCAAGACCTAAGACCCCTGGAGAGA
    GGGGTATGCTCCTGCCCCCACCCACCATAAGGGGAGTGAACT
    ATCCTAGGGGGCTGGCGACCTTGGGGAGACACCACATTACTG
    AGAGTGCTGAGCCCAGAAAAACTGACCGCCCTGTGTCCTGCC
    CACCTCCACACTCTAGAGCTATATTGAGAGGTGACAGTAGAT
    AGGGTGGGAGCTGGTAGCAGGGAGAGTGTTCCTGGGTGTGAG
    GGTGTAGGGGAAAGCCAGAGCAGGGGAGTCTGGCTTTGTCTC
    CTGAACACAATGTCTACTTAGTTATAACAGGCATGACCTGCT
    AAAGACCCAACATCTACGACCTCTGAAAAGACAGCAGCCCTG
    GAGGACAGGGGTTGTCTCTGAGCCTTGGGTGCTTGATGGTGC
    CACAAAGGAGGGCATGAGTGTGAGTATAAGGCCCCAGGAGCG
    TTAGAGAAGGGCACTTGGGAAGGGGTCAGTCTGCAGAGCCCC
    TATCCATGGAATCTGGAGCCTGGGGCCAACTGGTGTAAATCT
    CTGGGCCTGCCAGGCATTCAAAGCAGCACCTGCATCCTCTGG
    CAGCCTGGGGAGGCGGAAGGGAGCAACCCCCCACTTATACCC
    TTTCTCCCTCAGCCCCAGGATTAACACCTCTGGCCTTCCCCC
    TTCCCACCTCCCATCAGGAGTGGAGGGTTGCAGAGGGAGGGT
    AAAAACCTACATGTCCAAACATCATGGTGCACGATATATGGA
    TCAGTATGTGTAGAGGCAAGAAAGGAAATCTGCAGGCTTAAC
    TGGGTTAATGTGTAAAGTCTGTGTGCATGTGTGTGTGTCTGA
    CTGAAAACGGGCATGGCTGTGCAGCTGTTCAGTTCTGTGCGT
    GAGGTTACCAGACTGCAGGTTTGTGTGTAAATTGCCCAAGGC
    AAAGTGGGTGAATCCCTTCCATGGTTTAAAGAGATTGGATGA
    TGGCCTGCATCTCAAGGACCATGGAAAATAGAATGGACACTC
    TATATGTGTCTCTAAGCTAAGGTAGCAAGGTCTTTGGAGGAC
    ACCTGTCTAGAGATGTGGGCAACAGAGACTACAGACAGTATC
    TGTACAGAGTAAGGAGAGAGAGGAGGGGGTGTAGAATTCTCT
    TACTATCAAAGGGAAACTGAGTCGTGCACCTGCAAAGTGGAT
    GCTCTCCCTAGACATCATGACTTTGTCTCTGGGGAGCCAGCA
    CTGTGGAACTTCAGGTCTGAGAGAGTAGGAGGCTCCCCTCAG
    CCTGAAGCTATGCAGATAGCCAGGGTTGAAAGGGGGAAGGGA
    GAGCCTGGGATGGGAGCTTGTGTGTTGGAGGCAGGGGACAGA
    TATTAAGCCTGGAAGAGAAGGTGACCCTTACCCAGTTGTTCA
    ACTCACCCTTCAGATTAAAAATAACTGAGGTAAGGGCCTGGG
    TAGGGGAGGTGGTGTGAGACGCTCCTGTCTCTCCTCTATCTG
    CCCATCGGCCCTTTGGGGAGGAGGAATGTGCCCAAGGACTAA
    AAAAAGGCCATGGAGCCAGAGGGGCGAGGGCAACAGACCTTT
    CATGGGCAAACCTTGGGGCCCTGCTGTCCTCCTGTCACCTCC
    AGAGCCAAGGGATCAAAGGAGGAGGAGCCAGGACAGGAGGGA
    AGTGGGAGGGAGGGTCCCAGCAGAGGACTCCAAATTTAGGCA
    GCAGGCATATGGGATGGGATATAAAGGGGCTGGAGCACTGAG
    AGCTGTCAGAGATTTCTCCAACCCAGGTAAGAGGGAGTTTCG
    GGTGGGGGCTCTTCACCCACACCAGACCTCTCCCCACCTAGA
    AGGAAACTGCCTTTCCTGGAAGTGGGGTTCAGGCCGGTCAGA
    GATCTGACAGGGTGGCCTTCCACCAGCCTGGGAAGTTCTCAG
    TGGCAGGAGGTTTCCACAAGAAACACTGGATGCCCCTTCCCT
    TACGCTGTCTTCTCCATCTTCCTCCTGGGGATGCTCCTCCCC
    GTCTTGGTTTATCTTGGCTCTTCGTCTTCAGCAAGATTTGCC
    CTGTGCTGTCCACTCCATCTTTCTCTACTGTCTCCGTGCCTT
    GCCTTGCCTTCTTGCGTGTCCTTCCTTTCCACCCATTTCTCA
    CTTCACCTTTTCTCCCCTTCTCATTTGTATTCATCCTTCCTT
    CCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTTC
    TCCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTT
    CCTGTGTCAGAGTGCTGAGAATCACACCTGGGGTTCCCACCC
    TTATGTAAACAATCTTCCAGTGAGCCACAGCTTCAGTGCTGC
    TGGGTGCTCTCTTACCTTCCTCACCCCCTGGCTTGTCCTGTT
    CCATCCTGGTCAGGATCTCTAGATTGGTCTCCCAGCCTCTGC
    TACTCCTCTTCCTGCCTGTTCCTCTCTCTGTCCAGCTGCGCC
    ACTGTGGTGCCTCGTTCCAGCTGTGGTCCACATTCTTCAGGA
    TTCTCTGAAAAGTTAACCAGGTGAGAATGTTTCCCCTGTAGA
    CAGCAGATCACGATTCTCCCGGAAGTCAGGCTTCCAGCCCTC
    TCTTTCTCTGCCCAGCTGCCCGGCACTCTTAGCAAACCTCAG
    GCACCCTTACCCCACATAGACCTCTGACAGAGAAGCAGGCAC
    TTTACATGGAGTCCTGGTGGGAGAGCCATAGGCTACGGTGTA
    AAAGAGGCAGGGAAGTGGTGGTGTAGGAAAGTCAGGACTTCA
    CATAGAAGCCTAGCCCACACCAGAAATGACAGACAGATCCCT
    CCTATCTCCCCCATAAGAGTTTGAGTCGACCCGCGGCCCCGA
    ATTG
    Chicken cardiac GGGATAAAAGCAGTCTGGGCTTTCACATGACAGCATCTGGGG 35
    troponin T CTGCGGCAGAGGGTCGGGTCCGAAGCGCTGCCTTATCAGCGT
    promoter CCCCAGCCCTGGGAGGTGACAGCTGGCTGGCTTGTGTCAGCC
    (cTnT) CCTCGGGCACTCACGTATCTCCGTCCGACGGGTTTAAAATAG
    CAAAACTCTGAGGCCACACAATAGCTTGGGCTTATATGGGCT
    CCTGTGGGGGAAGGGGGAGCACGGAGGGGGCCGGGGCCGCTG
    CTGCCAAAATAGCAGCTCACAAGTGTTGCATTCCTCTCTGGG
    CGCCGGGCACATTCCTGCTGGCTCTGCCCGCCCCGGGGTGGG
    CGCCGGGGGGACCTTAAAGCCTCTGCCCCCCAAGGAGCCCTT
    CCCAGACAGCCGCCGGCACCCACCGCTCCGTGGGA
    Human Creatine CTCTCAGCCCTGGAAGTCCTTGCTCACAGCCGAGGCGCCGAG 36
    Kinase M AGCGCTTGCTCTGCCCAGATCTGCGCGAGTCTGGCGCCCGCG
    (hCKM) CTCTGAACGGCGTCGCTGCCCAGCCCCCTTCCCCGGGAGGTG
    GGAGCGGCCACCCAGGGCCCCGTGGCTGCCCTTGTAAGGAGG
    CGAGGCCCGAGGACACCCGAGACGCCCGGTTATAATTAACCA
    GGACACGTGGCGAACCCCCCTCCAACACCTGCCCCCGAACCC
    CCCCATACCCAGCGCCTCGGGTCTCGGCCTTTGCGGCAGAGG
    AGACAGCAAAGCGCCCTCTAAAAATAACTCCTTTCCCGGCGA
    CCGAGACCCTCCCTGTCCCCCGCACAGCGGAAATCTCCCAGT
    GGCACCGAGGGGGCGAGGGTTAAGTGGGGGGGAGGGTGACCA
    CCGCCTCCCACCCTTGCCCTGAGTTTGAATCTCTCCAACTCA
    GCCAGCCTCAGTTTCCCCTCCACTCAGTCCCTAGGAGGAAGG
    GGCGCCCAAGCGCGGGTTTCTGGGGTTAGACTGCCCTCCATT
    GCAATTGGTCCTTCTCCCGGCCTCTGCTTCCTCCAGCTCACA
    GGGTATCTGCTCCTCCTGGAGCCACACCTTGGTTCCCCGAGG
    TGCCGCTGGGACTCGGGTAGGGGTGAGGGCCCAGGGGGCACA
    GGGGGAGCCGAGGGCCACAGGAAGGGCTGGTGGCTGAAGGAG
    ACTCAGGGGCCAGGGGACGGTGGCTTCTACGTGCTTGGGACG
    TTCCCAGCCACCGTCCCATGTTCCCGGCGGGGGGCCAGCTGT
    CCCCACCGCCAGCCCAACTCAGCACTTGGTCAGGGTATCAGC
    TTGGTGGGGGGGCGTGAGCCCAGCCCCTGGGGCGGCTCAGCC
    CATACAAGGCCATGGGGCTGGGCGCAAAGCATGCCTGGGTTC
    AGGGTGGGTATGGTGCGGGAGCAGGGAGGTGAGAGGCTCAGC
    TGCCCTCCAGAACTCCTCCCTGGGGACAACCCCTCCCAGCCA
    ATAGCACAGCCTAGGTCCCCCTATATAAGGCCACGGCTGCTG
    GCCCTTCCTTTGGGTCAGTGTCACCTCCAGGATACAGACA
    Human beta- GCCCAGCACCCCAAGGCGGCCAACGCCAAAACTCTCCCTCCT 37
    actin (HuBa) CCTCTTCCTCAATCTCGCTCTCGCTCTTTTTTTTTTTCGCAA
    AAGGAGGGGAGAGGGGGTAAAAAAATGCTGCACTGTGCGGCG
    AAGCCGGTGAGTGAGCGGCGCGGGGCCAATCAGCGTGCGCCG
    TTCCGAAAGTTGCCTTTTATGGCTCGAGCGGCCGCGGCGGCG
    CCCTATAAAACCCAGCGGCGCGACGCGCCACCACCGCCGAGT
    C
    Chicken beta- GGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTC 38
    actin (CBA) CCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTA
    GGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTC
    CCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTA
    ATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCG
    CGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA
    GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCC
    GAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCT
    ATAAAAAGCGAAGCGCGCGGCGGGCGGGA
    Cytomegalovirus TGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGC 39
    (CMV) GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACG
    TGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGC
    GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACG
    TCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTC
    CAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCG
    GTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTT
    TAGTGAACCG
    Cytomegalovirus TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAG 40
    (CMV) CCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGG
    (second CCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTC
