WO2024215655A1 - Thérapies cardioprotectrices par bag3 - Google Patents

Thérapies cardioprotectrices par bag3 Download PDF

Info

Publication number
WO2024215655A1
WO2024215655A1 PCT/US2024/023699 US2024023699W WO2024215655A1 WO 2024215655 A1 WO2024215655 A1 WO 2024215655A1 US 2024023699 W US2024023699 W US 2024023699W WO 2024215655 A1 WO2024215655 A1 WO 2024215655A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
group
seq
virion
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/023699
Other languages
English (en)
Inventor
James Rush PRIEST
Yanchun XU
Jonathan Hung Yuan Tsui
Kathryn N. Ivey
Timothy C. Hoey
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tenaya Therapeutics Inc
Original Assignee
Tenaya Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tenaya Therapeutics Inc filed Critical Tenaya Therapeutics Inc
Publication of WO2024215655A1 publication Critical patent/WO2024215655A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • 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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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
    • C12N15/11—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
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • 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
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • 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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • 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
    • 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

Definitions

  • the Sequence Listing contained in the XML file is herein incorporated by reference in its entirety.
  • TECHNICAL FIELD [0003] The present disclosure relates to expression cassettes, vectors, virions, pharmaceutical compositions and cells comprising a nucleic acid encoding a BAG3 polypeptide or a cardioprotective variant thereof, such as BAG3-C151R.
  • the present disclosure also relates to the prevention and treatment of heart disease (e.g., cardiomyopathy, heart failure and related disorders) using such expression cassettes, vectors, virions, compositions and cells.
  • BACKGROUND [0004] Cardiomyopathy is responsible for about half of cardiac-related deaths. It is estimated that about 1 in 250 to 1 in 10,000 adults are affected by some form of cardiomyopathy.
  • Cardiomyopathy refers to a collection of conditions of the heart that occur when its ability to pump blood is reduced. Reduction in proper functioning, such as a contractile dysfunction, of the heart muscle can lead to myocardial infarction, heart failure, blood clots, valve problems, and cardiac arrest. Cardiomyopathies can be separated into primary and secondary categories that result in varied phenotypes. See McKenna et al. Circ Res.
  • Primary cardiomyopathies can be genetic, acquired, or mixed in etiology. Genetic cardiomyopathies are inherited and include arrhythmogenic right ventricular dysplasia, hypertrophic, ion channel disorders, left ventricular compaction, and mitochondrial myopathies. Acquired cardiomyopathies are due primarily to non-secondary, non-genetic causes that lead to cardiac complications and include myocarditis, peripartum, tachycardia- induced cardiomyopathy, and stress-induced cardiomyopathy. Cardiomyopathies with mixed etiology are caused by a combination of non-genetic and genetic factors, and include dilated cardiomyopathy and restrictive cardiomyopathy.
  • Secondary cardiomyopathies refer to heart disease resulting from an extra cardiovascular cause.
  • the underlying causes of secondary cardiomyopathies can be endocrine, infection, exposure to toxins, autoimmune related, nutritional, and/or neuromuscular.
  • DCM Dilated cardiomyopathy
  • Genetic mutations are an important cause of dilated cardiomyopathy.
  • Current treatment for DCM is limited to standard heart failure therapies and heart transplantation.
  • BAG family molecular chaperone regulator 3 (BAG3) is critical for normal heart function, playing a key role in formation of chaperone complexes and the stability of sHSPs.
  • Loss of function variants of BAG3 are associated with genetic DCM (haploinsufficiency).
  • knock out of BAG3 and E455K mutant of BAG3 lead to dysfunction of the chaperone complex and destabilization of sHSPs, resulting in increased insolubility of BAG3 complex substrates and DCM.
  • DCM genetic DCM
  • the disclosure provides an expression cassette comprising a polynucleotide encoding a BAG3 protein operably linked to a TNNT2 promoter.
  • the BAG3 protein is a wild type human BAG3 protein comprising the amino acid sequence of SEQ ID NO: 16.
  • the polynucleotide encoding the wild type human BAG3 protein is a codon-optimized polynucleotide.
  • the polynucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 15.
  • the BAG3 protein is a human BAG3 protein having a C151R mutation.
  • the polynucleotide encoding the human BAG3 protein having a C151R mutation is a codon-optimized polynucleotide.
  • the polynucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to SEQ ID NO: 19.
  • the human BAG3 protein having a C151R mutation has an amino acid sequence sharing at least 90%, at least 95%, at least 98% or 100% identity to SEQ ID NO: 20.
  • the TNNT2 promoter is a human TNNT2 promoter.
  • the TNNT2 promoter comprises a sequence sharing at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to SEQ ID NO: 1.
  • the human TNNT2 promoter is a wild-type human TNNT2 promoter of 600 bp, optionally having 100% identity to SEQ ID NO: 1.
  • the TNNT2 promoter has a sequence sharing at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to SEQ ID NO: 3.
  • the human TNNT2 promoter is a modified human TNNT2 promoter of 350 bp to 450 bp, or 350 bp to 500 bp.
  • the human TNNT2 promoter is a modified human TNNT2 promoter of about 400 bp of SEQ ID NO: 3.
  • the expression cassette comprises a polyadenylation sequence. [0011] In some embodiments, the polyadenylation sequence is a bGH poly(A) sequence.
  • the bGH poly(a) has a sequence at least 50%, at least 75%, at least 80%, at least 95% or 100% identical to SEQ ID NO: 11.
  • the expression cassette comprises a WPRE sequence.
  • the expression cassette does not comprise a WPRE sequence.
  • the disclosure provides a vector comprising any of the expression cassettes described herein.
  • the vector is a viral vector.
  • the disclosure provides a virion comprising any of the expression cassettes described herein.
  • the disclosure provides a virion comprising any of the vectors described herein.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) virion, comprising a vector genome comprising an expression cassette disclosed herein, wherein the expression cassette is flanked by a 5 ⁇ inverted terminal repeat (ITR) and a 3 ⁇ ITR.
  • the 5 ⁇ ITR comprise a sequence at least 95% or 100% identical to SEQ ID NOs: 13, and/or the 3 ⁇ ITR comprises a sequence that is at least 95% or 100% identical to SEQ ID NO: 14.
  • the virion is a wild type AAV9 virion.
  • the virion is an AAV9 virion variant comprising a capsid protein (such as any variant capsid protein described herein).
  • the capsid protein comprises a sequence sharing at least 75%, at least 80%, at least 85%, at least 95%, at least 98% or 100% identity to the AAV9 VP3 sequence of SEQ ID NO: 21.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) an amino acid insertion at position 584, or between positions 583 and 584, comprising one or more of an asparagine (N), a threonine (T), a tyrosine (Y), phenylalanine (F), and an alanine (A); (b) an amino acid insertion at position 585, or between positions 584 and 585, comprising one or more of a histidine (H) and a methionine (M); (c) an amino acid insertion at position 586, or between positions 585 and 586, comprising one or more of a histidine (H), a tyrosine (Y), a valine (V), a threonine (T), an alanine (A), an isoleucine (I), a tryptophan (W), a methionine (M), and a leucine (L); (d) an amino acid insertion at
  • the capsid protein comprises one, two, three, four or more substitutions or insertions in the VR-VIII site.
  • the capsid protein comprises, relative to reference SEQ ID NO: 22, one, two, three, four or more substitutions or insertions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four or more substitutions or insertions at positions from 585 to 590 in the VR-VIII site.
  • the capsid protein (i) is cardiotrophic, (ii) exhibits increased transduction efficiency in cardiac cells compared to the parental sequence, (iii) exhibits decreased transduction efficiency in liver cells compared to the parental sequence, and/or (iv) exhibits increased selectivity for the cardiac cells over liver cells compared to the parental sequence.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at position 452 an amino acid selected from the group consisting of: K and N.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, an amino acid substitution N452K.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 584 an amino acid selected from the group consisting of: R and H; (b) at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, L and Q; (c) at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, I and S; (d) at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, P and A; (e) at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, G and Q; (f) at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, Y and A; and/or (
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 452 an amino acid selected from the group consisting of: K and N; (b) at position 584 an amino acid selected from the group consisting of: R and H; (c) at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, L and Q; (d) at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, I and S; (e) at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, P and A; (f) at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, G and Q; (g) at position 589 an amino acid selected from the group consisting of: L, I, R, S, G
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 584 amino acid R; (b) at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H and, L; (c) at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, and I; (d) at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, and P; (e) at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, and G; (f) at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, and Y; and/or (g) at position 590 an amino acid selected from the group consisting of:
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four, five, six, seven or all eight of any of the following: (i) at position 452 amino acid K; (ii) at position 584 amino acid R; (iii)at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, and L; (iv) at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, and I; (v) at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, and P; (vi) at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, and G; (vii) at position 589 an amino acid selected from the group consisting of: L, I, R, S, G,
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V, T and Q; (b) at position 586 an amino acid selected from the group consisting of: N, T, M, G, D, and S; (c) at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N, V and A; (d) at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I, R and Q; (e) at position 589 an amino acid selected from the group consisting of: S, N, L, T, I, R and A; and/or (f) at position 590 an amino acid selected from the group consisting of: I, S, G, H, R and Q.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 452 an amino acid selected from the group consisting of: K and N; (b) at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V, T and Q; (c) at position 586 an amino acid selected from the group consisting of: N, T, M, G, D, and S; (d) at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N, V and A; (e) at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I, R and Q; (f) at position 589 an amino acid selected from the group consisting of: S, N, L, T, I, R and A; and (g) at position 590 an amino acid selected from the group consisting of: I, S, G, H, R and Q.