    version) AATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTT
    CCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCA
    CTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCC
    TATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGC
    CCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACAT
    CTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTG
    GCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG
    ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTT
    TTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA
    CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTG
    GGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT
    Cytomegalovirus CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC 41
    (CMV) (third CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC
    version) CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT
    GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT
    GTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGG
    TAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
    GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC
    TATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCG
    TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCC
    CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACG
    GGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCA
    AATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAG
    AGCT
    CAG promoter ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCC 42
    (first version) CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAAC
    GCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT
    ACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT
    GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC
    CGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCC
    TACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT
    GGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTC
    CCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTA
    ATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCG
    CGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGA
    GGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCC
    GAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCT
    ATAAAAAGCGAAGCGCGCGGCGGGCGG
    CAG promoter CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCC 43
    (second CAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC
    version) CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT
    GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT
    GTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGG
    TAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
    GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGC
    TATTACCATGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTC
    CCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT
    ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGG
    GGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGG
    CGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGG
    CGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCG
    GCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG
    Human EF1- CAACCTTTGGAGCTAAGCCAGCAATGGTAGAGGGAAGATTCT 44
    alpha (EF1-α) GCACGTCCCTTCCAGGCGGCCTCCCCGTCACCACCCCCCCCA
    ACCCGCCCCGACCGGAGCTGAGAGTAATTCATACAAAAGGAC
    TCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACTC
    CCACTAACGTAGAACCCAGAGATCGCTGCGTTCCCGCCCCCT
    CACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAATAGCATG
    CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGC
    CCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAA
    CGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTG
    ATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAG
    AACCGTATATAAGTGCAGTAGTCGCCGTGAACGTT
    Human ACTTGTGGACAAAGTTTGCTCTATTCCACCTCCTCCAGGCCC 48
    CamKIIa TCCTTGGGTCCATCACCCCAGGGGTGCTGGGTCCATCCCACC
    (CaMKIIa) CCCAGGCCCACACAGGCTTGCAGTATTGTGTGCGGTATGGTC
    AGGGCGTCCGAGAGCAGGTTTCGCAGTGGAAGGCAGGCAGGT
    GTTGGGGAGGCAGTTACCGGGGCAACGGGAACAGGGCGTTTT
    GGAGGTGGTTGCCATGGGGACCTGGATGCTGACGAAGGCTCG
    CGAGGCTGTGAGCAGCCACAGTGCCCTGC
  • In a certain embodiment, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In a certain embodiment, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In a certain embodiment, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33.
  • Further illustrative examples of promoters are the SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements. A large variety of other promoters are known and generally available in the art, and the sequences of many such promoters are available in sequence databases such as the GenBank database.
  • In some cases, vectors of the present disclosure further comprise one or more regulatory elements selected from the group consisting of an enhancer, an intron, a poly-A signal, a 2A peptide encoding sequence, a WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element), and a HPRE (Hepatitis B posttranscriptional regulatory element).
  • In some embodiments, the vector comprises a CMV enhancer.
  • In certain embodiments, the vectors comprise one or more enhancers. In particular embodiments, the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a (3-actin enhancer sequence, or an EF1-α enhancer sequence. Sequences of the foregoing are known in the art. For example, the sequence of the CMV immediate early (IE) enhancer is SEQ ID NO: 50.
  • In certain embodiments, the vectors comprise one or more introns. In particular embodiments, the intron is a rabbit globin intron sequence, a chicken β-actin intron sequence, a synthetic intron sequence, an SV40 intron, or an EF1-α intron sequence.
  • In certain embodiments, the vectors comprise a polyA sequence. In particular embodiments, the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence. In some embodiments, the poly-A signal may be a bovine growth hormone polyadenylation signal (bGHpA).
  • In certain embodiments, the vectors comprise one or more transcript stabilizing element. In particular embodiments, the transcript stabilizing element is a WPRE sequence, a HPRE sequence, a scaffold-attachment region, a 3′ UTR, or a 5′ UTR. In particular embodiments, the vectors comprise both a 5′ UTR and a 3′ UTR.
  • In some embodiments, the vector comprises a 5′ untranslated region (UTR) selected from Table 4. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 51-61.