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V and T; (b) at position 586 an amino acid selected from the group consisting of: N, T, M, G, and D; (c) at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N and V; (d) at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I and R; (e) at position 589 an amino acid selected from the group consisting of: S, N, L, T, I and R; and/or (f) at position 590 an amino acid selected from the group consisting of: I, S, G, H and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four, five, six or all seven of any of the following: (i) at position 452 amino acid K; (ii) at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V and T; (iii)at position 586 an amino acid selected from the group consisting of: N, T, M, G, and D; (iv) at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N and V; (v) at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I and R; (vi) at position 589 an amino acid selected from the group consisting of: S, N, L, T, I and R; and (vii) at position 590 an amino acid selected from the group consisting of: I, S, G, H and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 585 an amino acid selected from the group consisting of: E, N, M, C, and Q; (b) at position 586 an amino acid selected from the group consisting of: A, M, G, D, N and S; (c) at position 587 an amino acid selected from the group consisting of: T, N, V and A; (d) at position 588 an amino acid selected from the group consisting of: V, Y, T, S, I and Q; (e) at position 589 an amino acid selected from the group consisting of: S, G, L, I, R and A; and/or (f) at position 590 an amino acid selected from the group consisting of: I, S, G, R and Q.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 452 an amino acid selected from the group consisting of: K and N; (b) at position 585 an amino acid selected from the group consisting of: E, N, M, C, and Q; (c) at position 586 an amino acid selected from the group consisting of: A, M, G, D, N and S; (d) at position 587 an amino acid selected from the group consisting of: T, N, V and A; (e) at position 588 an amino acid selected from the group consisting of: V, Y, T, S, I and Q; (f) at position 589 an amino acid selected from the group consisting of: S, G, L, I, R and A; and (g) at position 590 an amino acid selected from the group consisting of: I, S, G, R and Q.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 585 an amino acid selected from the group consisting of: E, N, M, and C; (b) at position 586 an amino acid selected from the group consisting of: A, M, G, D, and N; (c) at position 587 an amino acid selected from the group consisting of: T, N, and V; (d) at position 588 an amino acid selected from the group consisting of: V, Y, T, S, and I; (e) at position 589 an amino acid selected from the group consisting of: S, G, L, I and R; and/or (f) at position 590 an amino acid selected from the group consisting of: I, S, G, and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four, five, six or all seven of any of the following: (i) at position 452 amino acid K; (ii) at position 585 an amino acid selected from the group consisting of: E, N, M, and C; (iii)at position 586 an amino acid selected from the group consisting of: A, M, G, D, and N; (iv) at position 587 an amino acid selected from the group consisting of: T, N, and V; (v) at position 588 an amino acid selected from the group consisting of: V, Y, T, S, and I; (vi) at position 589 an amino acid selected from the group consisting of: S, G, L, I and R; and (vii) at position 590 an amino acid selected from the group consisting of: I, S, G, and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 452 an amino acid selected from the group consisting of: K and N; and (b) at position 587 amino acid substitution A587T; and optionally comprises amino acid N or R at one, two or more positions selected from the group consisting of: 584, 585, 586, 588, 589, and 590.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 452 an amino acid selected from the group consisting of: K and N; and (b) amino acid N or R at one, two or more positions selected from the group consisting of: 584, 585, 586, 588, 589, and 590.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 452 an amino acid selected from the group consisting of: K and N; and (b) amino acid S at two or more positions selected from the group consisting of: 585, 586, 587, 588, 589 and 590.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (a) at position 452 an amino acid selected from the group consisting of: K and N; and (b) at three, four, five or six positions in the region 585-590 of the VR-VIII site, amino acids selected from the group consisting of: N, S, T, R and I.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at three, four, five or six positions in the region 585-590 of the VR-VIII site, amino acids selected from the group consisting of: N, S, T, and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585E, S586N, A587T, Q588V, A589S, Q590I, and N452K. [0042] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions S586T, A587L, Q588F, A589N, Q590S, and N452K. [0043] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585N, A587T, Q588Y, A589L, Q590G, and N452K.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585G, A587I, Q588L, A589T, Q590H, and N452K. [0045] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585M, S586M, A587T, Q588T, and Q590R; and amino acid N at position 452. [0046] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585N, A587T, Q588Y, A589L, and Q590G; and amino acid N at position 452.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585C, A587T, Q588S, A589I, and Q590R; and amino acid N at position 452.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, and Q590S; and amino acid N at position 452.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, Q590S, and N452K. [0050] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions Q585N, S586N, A587V, Q588I, A589S, Q590G, and N452K. [0051] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions S586G and Q588Y; and amino acid N at position 452.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitutions S586A, A587N, Q588Y, A589G, and N452K. [0053] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO:22, amino acids ATN at positions 581-583, and amino acids AQTG at positions 591- 594. [0054] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO:22, amino acids ATNH at positions 581-584, and amino acids AQTG at positions 591- 594.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO:22: (i) amino acid sequence ATNHENTVSIAQTG (SEQ ID NO: 35) at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452; (ii) amino acid sequence ATNHQTLFNSAQTG (SEQ ID NO: 36) at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452; (iii)amino acid sequence ATNHNSTYLGAQTG (SEQ ID NO: 37) at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452; (iv) amino acid sequence ATNHGSILTHAQTG (SEQ ID NO: 38) at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452; (v) amino acid sequence ATNHMMTTARAQTG (SEQ ID NO: 39) at the VR-VIII positions 581-594, and amino acid N
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: (i) an amino acid insertion at position 584 consisting of a TY, FN, or AT; (ii) an amino acid insertion at position 585 consisting of MH; (iii)an amino acid insertion at position 586 consisting of HY, VT, AI, WM, or ML; (iv) an amino acid insertion at position 587 consisting of PI; and/or (v) an amino acid insertion at position 588 consisting of IT or PT.