  • TABLE 4
    5′ SEQ
    UNTRANSLATED ID
    REGION SEQUENCE NO:
    Human beta-actin CGCGTCCGCCCGCGAGCACAGAGCCTCGCCTTTGCCGATC 51
    exon/intron CGCCGCCCGTCCACACCCGCCGCCAGGTAAGCCCGGCCAG
    CCGACCGGGGCATGCGGCCGCGGCCCTTCGCCCGTGCAGA
    GCCGCCGTCTGGGCCGCAGCGGGGGGCGCATGGGGCGGAA
    CCGGACCGCCGTGGGGGGCGCGGGAGAAGCCCCTGGGCCT
    CCGGAGATGGGGGACACCCCACGCCAGTTCGCAGGCGCGA
    GGCCGCGCTCGGGCGGGCGCGCTCCGGGGGTGCCGCTCTC
    GGGGCGGGGGCAACCGGCGGGGTCTTTGTCTGAGCCGGGC
    TCTTGCCAATGGGGATCGCACGGTGGGCGCGGCGTAGCCC
    CCGTCAGGCCCGGTGGGGGCTGGGGCGCCATGCGCGTGCG
    CGCTGGTCCTTTGGGCGCTAACTGCGTGCGCGCTGGGAAT
    TGGCGCTAATTGCGCGTGCGCGCTGGGACTCAATGGCGCT
    AATCGCGCGTGCGTTCTGGGGCCCGGGCGCTTGCGCCACT
    TCCTGCCCGAGCCGCTGGCGCCCGAGGGTGTGGCCGCTGC
    GTGCGCGCGCGCGACCCGGTCGCTGTTTGAACCGGGCGGA
    GGCGGGGCTGGCGCCCGGTTGGGAGGGGGTTGGGGCCTGG
    CTTCCTGCCGCGCGCCGCGGGGACGCCTCCGACCAGTGTT
    TGCCTTTTATGGTAATAACGCGGCCGGCCCGGCTTCCTTT
    GTCCCCAATCTGGGCGCGCGCCGGCGCCCCCTGGCGGCCT
    AAGGACTCGGCGCGCCGGAAGTGGCCAGGGCGGCAGCGGC
    TGCTCTTGGCGGCCCCGAGGTGACTATAGCCTTCTTTTGT
    GTCTTGATAGTTCGCCAGCCTCTGCTAACCATGTTCATGC
    CTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTT
    ATTGTGCTGTCTCATCATTTTGGCAAAGAATTC
    Chicken beta-actin GTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCG 52
    exon/intron + rabbit CCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTT
    globin intron ACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG
    GGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTT
    TTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGG
    CCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTG
    TGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGC
    CCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTG
    TGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGC
    GGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAG
    GCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGG
    TGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCC
    CCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGG
    GGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGG
    CGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGG
    CCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCG
    GCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCG
    CAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAG
    GGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTG
    GGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAA
    GCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGC
    CTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCC
    AGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGG
    GGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACC
    GGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCT
    TCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGT
    GCTGTCTCATCATTTTGGCAAAGAATTC
    SV40 intron GGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGA 53
    (Chimeric intron TCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGA
    sequence) TGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTC
    Shown in FIG. 14 TGCTCTAAAAGCTGCGGAATTGTACCCGC
    5′UTR-Syn1 Hs AGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAG 54
    AGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGCCCCCG
    CGGCTCCTGGCCAGACCACCCCTAGGACCCCCTGCCCCAA
    GTCGCA
    CMV IE exon TCAGATCGCCTGGAGAGGCCATCCACGCTGTTTTGACCTC 55
    CATAGTGGACACCGGGACCGATCCAGCCTCCGCGGCCGGG
    AACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGA
    C
    TPL-ePKP2 CTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGT 56
    (adenovirus derived TGGGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCA
    enhancer element) GTACTCTTGGATCGGAAACCCGTCGGCCTCCGAACGGTAC
    TCCGCCACCGAGGGACCTGAGCGAGTCCGCATCGACCGGA
    TCGGAAAACCTCTCGAGAAAGGCGTCTAACCAGTCACAGT
    CGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCAGCGGGTG
    GCGGTCGGGGTTGTTTCTGGCGGAGGTGCTGCTGATGATG
    TAATTAAAGTAGGCGGTCTTGAGACGGCGGATGGTCGAGG
    TGAGGTGTGGCAGGCTTGAGATCCAGCTGTTGGGGTGAGT
    ACTCCCTCTCAAAAGCGGGCATTACTTCTGCGCTAAGATT
    GTCAGTTTCCAAAAACGAGGAGGATTTGATATTCACCTGG
    CCCGATCTGGCCATACACTTGAGTGACAATGACATCCACT
    TTGCCTTTCTCTCCACAGGTGTCCACTCCCAG
    Human EF1-α CTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTG 57
    intron/exon CCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTA
    TGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTCCAG
    TACGTGATTCTTGATCCCGAGCTGGAGCCAGGGGCGGGCC
    TTGCGCTTTAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAG
    GCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGT
    GGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCT
    AGCCATTTAAAATTTTTGATGACGTGCTGCGACGCTTTTT
    TTCTGGCAAGATAGTCTTGTAAATGCGGGCCAGGATCTGC
    ACACTGGTATTTCGGTTTTTGGGCCCGCGGCCGGCGACGG
    GGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGC
    CTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTC
    AAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCC
    GTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCG
    GCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCC
    CTGCTCCAGGGGGCTCAAAATGGAGGACGCGGCGCTCGGG
    AGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCC
    TTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGT
    ACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTGGAGCTT
    TTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTAT
    GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAG
    TTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATT
    TGGCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCT
    CAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAG
    HumanEF1-α, GTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTA 58
    intron A CGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTG 
    GCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGA
    AGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCC
    CTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTG
    GGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGT
    CTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTT
    GATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCT
    TGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTT
    TTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGC
    GCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACC
    GAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCT
    CTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCT
    GGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGC
    GGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCA
    AAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGT
    CACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGT
    CGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGG
    CACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTT
    TAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCA
    CACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCAC
    TTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTG
    GATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG
    TTTTTTTCTTCCATTTCAG
    5′UTR human TCAGAAGCCCCGGGCTCGTCAGTCAAACCGGTTCTCTGTT 59
    CamKIIa TGCACTCGGCAGCACGGGCAGGCAAGTGGTCCCTAGGTTC
    GGG
    B-globin intron GTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTT 60
    TTCTATGGTTAAGTTCATGTCATAGGAAGGGGAGAAGTAA
    CAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATT
    TGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTT
    TTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCT
    TTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCA
    CCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGC
    AATAGCAATATTTCTGCATATAAATATTTCTGCATATAAA
    TTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGC
    TACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGG
    ATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTG
    CTAATCATGTTCATACCTCTTATCTTCCTCCCACAG
    SV40 intron (long TCTAGAGGATCCGGTACTCGAGGAACTGAAAAACCAGAAA 61
    form; underlined 5′ GTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGG
    and 3′ extensions) TCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCT
    CAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTG
    TTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGC
  • In some embodiments, the vector comprises a 3′ untranslated region selected from Table 5. In some embodiments, the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 62-70.