  • the capsid protein shares, or comprises a sequence sharing, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% amino acid sequence identity to: (i) the AAV9 VP1 sequence according to SEQ ID NO: 22, and/or (ii) the AAV9 VP3 sequence according to SEQ ID NO: 21.
  • the virion has a cardioprotective effect, wherein the cardioprotective effect is protection against and/or amelioration of decrease in sarcomere count or sarcomere dysfunction or disarray in cells, optionally wherein the cells are cardiomyocytes, and optionally wherein the cardioprotective effect is observed in a BAG3 haploinsufficiency background in vitro.
  • the disclosure provides a pharmaceutical composition comprising a vector of disclosed herein or a virion of disclosed herein and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises the virion in the amount of about or less than 1x10 14 vg/kg, about or less than 1x10 13 vg/kg, or about or less than 6x10 12 vg/kg, or in an amount from about 1x10 12 to 1x10 14 vg/kg.
  • the disclosure provides a method of transducing a cell, comprising contacting the cell with the vector or virion disclosed herein, wherein the vector or virion transduces the cell.
  • the disclosure provides a cell comprising an expression cassette, vector, or virion disclosed herein.
  • the cell is a cardiac cell, optionally wherein the cardiac cell is a cardiomyocyte.
  • the disclosure provides a method of treating and/or preventing heart disease in a subject, comprising administering to a subject a vector, virion, pharmaceutical composition, or cell described herein.
  • the heart disease is associated with a deleterious or loss of function mutation in the BAG3 gene and/or a decreased BAG3 protein level in cardiac cells of the subject.
  • the deleterious or loss of function mutation in the BAG3 gene is any mutation described herein or known in the art, e.g., E455K.
  • the subject is a human.
  • the method is a method of treating heart disease in a subject in need thereof, optionally wherein the subject has been diagnosed with the heart disease.
  • the heart disease is cardiomyopathy. In some embodiments, the cardiomyopathy is dilated cardiomyopathy or hypertrophic cardiomyopathy. In some embodiments, the heart disease is heart failure. In some embodiments, the heart failure is a heart failure with reduced ejection fraction. In some embodiments, the heart failure is an ischemic heart failure.
  • the administering improves cardiac function, optionally wherein the administering decreases left ventricular internal diameter (LVID) and/or increases ejection fraction. In some embodiments, the administering ameliorates decrease in sarcomere count or sarcomere dysfunction or disarray in cardiac cells of the subject.
  • LVID left ventricular internal diameter
  • the administering ameliorates decrease in sarcomere count or sarcomere dysfunction or disarray in cardiac cells of the subject.
  • the administering is systemic administration or local administration to the heart.
  • the systemic administration is intravenous administration.
  • the local administration is by direct injection into the heart or cardiac tissue, intracoronary administration or retrograde coronary sinus infusion.
  • the administering is administering the virion in the amount of about or less than 1x10 14 vg/kg, about or less than 1x10 13 vg/kg, or about or less than 6x10 12 vg/kg, or in an amount from about 1x10 12 to 1x10 14 vg/kg.
  • FIG. 1 is a graph demonstrating sarcomere count in cardiomyocytes treated with siBAG3 alone or with AAV BAG3 wild type, AAV BAG3 C151R or AAV GFP control.
  • High- content microscopy of a wild-type induced pluripotent stem cell (iPSC) treated with siBAG3 shows decreased normalized sarcomere count (p ⁇ 0.001) recapitulating a key cellular phenotype in BAG3 deficient cardiomyopathy.
  • FIG. 2 shows graphs measuring LV (left ventricular) dilation, cardiac function (Ejection Fraction), and survival rate of BAG3-cKO and BAG4-E455K mice compared to wild-type control mice.
  • FIGs. 3A-3D are graphs showing ejection fraction (FIG. 3A), left ventricular internal diameter at systole (LVIDs) (FIG. 3B), left ventricular internal diameter at diastole (LVIDd) (FIG. 3C), and delta ejection fraction (FIG.