  • TABLE 5
    SEQ
    3′ UNTRANSLATED ID
    REGION SEQUENCE NO:
    WPRE(x) (mutated AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG 62
    woodchuck hepatitis GTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATA
    regulatory element - CGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGT
    version 1) ATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGC
    TGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACG
    TGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACT
    GGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGA
    CTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCAT
    CGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTG
    TTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCAT
    CGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGAT
    TCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTC
    AATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTC
    TGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAG
    TCGGATCTCCCTTTGGGCCGCCTCCCCGC
    WPRE(x) (mutated TCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGT 63
    woodchuck hepatitis ATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACG
    regulatory element - CTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTAT
    version 2) GGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTG
    TCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTG
    GCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGG
    TTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACT
    TTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCG
    CCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTT
    GGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCG
    TCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTC
    TGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAA
    TCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTG
    CGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTC
    GGATCTCCCTTTGGGCCGCCTCCCCGCA
    WPRE(x) (mutated TTCCTGTTAATCAACCTCTGGATTACAAAATTTGTGAAAG 64
    woodchuck hepatitis ATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTA
    regulatory element - TGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTG
    version 3) CTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATC
    CTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTC
    AGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAA
    CCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCT
    TTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCG
    GAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGG
    CTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGG
    GAAGCTGACGTCCTTTCCGCGGCTGCTCGCCTGTGTTGCC
    ACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTT
    CGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCT
    GCCGGCTCTGCGGCCTCTTCCGCCTCTTCGCCTTCGCCCT
    CAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCCA
    TGTATCTTTTTCACCTGTGCCTTGTTTTTGCCTGTGTTCC
    GCGTCCTACTTTTCAAGCCTCCAAGCTGTGCCTTGGGCGG
    CTTTGGGGCATGGACATAGATCCCTATAAAGAATTTGGTT
    CATCTTATCAGTTGTTGAATTTTCTTCCTTTGGAC
    CAAX TGTGTGATAATG 65
    EES CTGTTCTCATCACATCATATCAAGGTTATATACCATCAAT 66
    ATTGCCACAGATGTTACTTAGCCTTTTAATATTTCTCTAA
    TTTAGTGTATATGCAATGATAGTTCTCTGATTTCTGAGAT
    TGAGTTTCTCATGTGTAATGATTATTTAGAGTTTCTCTTT
    CATCTGTTCAAATTTTTGTCTAGTTTTATTTTTTACTGAT
    TTGTAAGACTTCTTTTTATAATCTGCATATTACAATTCTC
    TTTACTGGGGTGTTGCAAATATTTTCTGTCATTCTATGGC
    CTGACTTTTCTTAATGGTTTTTTAATTTTAAAAATAAGTC
    TTAATATTCATGCAATCTAATTAACAATCTTTTCTTTGTG
    GTTAGGACTTTGAGTCATAAGAAATTTTTCTCTACACTGA
    AGTCATGATGGCATGCTTCTATATTATTTTCTAAAAGATT
    TAAAGTTTTGCCTTCTCCATTTAGACTTATAATTCACTGG
    AATTTTTTTGTGTGTATGGTATGACATATGGGTTCCCTTT
    TATTTTTTACATATAAATATATTTCCCTGTTTTTCTAAAA
    AAGAAAAAGATCATCATTTTCCCATTGTAAAATGCCATAT
    TTTTTTCATAGGTCACTTACATATATCAATGGGTCTGTTT
    CTGAGCTCTACTCTATTTTATCAGCCTCACTGTCTATCCC
    CACACATCTCATGCTTTGCTCTAAATCTTGATATTTAGTG
    GAACATTCTTTCCCATTTTGTTCTACAAGAATATTTTTGT
    TATTGTCTTTGGGCTTTCTATATACATTTTGAAATGAGGT
    TGACAAGTTA
    HPRE ATAACAGGCCTATTGATTGGAAAGTTTGTCAACGAATTGT 67
    GGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAATGTGGA
    TATCCTGCTTTAATGCCTTTATATGCATGTATACAAGCAA
    AACAGGCTTTTACTTTCTCGCCAACTTACAAGGCCTTTCT
    CAGTAAACAGTATATGACCCTTTACCCCGTTGCTCGGCAA
    CGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCA
    CTGGTTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGG
    AACCTTTGTGTCTCCTCTGCCGATCCATACTGCGGAACTC
    CTAGCCGCTTGTTTTGCTCGCAGCAGGTCTGGAGCAAACC
    TCATCGGGACCGACAATTCTGTCGTACTCTCCCGCAAGTA
    TACATCGTTTCCATGGCTGCTAGGCTGTGCTGCCAACTGG
    ATCCTGCGCGGGACGTCCTTTGTTTACGTCCCGTCGGCGC
    TGAATCCCGCGGACGACCCCTCCCGGGGCCGCTTGGGGCT
    CTACCGCCCGCTTCTCCGTCTGCCGTACCGTCCGACCACG
    GGGCGCACCTCTCTTTACGCGGACTCCCCGTCTGTGCCTT
    CTCATCTGCCGGACCGTGTGCACTTCGCTTCACCTCTGCA
    CGTCGCATGGAGGCCACCGTGAACGCCCACCGGAACCTGC
    CCAAGGTCTTGCATAAGAGGACTCTTGGACTTTCAGCAAT
    GTCATC
    R2V17 (HepB derived TTCCTGTAAACAGGCCTATTGATTGGAAAGTTTGTCAACG 68
    enhancer element) AATTGTGGGTCTTTTGGGGTTTGCTGCCCCTTTTACGCAA
    TGTGGATATCCTGCTTTAATGCCTTTATATGCATGTATAC
    AAGCAAAACAGGCTTTTACTTTCTCGCCAACTTACAAGGC
    CTTTCTCAGTAAACAGTATATGACCCTTTACCCCGTTGCT
    CGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAA
    CCCCCACTGGTTGGGGCTTGGCCATAGGCCATCAGCGCAT
    GCGTGGAACCTTTGTGTCTCCTCTGCCGATCCATACTGCG
    GAACTCCTAGCCGCTTGTTTTGCTCGCAGCTGGACTGGAG
    CAAACCTCATCGGGACCGACAATTCTGTCGTACTCTCCCG
    CAAGCACTCACCGTTTCCGCGGCTGCTCGCCTGTGTTGCC
    ACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTT
    CGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCT
    GCCGGCTCTGCGGCCTCTTCCGCCTCTTCGCCTTCGCCCT
    CAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCCA
    TGTATCTTTTTCACCTGTGCCTTGTTTTTGCCTGTGTTCC
    GCGTCCTACTTTTCAAGCCTCCAAGCTGTGCCTTGGGCGG
    CTTTGGGGCATGGACATAGATCCCTATAAAGAATTTGGTT
    CATCTTATCAGTTGTTGAATTTTCTTCCTTTGGAC
    3′UTR(globin) GCTGGAGCCTCGGTAGCCGTTCCTCCTGCCCGCTGGGCCT 69
    CCCAACGGGCCCTCCTCCCCTCCTTGCACCGGCCCTTCCT
    GGTCTTTGAATAAA
    WPRE(r) ATTCGAGCATCTTACCGCCATTTATTCCCATATTTGTTCT 70
    GTTTTTCTTGATTTGGGTATACATTTAAATGTTAATAAAA
    CAAAATGGTGGGGCAATCATTTACATTTTTAGGGATATGT
    AATTACTAGTTCAGGTGTATTGCCACAAGACAAACATGTT
    AAGAAACTTTCCCGTTATTTACGCTCTGTTCCTGTTAATC
    AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGATAT
    TCTTAACTATGTTGCTCCTTTTACGCTGTGTGGATATGCT
    GCTTTAATGCCTCTGTATCATGCTATTGCTTCCCGTACGG
    CTTTCGTTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTC
    TCTTTATGAGGAGTTGTGGCCCGTTGTCCGTCAACGTGGC
    GTGGTGTGCTCTGTGTTTGCTGACGCAACCCCCACTGGCT
    GGGGCATTGCCACCACCTGTCAACTCCTTTCTGGGACTTT
    CGCTTTCCCCCTCCCGATCGCCACGGCAGAACTCATCGCC
    GCCTGCCTTGCCCGCTGCTGGACAGGGGCTAGGTTGCTGG
    GCACTGATAATTCCGTGGTGTTGTCGGGGAAGGGCC
  • In some embodiments, the vector comprises a polyadenylation (polyA) signal selected from Table 6. In some embodiments, the polyA signal comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 71-75.
  • TABLE 6
    POLY- SEQ
    ADENYLATION ID
    SITE SEQUENCE NO:
    Rabbit globin TGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTG 71
    (pAGlobin-Oc) TTGGAATTTTTTGTGTCTCTCACTCGGAAGAACATATGG
    GAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGT
    TTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCAT
    GAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAA
    ACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGC
    CTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGT
    GTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACA
    TGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACT
    CCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC
    Bovine growth TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTC 72
    hormone (pAGH-Bt - CTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTA
    version 1) ATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTG
    TCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAA
    GGGGGAGGATTGGGAATACAATAGCAGGCATGCTGGGGA
    TGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTG
    ACCCGGTTCCTCCTGGG
    Bovine growth TTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTC 73
    hormone (pAGH-Bt - CTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTA
    version 2) ATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTG
    TCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAA
    GGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA
    TGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTG
    ACCCGGTTCCTCCTGGG
    Bovine growth CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCT 74
    hormone (pAGH-Bt - CCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCA
    version 3) CTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT
    GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGG
    GGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCA
    GGCATGCTGGGGATGCGGTGGGCTCTATGG
    Human growth CTGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCT 75
    hormone (pAGH-Hs) CTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGC
    CTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGAC
    TAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTG
    GTATGGAGCAAGGGGCCCAAGTTGGGAAGAAACCTGTAG
    GGCCTGC
  • Illustrative vector genomes are depicted in FIGS. 1-4 and 11-14; and provided as SEQ ID NOs: 12-15 and 89-92. The expression cassette of each sequence is SEQ ID NOs: 8-11 or 93-96. In some embodiments, the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 12-15 and 89-92, optionally with or without the ITR sequences. In some embodiments, the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 8-11 and 93-96.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; a PKPa transgene; an WPRE(x) element; an pAGH-HS sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; any one of SEQ ID NOs: 3, 6, and 87; SEQ ID NO: 63; and SEQ ID NO: 75; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; a PKPa transgene; an WPRE(x) element; an pAGH-HS sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 3, 6, and 87; SEQ ID NO: 63; and SEQ ID NO: 75; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; a PKPb transgene; an WPRE(x) element; an pAGH-HS sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; any one of SEQ ID NOs: 4, 7, and 88; SEQ ID NO: 63; and SEQ ID NO: 75; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; a PKPb transgene; an WPRE(x) element; an pAGH-HS sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 4, 7, and 88; SEQ ID NO: 63; and SEQ ID NO: 75; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; a PKPa transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; any one of SEQ ID NOs: 3, 6, and 87; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; a PKPa transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 3, 6, and 87; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; a PKPb transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; any one of SEQ ID NOs: 4, 7, and 88; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; a PKPb transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; any one of SEQ ID NOs: 4, 7, and 88; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; SV40 intron; a PKPa transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 3, 6, and 87; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; SV40 intron; a PKPa transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 3, 6, and 87; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2a transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; an MHCK7 promoter; SV40 intron; a PKPb transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 31; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 4, 7, and 88; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In a certain embodiment, the vector genome comprises, in 5′ to 3′ order, a 5′ ITR; a hTnnT2 promoter; SV40 intron; a PKPb transgene; optionally a WPRE element; a polyadenylation sequence; and a 3′ ITR. The vector genome may comprise, in 5′ to 3′ order, the polynucleotide sequences SEQ ID NO: 32 or 33; SEQ ID NO: 53 or 61; any one of SEQ ID NOs: 4, 7, and 88; or polynucleotide sequences sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to each of the foregoing. In certain embodiments, this vector genome is packaged in an AAV9 or AAVrh74 vector. The PKP2b transgene of this embodiment is a full length transgene, i.e. a transgene encoding a PKP of at least 800 or at least 830 amino acids.
  • In each case the optionally WPRE element may be present or absent.
    • Adeno-Associated Virus Vector
  • AAV vectors useful in the practice of the present invention can be packaged into AAV virions (viral particles) using various systems including adenovirus-based and helper-free systems. Standard methods in AAV biology include those described in Kwon and Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) 14(3):316-27. Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimm et al. Curr Gene Ther. (2003) 3(4):281-304; Deyle D R, Russell D W. Curr Opin Mol Ther. (2009) 11(4):442-447; McCarty et al. Gene Ther. (2001) 8(16):1248-54; and Duan et al. Mol Ther. (2001) 4(4):383-91. Helper-free systems included those described in U.S. Pat. Nos. 6,004,797; 7,588,772; and 7,094,604;
  • AAV DNA in the rAAV genomes may be from any AAV variant or serotype for which a recombinant virus can be derived including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAVrh10. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. 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.
  • In some cases, the rAAV comprises a self-complementary genome. As defined herein, an rAAV comprising a “self-complementary” or “double stranded” genome refers to an rAAV which has been engineered such that the coding region of the rAAV is configured to form an intra-molecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promoter efficient transduction independently of DNA synthesis. Gene Therapy. 8 (16): 1248-54 (2001). The present disclosure contemplates the use, in some cases, of an rAAV comprising a self-complementary genome because upon infection (such transduction), rather than waiting for cell mediated synthesis of the second strand of the rAAV genome, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. It will be understood that instead of the full coding capacity found in rAAV (4.7-6 kb), rAAV comprising a self-complementary genome can only hold about half of that amount (≈2.4 kb).
  • In other cases, the rAAV vector comprises a single stranded genome. As defined herein, a “single standard” genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAVs have singled stranded DNA genomes. There have been some indications that rAAVs should be scAAVs to achieve efficient transduction of cells. The present disclosure contemplates, however, rAAV vectors that maybe have singled stranded genomes, rather than self-complementary genomes, with the understanding that other genetic modifications of the rAAV vector may be beneficial to obtain optimal gene transcription in target cells. In some cases, the present disclosure relates to single-stranded rAAV vectors capable of achieving efficient gene transfer to anterior segment in the mouse eye. See Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE 12(8): e0182473 (2017).
  • In some cases, the rAAV vector is of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). In some cases, the rAAV vector is of the serotype AAV9. In some embodiments, said rAAV vector is of serotype AAV9 and comprises a single stranded genome. In some embodiments, said rAAV vector is of serotype AAV9 and comprises a self-complementary genome. In some embodiments, a rAAV vector comprises the inverted terminal repeat (ITR) sequences of AAV2. In some embodiments, the rAAV vector comprises an AAV2 genome, such that the rAAV vector is an AAV-2/9 vector, an AAV-2/6 vector, or an AAV-2/8 vector.
  • Full-length sequences and sequences for capsid genes for most known AAVs are provided in U.S. Pat. No. 8,524,446, which is incorporated herein in its entirety.
  • AAV vectors may comprise wild-type AAV sequence or they may comprise one or more modifications to a wild-type AAV sequence. In certain embodiments, an AAV vector comprises one or more amino acid modifications, optionally substitutions, deletions, or insertions, within a capsid protein, optionally VP1, VP2 and/or VP3. In particular embodiments, the modification provides for reduced immunogenicity when the AAV vector is provided to a subject.
  • Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as endothelial cells or more particularly endothelial tip cells. In some embodiments, the rAAV is directly injected into the intracerebroventricular space of the subject.
  • In some embodiments, the rAAV virion is an AAV2 rAAV virion. The capsid many be an AAV2 capsid or functional variant thereof. In some embodiments, the AAV2 capsid shares at least 98%, 99%, or 100% identity to a reference AAV2 capsid, e.g., SEQ ID NO: 76.
  • In some embodiments, the rAAV virion is an AAV9 rAAV virion. The capsid many be an AAV9 capsid or functional variant thereof. In some embodiments, the AAV9 capsid shares at least 98%, 99%, or 100% identity to a reference AAV9 capsid, e.g., SEQ ID NO: 77.
  • In some embodiments, the rAAV virion is an AAV6 rAAV virion. The capsid many be an AAV9 capsid or functional variant thereof. In some embodiments, the AAV6 capsid shares at least 98%, 99%, or 100% identity to a reference AAV6 capsid, e.g., SEQ ID NO: 78.
  • In some embodiments, the rAAV virion is an AAVrh.10 rAAV virion. The capsid many be an AAV9 capsid or functional variant thereof. In some embodiments, the AAVrh.10 capsid shares at least 98%, 99%, or 100% identity to a reference AAVrh.10 capsid, e.g., SEQ ID NO: 79.
  • In some embodiments, the capsid protein is encoded by a polynucleotide supplied on a plasmid in trans to the transfer plasmid. The polynucleotide sequence of wild-type AAVrh74 cap is provided as SEQ ID NO: 80.
  • The disclosure further provides protein sequences for AAVrh74 VP1, VP2, and VP3, including SEQ ID NOs: 81-83, and homologs or functional variants thereof.
  • In certain cases, the AAVrh74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh74 VP1 which is set forth in SEQ ID NO: 81. In some embodiments, the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh74 VP2 which is set forth in SEQ ID NO: 82. In some embodiments, the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh74 VP3 which is set forth in SEQ ID NO: 83.
  • In some embodiments, the rAAV virion is an AAV-PHP.B rAAV virion or a neutrotrophic variant thereof, such as, without limitation, those disclosed in Int'l Pat. Pub. Nos. WO 2015/038958 A1 and WO 2017/100671 A1. For example, the AAV capsid may comprise at least 4 contiguous amino acids from the sequence TLAVPFK (SEQ ID NO:85) or KFPVALT (SEQ ID NO:86), e.g., inserted between a sequence encoding for amino acids 588 and 589 of AAV9.
  • The capsid many be an AAV-PHP.B capsid or functional variant thereof. In some embodiments, the AAV-PHP.B capsid shares at least 98%, 99%, or 100% identity to a reference AAV-PHP.B capsid, e.g., SEQ ID NO: 84.
  • Further AAV capsids used in the rAAV virions of the disclosure include those disclosed in Pat. Pub. Nos. WO 2009/012176 A2 and WO 2015/168666 A2.
  • Without being bound by theory, the present inventors have determined that an AAV9 vector, AAVrh.74, or an AAVrh.10 vector will confer desirable cardiac tropism on the vector. Without being bound by theory, the present inventors have further determined that an AAV9 vector, AAVrh.74, or an AAVrh.10 vector may provide desired specificity to cardiac cells.