  • expression cassettes comprising a nucleic acid encoding BAG3 or a variant thereof (operably linked to a cardiac specific-promoter, in particular a TNNT2 promoter described herein (e.g., TNNT2 promoter of SEQ ID NO: 1 or SEQ ID NO: 3, or a promoter having at least 90% or 95% identity thereto).
  • a cardiac specific-promoter in particular a TNNT2 promoter described herein (e.g., TNNT2 promoter of SEQ ID NO: 1 or SEQ ID NO: 3, or a promoter having at least 90% or 95% identity thereto).
  • expression cassettes comprising a nucleic acid encoding a cardioprotective variant of BAG3, such as BAG-C151R, operably linked to a cardiac specific-promoter, in particular a TNNT2 promoter described herein (e.g., TNNT2 promoter of SEQ ID NO: 1 or SEQ ID NO: 3, or a promoter having at least 90% or 95% identity thereto).
  • a TNNT2 promoter described herein e.g., TNNT2 promoter of SEQ ID NO: 1 or SEQ ID NO: 3, or a promoter having at least 90% or 95% identity thereto.
  • vectors comprising an expression cassette described herein.
  • the vectors are AAV vectors such as AAV9 vectors.
  • AAV virions comprising any of the expression cassettes described herein and an AAV capsid protein, wherein the capsid protein can be a wild-type capsid protein (e.g., wild type AAV9 capsid protein) or a variant capsid protein (such as any of the variant capsid proteins described herein).
  • cells comprising a polynucleotide, an expression cassette, a vector or a virion described herein.
  • compositions comprising a polynucleotide, an expression cassette, a vector or a virion described herein, and a pharmaceutically acceptable carrier.
  • methods of treatment of heart disease comprising administering to a subject in need thereof a polynucleotide, an expression cassette, a vector, a virion, a pharmaceutical composition or a cell described herein.
  • kits for treatment of heart disease comprising administering to a subject in need thereof a polynucleotide, an expression cassette, a vector, a virion, a pharmaceutical composition or a cell comprising a nucleic acid encoding BAG3 or a cardioprotective variant of BAG3 (e.g., C151R variant of BAG3) operably linked to a promoter, such as a cardiac- specific promoter, e.g., a TNNT2 promoter (such as any of those described herein).
  • the administering is intravenous (IV) or by local delivery to the heart (e.g., by intracardiac injection or intracardiac catheterization).
  • the methods described herein are effective in treating cardiomyopathy (e.g., hypertrophic cardiomyopathy or dilated cardiomyopathy) or heart failure (e.g., heart failure with reduced ejection fraction). In some embodiments, the methods described herein are effective in increasing ejection fraction, decreasing left ventricular internal diameter and/or increasing survival. In some embodiments, a single administration of a vector or virion described herein is effective in maintaining or improving heart function in the treated subject for at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, or more.
  • cardiomyopathy e.g., hypertrophic cardiomyopathy or dilated cardiomyopathy
  • heart failure e.g., heart failure with reduced ejection fraction
  • the methods described herein are effective in increasing ejection fraction, decreasing left ventricular internal diameter and/or increasing survival.
  • a single administration of a vector or virion described herein is effective in maintaining or improving heart function in the treated subject for at least 2 weeks, 3 weeks,
  • the subject being treated has a deleterious or loss of function mutation in BAG3 and/or haploinsufficiency or decreased protein level of BAG3 (e.g., in cardiac cells).
  • the polynucleotides, expression cassettes, vectors and virions described herein when delivered to a cell or a subject, are effective to protect against and/or ameliorate sarcomere dysfunction and/or disarray (such as that caused by BAG3 knockdown, loss of function or haploinsufficiency).
  • any feature or combination of features set forth herein can be excluded or omitted.
  • any feature or combination of features set forth herein can be excluded or omitted.
  • the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
  • the term “and/or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.
  • the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents. [0074] “AAV” is an abbreviation for adeno-associated virus.
  • AAV recombinant adeno-associated virus
  • AAV includes AAV or any subtype.
  • AAV5 refers to AAV subtype 5.
  • AAV9 refers to AAV subtype 9.
  • AAV9 GenBank Accession Numbers NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), NC_006261 (AAV8), and AY530579 (AAV9).
  • Publications describing AAV include Srivistava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol.71:6823; Chiorini et al.
  • rAAV vector refers either to the DNA packaged into in the rAAV virion or to the rAAV virion itself, depending on context.
  • rAAV vector refers to a nucleic acid (typically a plasmid) comprising a polynucleotide sequence capable of being packaged into an rAAV virion, but with the capsid or other proteins of the rAAV virion.
  • an rAAV vector comprises a heterologous polynucleotide sequence (i.e., a polynucleotide not of AAV origin) and one or two AAV inverted terminal repeat sequences (ITRs) flanking the heterologous polynucleotide sequence. Only one of the two ITRs may be packaged into the rAAV and yet infectivity of the resulting rAAV virion may be maintained.