  • In an aspect, the disclosure provides pharmaceutical compositions comprising the rAAV virion of the disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • For purposes of administration, optionally by injection, various solutions can be employed, such as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as Poloxamer 188, e.g., at 0.001% or 0.01%. A dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
  • The pharmaceutical forms suitable for injectable use include but are not limited to sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form is sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the certain methods of preparation are vacuum drying and the freeze-drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • In another aspect, the disclosure comprises a kit comprising an rAAV virion of the disclosure and instructions for use.
  • In an aspect, the disclosure provides a method of increasing PKP2 activity in a cell, comprising contacting the cell with an rAAV of the disclosure. In another aspect, the disclosure provides a method of increasing PKP2 activity in a subject, comprising administering to the subject an rAAV of the disclosure. In some embodiments, the cell and/or subject is deficient in PKP2 messenger RNA or PKP2 protein expression levels and/or activity and/or comprises a loss-of-function mutation in PKP2. The cell may be a cardiac cell, e.g. a cardiomyocyte cell. In particular embodiments, the subject is a mammal, e.g., a human.
  • In some embodiments, the method promotes survival of cardiac cell, e.g. a cardiomyocyte cell, in cell culture and/or in vivo. In some embodiments, the method promotes and/or restores function of the heart.
  • In another aspect, the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of an rAAV virion of the disclosure. In some embodiments, the disease or disorder is a cardiac disease or disorder. Illustrative cardiac disorders include heart failure, arrhythmogenic right ventricular cardiomyopathy (ARVC), Brugada syndrome (BrS) and idiopathic ventricular fibrillation. In certain embodiments, the subject suffers from or is at risk for arrhythmogenic right ventricular cardiomyopathy (ARVC). In particular embodiments, the subject is a mammal, e.g., a human, having a loss-of-function mutation in a PKP2 gene. In particular methods, treatment with the rAAV virion results in expression of the PKP2 protein encoded by the rAAV virion in the subject, e.g., in the subject's heart or cardiac tissue. In certain embodiments, treatment with the rAAV virion results in at least two-fold, at least five-fold, at least ten-fold, or more PKP2 protein levels detectable in the subject's heart.
  • The AAV-mediated delivery of PKP2 protein to the heart may increase life span, prevent or attenuate cardiac cell degeneration, heart failure, scarring, reduced ejection fraction, arrythmia, angina, exercise intolerance, angina (chest pain), sudden cardiac death, exertional myalgias and cramps. The AAV-mediated delivery of PKP2 protein to the heart may show improvement from, or prevent normal disease course detected by use of pathological electrocardiogram, cardiac MRI, heart biopsy, decrease in paroxysmal ventricular arrhythmias, decrease in sudden cardiac death, and/or decrease in or lack of further development of fibro-fatty deposits in right ventricular myocardium. The methods of the disclosure may prevent a decrease in, restore, and/or increase right ventricular ejection fraction (RVEF).
  • The methods disclosed herein may provide efficient biodistribution in the heart. They may result in sustained in expression in all, or a substantial fraction of, cardiac cells, e.g., cardiomyocytes. Notably, the methods disclosed herein may provide long-lasting expression of PKP2 protein throughout the life of the subject following AAV vector administration. In some embodiments, PKP2 protein expression in response to treatment lasts at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, or 40 years.
  • Combination therapies are also contemplated by the invention. Combinations of methods of the invention with standard medical treatments (e.g., corticosteroids or topical pressure reducing medications) are specifically contemplated, as are combinations with novel therapies. In some cases, a subject may be treated with a steroid and/or combination of immune suppressing agents to prevent or to reduce an immune response to administration of a rAAV described herein.
  • In some embodiments, the AAV vector is administered at a dose of between about 1×1012 and 5×1014 vector genomes (vg) or between about 1×1012 and 6×1014 vg of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered at a dose of between about 1×1013 and 5×1014 vg/kg. In some embodiments, the AAV vector is administered at a dose of between about 5×1013 and 3×1014 vg/kg. In some embodiments, the AAV vector is administered at a dose of between about 5×1013 and 1×1014 vg/kg. In some embodiments, the AAV vector is administered at a dose of less than about 1×1012 vg/kg, less than about 3×1012 vg/kg, less than about 5×1012 vg/kg, less than about 7×1012 vg/kg, less than about 1×1013 vg/kg, less than about 3×1013 vg/kg, less than about 5×1013 vg/kg, less than about 7×1013 vg/kg, less than about 1×1013 vg/kg, less than about 3×1014 vg/kg, less than about 5×1014 vg/kg, less than about 7×1014 vg/kg, less than about 1×1015 vg/kg, less than about 3×1015 vg/kg, less than about 5×1015 vg/kg, or less than about 7×1015 vg/kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector. In some cases, it may be advantageous to use a higher dose for an AAV rh74 vector than for an AAV9 vector.
  • In some embodiments, the AAV vector is administered at a dose of about 1×1012 vg/kg, about 3×1012 vg/kg, about 5×1012 vg/kg, about 7×1012 vg/kg, about 1×1013 vg/kg, about 3×1013 vg/kg, about 5×1013 vg/kg, about 7×1013 vg/kg, about 1×1014 vg/kg, about 3×1014 vg/kg, about 5×1014 vg/kg, about 7×1014 vg/kg, about 1×1015 vg/kg, about 3×1015 vg/kg, about 5×1015 vg/kg, or about 7×1015 vg/kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • In some embodiments, the AAV vector is administered at a dose of 1×1012 vg/kg, 3×1012 vg/kg, 5×1012 vg/kg, 7×1012 vg/kg, 1×1013 vg/kg, 3×1013 vg/kg, 5×1013 vg/kg, 7×1013 vg/kg, 1×1014 vg/kg, 3×1014 vg/kg, 5×1014 vg/kg, 7×1014 vg/kg, 1×1015 vg/kg, 3×1015 vg/kg, 5×1015 vg/kg, or 7×1015 vg/kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • In some embodiments, the AAV vector is administered systemically at a dose of between about 1×1012 and 5×1014 vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered systemically at a dose of between about 1×1013 and 5×1014 vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of between about 5×1013 and 3×1014 vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of between about 5×1013 and 1×1014 vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of less than about 1×1012 vg/kg, less than about 3×1012 vg/kg, less than about 5×1012 vg/kg, less than about 7×1012 vg/kg, less than about 1×1013 vg/kg, less than about 3×1013 vg/kg, less than about 5×1013 vg/kg, less than about 7×1013 vg/kg, less than about 1×1014 vg/kg, less than about 3×1014 vg/kg, less than about 5×1014 vg/kg, less than about 7×1014 vg/kg, less than about 1×1015 vg/kg, less than about 3×1015 vg/kg, less than about 5×1015 vg/kg, or less than about 7×1015 vg/kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • In some embodiments, the AAV vector is administered systemically at a dose of about 1×1012 vg/kg, about 3×1012 vg/kg, about 5×1012 vg/kg, about 7×1012 vg/kg, about 1×1013 vg/kg, about 3×1013 vg/kg, about 5×1013 vg/kg, about 7×1013 vg/kg, about 1×1014 vg/kg, about 3×1014 vg/kg, about 5×1014 vg/kg, about 7×1014 vg/kg, about 1×1015 vg/kg, about 3×1015 vg/kg, about 5×1015 vg/kg, or about 7×1015 vg/kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • In some embodiments, the AAV vector is administered systemically at a dose of 1×1012 vg/kg, 3×1012 vg/kg, 5×1012 vg/kg, 7×1012 vg/kg, 1×1013 vg/kg, 3×1013 vg/kg, 5×1013 vg/kg, 7×1013 vg/kg, 1×1014 vg/kg, 3×1014 vg/kg, 5×1014 vg/kg, 7×1014 vg/kg, 1×1015 vg/kg, 3×1015 vg/kg, 5×1015 vg/kg, or 7×1015 vg/kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • In some embodiments, the AAV vector is administered intravenously at a dose of between about 1×1012 and 5×1014 vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered intravenously at a dose of between about 1×1013 and 5×1014 vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of between about 5×1013 and 3×1014 vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of between about 5×1013 and 1×1014 vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of less than about 1×1012 vg/kg, less than about 3×1012 vg/kg, less than about 5×1012 vg/kg, less than about 7×1012 vg/kg, less than about 1×1013 vg/kg, less than about 3×1013 vg/kg, less than about 5×1013 vg/kg, less than about 7×1013 vg/kg, less than about 1×1014 vg/kg, less than about 3×1014 vg/kg, less than about 5×1014 vg/kg, less than about 7×1014 vg/kg, less than about 1×1015 vg/kg, less than about 3×1015 vg/kg, less than about 5×1015 vg/kg, or less than about 7×1015 vg/kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • In some embodiments, the AAV vector is administered intravenously at a dose of about 1×1012 vg/kg, about 3×1012 vg/kg, about 5×1012 vg/kg, about 7×1012 vg/kg, about 1×1013 vg/kg, about 3×1013 vg/kg, about 5×1013 vg/kg, about 7×1013 vg/kg, about 1×1014 vg/kg, about 3×1014 vg/kg, about 5×1014 vg/kg, about 7×1014 vg/kg, about 1×1015 vg/kg, about 3×1015 vg/kg, about 5×1015 vg/kg, or about 7×1015 vg/kg.
  • In some embodiments, the AAV vector is administered intravenously at a dose of 1×1012 vg/kg, 3×1012 vg/kg, 5×1012 vg/kg, 7×1012 vg/kg, 1×1013 vg/kg, 3×1013 vg/kg, 5×1013 vg/kg, 7×1013 vg/kg, 1×1014 vg/kg, 3×1014 vg/kg, 5×1014 vg/kg, 7×1014 vg/kg, 1×1015 vg/kg, 3×1015 vg/kg, 5×1015 vg/kg, or 7×1015 vg/kg. In certain embodiments, the AAV vector delivered at any of these doses is an AAV9 vector or an AAV rh74 vector.
  • Evidence of functional improvement, clinical benefit or efficacy in patients may be revealed by change in New York Heart Association functional classification (NYHA Class), pathological electrocardiogram, cardiac MRI, heart biopsy, decrease in paroxysmal ventricular arrhythmias, decrease in sudden cardiac death, and/or decrease in or lack of further development of fibro-fatty deposits in right ventricular myocardium. Benefit may be observed in electrocardiographic features normally associated with arrhythmogenic right ventricular cardiomyopathy such as T wave inversion, prolonged S-wave upstroke, localized QRS widening, and/or paroxysmal episodes of ventricular tachycardia.
  • In some embodiments, the method prevents or reduces a decrease in left ventricle ejection fraction percentage (LVEF %), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • In some embodiments, the method prevents or reduces a decrease in left ventricle fractional shortening percentage (FS %), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • In some embodiments, the method prevents or reduces an increase in right ventricle area in millimeters squared (RV Area (mm2), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • In some embodiments, the method prevents or reduces a decrease in right ventricle velocity time integral in millimeters per second (RV VTI (mm/sec), optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the decrease observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • In some embodiments, the method prevents or reduces an increase in left ventricle or right ventricle fibrosis, optionally by about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to the increase observed in an untreated subject suffering from or at risk for disease or disorder related to or caused by loss of function in PKP2.
  • Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, systemic, local, direct injection, intravenous, intracardiac administration. In some cases, administration comprises systemic, local, direct injection, intravenous, intracardiac injection. Administration may be performed by cardiac catheterization.
  • In some embodiments, the disclosure provides for local administration and systemic administration of an effective dose of rAAV and compositions of the invention. For example, systemic administration may be administration into the circulatory system so that the entire body is affected. Systemic administration includes parental administration through injection, infusion or implantation. Routes of administration for the compositions disclosed herein include intravenous (“IV”) administration, intraperitoneal (“IP”) administration, intramuscular (“IM”) administration, intralesional administration, or subcutaneous (“SC”) administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, a depot formulation, etc. In some embodiments, the methods of the disclosure comprise administering an AAV vector of the disclosure, or pharmaceutical composition thereof by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic or intracranial administration.
  • In particular, administration of rAAV of the present invention may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal. Administration includes, but is not limited to, injection into the heart.
  • In some embodiments, the methods of the disclosure comprise intracardiac delivery. Infusion may be performed using specialized cannula, catheter, syringe/needle using an infusion pump. Administration may comprise delivery of an effective amount of the rAAV virion, or a pharmaceutical composition comprising the rAAV virion, to the heart. These may be achieved, e.g., via intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic or intracranial administration. The compositions of the disclosure may further be administered intravenously.
  • The method of treatment disclosed herein may reduce and/or prevent one or more symptoms including but not limited to ventricular hypertrophy, ventricular tachycardia, exercise intolerance, angina, and reduced RVEF.
    • EXAMPLES Example 1: Pre-Clinical Bioactivity and Efficacy
  • Vectors illustrated in FIGS. 1-4 are tested. AAV vectors or respective expression cassettes are tested in vitro using cultured cardiomyocytes (e.g., induced pluripotent stem cell cardiomyocytes, iPSC-CMs) or other cells amenable to transfection or transduction with these constructs. Expression of PKP2 is assessed by immunofluorescence and Western blot. Cell-based studies employing patient iPSC-derived cardiomyocytes will reveal benefit of overexpression of PKP2 transgene (either following AAV vector transduction and/or transfection with vector plasmids) by a decreased adipogenic potential (e.g. less lipid accumulation), decreased upregulation or abnormal peroxisome proliferator-activated receptor gamma activation, associated with increased density of PKP2.
  • Selected vectors are tested in vivo using mutant mouse models of cardiomyopathy (e.g., PKP2-cKO, among others). Evidence of benefit of AAV mediated overexpression of PKP2 may be revealed using a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line, referred to as “PKP2-cKO”. This mouse model allows control of the onset of PKP2 loss of expression, limits loss of PKP2 to adult myocytes, and initiates a progression of molecular and functional events leading to an arrhythmogenic cardiomyopathy, with right ventricular predominance in this mouse. Additional mouse models that result in similar course of pathology may also be utilized to reveal benefit of AAV-mediated overexpression of PKP2 in cardiomyocytes. Benefit of AAV-mediated PKP2 overexpression would be evidenced by increase in survival, mitigation of the normal progression of cardiomyopathy observed on echocardiograms from left and/or right ventricle (e.g. greater left ventricular ejection fraction, greater left ventricle fractional shortening, and greater right ventricle velocity time interval, compared to PKP cKO formulation buffer control animals).
  • Electrophysiological evidence of functional benefit of AAV-mediated delivery of PKP2 protein is demonstrated by mitigation of disease-related disrupted calcium dynamics in affected cardiomyocytes, most notably on measures of L-type calcium current, sarcoplasmic reticulum calcium leak, diastolic calcium leak, as well as standard measures of calcium transients in affected (e.g., PKP2-deficient) cardiomyocytes such as time to peak amplitude and relaxation time constants. Histological analyses will reveal benefit of AAV-mediated PKP2 overexpression by diminished appearance of disease-related collagen deposition (e.g., via trichrome stain) in various regions of the heart including ventricles, compared to cKO formulation buffer injected controls. Additional benefit will also be revealed by evaluating cardiomyocyte ventricular proteins involved in calcium signaling pathways, measured by increased (i.e., normalized) relative levels of Casq2, and/or Trdn, and/or Cav 1.2, and/or AnkB and/or RyR2, relative to non AAV-PKP2 treated, PKP2-cKO diseased controls.
    • Example 2: In Vitro Testing of Adeno-Associated Virus Vectors
  • AAV vectors are described herein (see FIGS. 11-12) were prepared and used to transduce differentiated AC16 cells, a human cardiomyocyte cell line. Expression levels of PKP2 (PKP2a isoform) were assessed by Western Blot (FIGS. 5A-5B). Surprisingly, the MHCK7 promoter causes robust expression of PKP2 in cardiomyocytes, whereas the hTnnT2 promoter (“hTnT”) generates marginal PKP2 levels above background under the current testing conditions. The AAVrh.74 serotype induced higher expression of PKP2 than the AAV9 serotype vector.
  • Based on these results, we conclude that AAV9 vectors or AAVrh74 vectors can effectively be used to express PKP2 in cardiomyocytes, and that the MHCK7 promoter is superior to the hTnnT2 promoter when solely evaluating the relative levels of PKP2 expression.
    • Example 3: In Vivo Efficacy of Adeno-Associated Virus Vectors
  • A “PKP2-cKO” mouse model of PKP2-deficiency, as described in Cerrone et al., Nat Comm., 2017 was obtained. This cardiomyocyte-specific, tamoxifen-activated PKP2 knockout murine line (aMHC-Cre-ER(T2)/Pkp2 fl/fl; referred to as “PKP2-cKO”) was utilized to control the onset of PKP2 loss of expression (see Cerrone et al., Nat Comm, 2017). The conditional loss of PKP2 expression in this mouse model is limited to adult myocytes and the temporal progression of the molecular, structural and functional events as a consequence of PKP2-cKO have been established (Cerrone et al., Nat Comm, 2017). PKP2 deficiency in adult ventricular myocytes is sufficient to cause an arrhythmogenic cardiomyopathy of RV predominance, which includes the ‘hallmark’ functional, molecular, and structural indices consistent with the disease phenotype of ARVC.