  • ITRs inverted terminal repeat sequences
  • An rAAV vector may be designed to generate either single-stranded (ssAAV) or self- complementary (scAAV). See McCarty D. (2008) Mo. Ther.
  • An “AAV particle” refers to an extracellular viral particle including at least one viral capsid protein (e.g. VP1) and an encapsidated AAV vector (or fragment thereof), including the capsid proteins.
  • VP1 viral capsid protein
  • encapsidated AAV vector or fragment thereof
  • capsid proteins of AAV.
  • VP1, VP2, and VP3 are expressed from the same open reading frame, engineering of the sequence that encodes VP3 inevitably alters the sequences of the C-terminal domain of VP1 and VP2.
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of more than about 100 nucleotides, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non- natural, or derivatized nucleotide bases.
  • Oligonucleotide generally refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA or RNA. However, for the purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides are also known as “oligomers” or “oligos” and may be isolated from genes, or chemically synthesized by methods known in the art. The terms “polynucleotide” and “nucleic acid” should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
  • promoter refers a polynucleotide sequence that has one or more recognition site(s) to which an RNA polymerase binds, such that in a host or target cell, an RNA polymerase may initiate and transcribe a polynucleotide sequence “downstream” of the promoter into an RNA.
  • a “promoter” is operably linked or operatively linked to a polynucleotide sequence if in a host or target cell in which the promoter is active, an RNA polymerase initiates transcription of the polynucleotide at a transcription state site.
  • Promoters operative in mammalian cells generally comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.
  • upstream and upstream end refer to a portion of a polynucleotide that is, with reference to a transcription start site (TSS), 5 ⁇ to the TSS on the sense strand (or coding strand) of the polynucleotide; and 3 ⁇ to the TSS on the antisense strand of the polynucleotide.
  • TSS transcription start site
  • downstream and downstream end refer to a portion of a polynucleotide that is, with reference to a TSS, 3 ⁇ to TSS on the sense strand (or coding strand) of the polynucleotide; and 5 ⁇ to the TSS on the antisense strand of the polynucleotide.
  • a deletion from the upstream end of a promoter is a deletion of one or more base pairs in the non-transcribed region of the polynucleotide, 5 ⁇ to the TSS on the sense strand (or equivalently, 3 ⁇ to the TSS on the antisense strand).
  • a deletion from the downstream end of a promoter is a deletion of one or more base pairs in the transcribed region of the polynucleotide, 3 ⁇ to the TSS on the sense strand (or equivalently, 5 ⁇ to the TSS on the antisense strand).
  • transgene refers to a nucleic acid sequence encoding a protein or RNA (e.g., a therapeutic protein), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal’s genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a protein or RNA e.g., a therapeutic protein
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • sequence identity refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • reference sequence refers to a molecule to which a test sequence is compared.
  • the determination of the percentage of sequence identity may take place after a local alignment.
  • Such alignments are well known in the art, for instance the service EMBOSS Matcher identifies local similarities between two sequences using an algorithm based on the LALIGN application, version 2.0u4.
  • the identity between two nucleic acid sequences may be calculated using the service Matcher (EMBOSS) set to the default parameters, e.g. matrix (DNAfull), gap open (16), gap extend (4), alternative matches (1).
  • EMBOSS service Matcher
  • expression cassette refers to a polynucleotide cassette comprising a coding sequence which encodes a gene product of interest used to effect the expression of the gene product in target cells, which is operably linked to a promoter.
  • the expression cassette of an AAV vector includes only the polynucleotides between (and not including) the ITRs.
  • “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature, or that the polynucleotide is assembled from synthetic oligonucleotides.
  • a “recombinant” protein is a protein produced from a recombinant polypeptide.
  • a recombinant virion is a virion that comprises a recombinant polynucleotide and/or a recombinant protein, e.g. a recombinant capsid protein.
  • the term “delivery”, which is used interchangeably with “transduction,” refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids.
  • the term “modified” refers to a substance or compound (e.g., a cell, a polynucleotide sequence, and/or a polypeptide sequence) that has been altered or changed as compared to the corresponding unmodified substance or compound.
  • sample refers to a biological composition (e.g., a cell or a portion of a tissue) that is subjected to analysis and/or genetic modification.
  • a sample is a “primary sample” in that it is obtained directly from a subject; in some embodiments, a “sample” is the result of processing of a primary sample, for example to remove certain components and/or to isolate or purify certain components of interest.
  • transfection refers to the uptake of foreign DNA by a cell. A cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art.
  • RNA Ribonucleic acid
  • expression refers to the process by which a nucleic acid is translated into peptides or is transcribed into RNA, which, for example, can be translated into peptides, polypeptides or proteins.
  • nucleic acid is derived from genomic DNA
  • expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.
  • heterologous nucleic acid to be expressed in a host cell it must initially be delivered into the cell and then, once in the cell, ultimately reside in the nucleus.
  • a “heterologous” polynucleotide or nucleic acid refers to a polynucleotide or portion of a polynucleotide derived from a source other than the host organism or, for a viral vector, the native, non-recombinant virus.
  • heterologous DNA examples include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies.
  • wild type refers to the naturally-occurring polynucleotide sequence encoding a protein, or a portion thereof, or protein sequence, or portion thereof, respectively, as it normally exists in vivo in a normal or healthy subject.
  • variant refers to a protein or nucleic acid having one or more genetic changes (e.g.
  • a variant of a therapeutic protein retains the same or substantially the same activity and/or provides the same or substantially the same therapeutic benefit to a subject in need thereof.
  • a variant of a promoter sequence retains the ability to initiate transcription at the same or substantially the same level as the reference promoter, and retains the same or substantially the same cell type specificity.
  • polynucleotides variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.
  • protein variants have at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.
  • subject includes animals, such as e.g. mammals.
  • the mammal is a primate.
  • the mammal is a human.
  • subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; or domesticated animals such as dogs and cats.
  • subjects are rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.
  • rodents e.g., mice, rats, hamsters
  • rabbits primates, or swine such as inbred pigs and the like.
  • subject and “patient” are used interchangeably herein.
  • the term “administering” to a subject is a procedure by which one or more delivery agents, together or separately, are introduced into or applied onto a subject such that target cells which are present in the subject are eventually contacted with the agent.
  • the term “gene product” refers to a protein or nucleic acid produced by the transcription of a polynucleotide and, in the case of a protein gene product, the subsequent translation of transcript into a protein.
  • the term “cardiomyopathy” refers to the deterioration of the function of the myocardium (i.e., the actual heart muscle) for any reason. Subjects with cardiomyopathy are often at risk of arrhythmia or sudden cardiac death or both.
  • the term “dilated cardiomyopathy” includes, but is not limited to, cardiomyopathy caused by truncating variants in the TTN Gene.
  • hypotrophic cardiomyopathy refers to a disease of the heart and myocardium in which a portion of the myocardium is hypertrophied.
  • familial hypertrophic cardiomyopathy refers to a genetic disorder characterized by increased growth (i.e., hypertrophy) in thickness of the wall of the left ventricle.
  • left ventricular internal diameter at diastole or “LVIDd” refers to left ventricular size at diastole.
  • the term “left ventricular internal diameter at systole” or “LVIDs” refers to left ventricular size at systole.
  • the term “left ventricular mass” refers to the weight of the left ventricle.
  • the term “ejection fraction” refers to the amount of blood being bumped out of the left ventricle each time it contracts, expressed as a percentage to the total amount of blood in left ventricle.
  • the term “effective amount” refers to the minimum amount of an agent or composition required to result in a particular physiological effect.
  • the effective amount of a particular agent may be represented in a variety of ways based on the nature of the agent, such as mass/volume, # of cells/volume, particles/volume, (mass of the agent)/(mass of the subject), # of cells/(mass of subject), or particles/(mass of subject).
  • the effective amount of a particular agent may also be expressed as the half-maximal effective concentration (EC50), which refers to the concentration of an agent that results in a magnitude of a particular physiological response that is half-way between a reference level and a maximum response level.
  • EC50 half-maximal effective concentration
  • BAG3 POLYNUCLEOTIDES, EXPRESSION CASSETTES, VECTORS, VIRIONS AND COMPOSITIONS [0109]
  • described herein are polynucleotides encoding C151R mutant of BAG3, and expression cassettes vectors, and virions comprising the same.
  • described herein are polynucleotides encoding BAG3 or a cardioprotective mutant or variant thereof operably linked to a promoter, such as TNNT2 promoter, and expression cassettes, vectors and virions comprising the same.
  • described herein are polynucleotides encoding C151R mutant of BAG3 operably linked to a promoter, such as TNNT2 promoter, and expression cassettes vectors, and virions comprising the same.
  • the expression cassettes, vectors and virions described herein, when delivered to a subject are effective to protect against and/or ameliorate sarcomere dysfunction and/or disarray.
  • the expression cassettes, vectors or virions described herein, when delivered to a subject are effective to treat a heart disease (e.g., cardiomyopathy).
  • the vectors or virions described herein, when delivered to a subject are effective to treat dilated cardiomyopathy (DCM).
  • DCM dilated cardiomyopathy
  • the vectors or virions described herein, when delivered to a subject are effective to treat heart failure.
  • treatment with a vectors or virion described herein provides a therapeutic physiological effect or benefit to a subject with heart disease (e.g., a subject with cardiomyopathy).
  • BAG3 and cardioprotective mutants of BAG3, in particular BAG-C151R are wild type human BAG3 gene.