  • PKP2-Cko mice were injected with tamoxifen, causing myocyte-specific knockout of PKP2. Mice were injected with AAV vectors (as described below) at 3×1013 vg/kg by intravenous (tail vein) injection. Four weeks later, myocyte-specific knockout of PKP2 was induced by treatment of the mice with tamoxifen. The vector genomes used were:
  • 5′ ITR; MHCK7 promoter (with its enhancer element); SV40 intron; Kozak sequence; PKPa transgene; WPRE(x); hGH polyadenylation sequence); 3′ ITR—shown in FIG. 11
  • 5′ ITR; hTnnT2 promoter (with exon 1); Kozak sequence; PKPa transgene; WPRE(x); hGH polyadenylation sequence); 3′ ITR—shown in FIG. 12.
  • Each vector genome was tested in a AAV9 serotype or AAVrh74 serotype vector.
  • At 21 or 28 days after tamoxifen treatment, which is 25 or 32 weeks after AAV treatment, mice were evaluated for various physiology parameters, essentially as described in Cerrone et al., Nat Comm, 2017 or using standard methodologies known in the art. Efficacy in treating disease was assessed by left ventricle ejection fraction percentage (LVEF %) (FIGS. 6A-6D), left ventricle fractional shortening percentage (FS %) (FIGS. 7A-7D), right ventricle area in millimeters squared (RV Area (mm2)) (FIGS. 8A-8D), right ventricle velocity time integral in millimeters per second (RV VTI (mm/sec)) (FIGS. 9A-9D), and degree of fibrosis (FIGS. 10A-10B). These measures are appropriate functional and morphological indices to evaluate potential efficacy of AAV-mediated PKP2 overexpression in cardiomyocytes as they are among key parameters indicative of ARVC in human disease. Generally, a right ventricle normally has slightly greater amount of fibrosis (irrespective of disease); and this is further exacerbated with lack of PKP2 in the cKO model. Progressive deterioration of these parameters was observed within 21 days of tamoxifen injection, because tamoxifen injection causes myocyte-specific knockout of the PKP gene.
  • Evidence for mitigation of the disease phenotype was observed following both AAV9- and AAVrh.74-mediated PKP expression, to varying degrees. With the dose studied to date (3×1013 vg/kg) using a pre-treatment paradigm (AAV 4 weeks prior to tamoxifen-induced PKP cKO), AAV9 surprisingly produced the most robust effects on all parameters. Nevertheless, given the cardiotropism of AAVrh74 and given that biological effects were observed with AAVrh.74-mediated overexpression of PKP2 in this model (e.g. LVEF %, FS %, and right ventricular area), optimization of the dose of AAVrh.74 in combination with the appropriate promoter (i.e., either MHCK7 or hTnnT2) could enable robust therapeutic potential for this vector.
  • These results demonstrate both AAV9 and AAVrh.74 can be used to treat PKP2-related diseases, such as Arrhythmogenic right ventricular cardiomyopathy (ARVC) [also known as Arrhythmogenic Right Ventricular Dysplasia (ARVD) or Arrhythmogenic Cardiomyopathy (ACM)] for which the PKP-cKO mouse is considered an appropriate model. Additionally, vectors with either MHCK7 promoter or hTnnT2 promoter have been demonstrated to be effective in treating PKP2-related disease.

Claims (31)

1.-91. (canceled)
92. A method of treating arrhythmogenic right ventricular cardiomyopathy (ARVC) in a subject having a mutation in a PKP2 gene, comprising:
administering to the subject a recombinant adeno-associated virus (rAAV) vector comprising a vector genome,
wherein the vector genome comprises an expression cassette and flanking AAV inverted terminal repeats (ITRs), the expression cassette comprising a polynucleotide comprising:
a polynucleotide sequence encoding a Plakophilin-2 (PKP2);
a promoter sequence, wherein the promoter sequence is operatively linked to the polynucleotide sequence encoding the PKP2; and
a polyA sequence,
wherein the rAAV vector is an AAV9 vector, and
wherein the method treats one or more symptoms of ARVC.
93. The method of claim 92, wherein the rAAV vector comprises a capsid protein that shares at least 95% polypeptide sequence identity to SEQ ID NO: 77.
94. The method of claim 92, wherein the rAAV vector comprises a capsid protein that shares 99% polypeptide sequence identity to SEQ ID NO: 77.
95. The method of claim 92, wherein the rAAV vector comprises a capsid protein that shares 100% polypeptide sequence identity to SEQ ID NO: 77.
96. The method of claim 92, wherein the promoter is a cardiac-specific promoter.
97. The method of claim 92, wherein the promoter is a muscle-specific promoter.
98. The method of claim 92, wherein the promoter is a cardiomyocyte-specific promoter.
99. The method of claim 98, wherein the promoter is a Myosin Heavy-chain Creatine Kinase 7 (MHCK7) promoter.
100. The method of claim 99, wherein the MHCK7 promoter shares at least 90% polynucleotide sequence identity with SEQ ID NO: 31.
101. The method of claim 99, wherein the MHCK7 promoter shares at least 95% polynucleotide sequence identity with SEQ ID NO: 31.
102. The method of claim 98, wherein the promoter is a cardiac troponin T (hTNNT2) promoter.
103. The method of claim 102, wherein the hTNNT2 promoter shares at least 90% polynucleotide sequence identity with SEQ ID NO: 33.
104. The method of claim 102, wherein the hTNNT2 promoter shares at least 95% polynucleotide sequence identity with SEQ ID NO: 33.
105. The method of claim 102, wherein the expression cassette comprises exon 1 of the cardiac troponin T (hTNNT2) gene.
106. The method of claim 105, where the hTNNT2 promoter and exon 1 together share at least 90% polynucleotide sequence identity with SEQ ID NO: 32.
107. The method of claim 102, wherein the polyA signal is a human growth hormone (hGH) polyA.
108. The method of claim 107, wherein the expression cassette comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
109. The method of claim 102, wherein the PKP2 is PKP2 isoform A.
110. The method of claim 109, wherein the PKP2 isoform A shares at least 90% polypeptide sequence identity with SEQ ID NO: 1.
111. The method of claim 102, wherein the PKP2 is PKP2 isoform B.
112. The method of claim 111, wherein the PKP2 isoform B shares at least 90% polypeptide sequence identity with SEQ ID NO: 2.
113. The method of claim 92, wherein the rAAV vector is administered by intravenous injection, intracardiac injection, intracardiac infusion, or cardiac catheterization.
114. The method of claim 92, wherein the rAAV vector is administered at a dose of between about 1×1013 and 5×1014 vg/kg.
115. The method of claim 92, wherein the rAAV vector is administered at a dose of between about 5×1013 and 3×1014 vg/kg.
116. The method of claim 92, wherein the rAAV vector is administered at a dose of about 2×1014 vg/kg.
117. The method of claim 92, wherein the method reduces a decrease in left ventricle ejection fraction percentage (LVEF %) compared to the decrease observed in an untreated subject identified as having a mutation in the PKP2 gene.
118. The method of claim 92, wherein the method reduces a decrease in left ventricle fractional shortening percentage (FS %) compared to the decrease observed in an untreated subject identified as having a mutation in the PKP2 gene.
119. The method of claim 92, wherein the method prevents an increase in right ventricle area in millimeters squared (RV Area (mm2)).
120. The method of claim 92, wherein the method increases survival compared to survival of untreated subjects identified as having a mutation in the PKP2 gene.
121. A recombinant adeno-associated virus (rAAV) vector comprising a vector genome,
wherein the vector genome comprises an expression cassette and flanking AAV inverted terminal repeats (ITRs), the expression cassette comprising a polynucleotide comprising:
a polynucleotide sequence encoding a Plakophilin-2 (PKP2);
a promoter sequence, wherein the promoter sequence is operatively linked to the polynucleotide sequence encoding the PKP2; and
a polyA sequence, and
wherein the rAAV vector is an AAV9 vector.
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