  • the transgene used in the vectors, virions, compositions and methods described herein is a cardioprotective mutant or variant of human BAG3 gene.
  • the transgene used in the vectors, virions, compositions and methods described herein is a genetic variant of the BAG3 gene which encodes a C151R mutant of BAG3.
  • the disclosure provides a vector comprising a polynucleotide sequence that encodes BAG3 protein (or a mutant, variant, or fragment thereof), operatively linked to a cardiac-specific promoter (e.g., a TNNT2 promoter).
  • the polynucleotide sequence is codon-optimized.
  • the disclosure provides a vector comprising a polynucleotide sequence that encodes BAG3-C151R mutant protein, operatively linked to a cardiac-specific promoter (e.g., a TNNT2 promoter).
  • a cardiac-specific promoter e.g., a TNNT2 promoter
  • the polynucleotide sequence is codon-optimized.
  • polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, for example polynucleotides that are optimized for human and/or primate codon selection. Further, alleles of the genes comprising the polynucleotide sequences provided herein may also be used. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. [0120] Polynucleotides can be prepared, manipulated and/or expressed using any of a variety of well-established techniques known and available in the art.
  • the polynucleotide sequence described herein encodes a BAG3 protein that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a wild-type human BAG3. In some embodiments, the polynucleotide sequence described herein encodes a BAG3 protein that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 16.
  • the polynucleotide sequence that encodes BAG3 shares at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a wild-type human BAG3 gene.
  • the polynucleotide sequence that encodes BAG3 is codon optimized.
  • the polynucleotide sequence that encodes BAG3 shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15.
  • a mutant, variant or fragment of BAG3 retains the function of BAG3.
  • a mutant, variant or fragment of BAG3 is cardioprotective.
  • a cardioprotective variant of BAG3 is used in the polynucleotides, expression cassettes, vectors, virions, cells and methods described herein.
  • the polynucleotide sequence described herein encodes a BAG3 C151R mutant or variant protein that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a naturally occurring BAG3 C151R variant.
  • the polynucleotide sequence described herein encodes a BAG3 C151R mutant or variant protein that shares at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 20. [0125] In some embodiments, the polynucleotide sequence that encodes BAG3 shares at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a naturally occurring BAG3 C151R variant. In some embodiments, the polynucleotide sequence that encodes a BAG3 C151R mutant or variant is codon optimized.
  • the polynucleotide sequence that encodes BAG3 C151R mutant or variant protein shares at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 19.
  • the disclosure provides a vector comprising a codon-optimized polynucleotide sequence that encodes BAG3 protein, operatively linked to a TNNT2 promoter.
  • the polynucleotide sequence that encodes codon-optimized BAG3 protein shares at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15.
  • the disclosure provides a vector comprising a codon-optimized polynucleotide sequence that encodes BAG3-C151R mutant protein, operatively linked to a TNNT2 promoter.
  • the polynucleotide sequence that encodes codon- optimized BAG3-C151R protein shares at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 19.
  • Illustrative BAG3 sequences are shown in Table 1 below. Table 1.
  • Illustrative BAG3 Sequences BAG3 Expression Cassettes [0129] In some embodiments, the present disclosure provides expression cassettes encoding a BAG3 protein or a variant thereof (e.g., BAG C151R) operably linked to a promoter.
  • the present disclosure provides expression cassettes encoding a BAG3 protein or a variant thereof (e.g., BAG C151R) operably linked to one, two or more promoters.
  • an expression cassette comprises one or more muscle-specific or cardiac- specific promoters operably linked to a gene encoding BAG3 protein or a variant, mutant or fragment thereof.
  • an expression cassette comprises one or more cardiac-specific promoters operably linked to a gene encoding BAG3-C151R protein.
  • polynucleotides and expression cassettes contemplated herein may be combined with other sequences, such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self- cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art.
  • promoters and/or enhancers such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP,
  • the disclosure provides an expression cassette comprising a transgene encoding a BAG3 protein, e.g., a wild type BAG3 or a cardioprotective variant thereof (e.g., BAG3- C151R).
  • the transgene polynucleotide sequence in an expression cassette can be, for example, an open reading frame encoding a protein.
  • the expression cassette may comprise, optionally, a promoter operatively linked to the transgene, optionally an intron region, optionally a polyadenylation (poly(A)) signal, optionally a woodchuck hepatitis virus post-transcriptional element (WPRE), and optionally a transcription termination signal.
  • the expression cassette may be flanked by one or more inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • An expression cassette flanked by one or more ITRs is herein referred to as a “viral genome.”
  • the ITRs in an expression cassette serve as markers used for viral packaging of the expression cassette (Clark et al. Hum Gene Ther. 6:1329-41 (1995)).
  • the expression cassette can be integrated into the host cell genome by, for example, infecting the host cell with an rAAV virion comprising capsid protein and a viral genome comprising an expression cassette, thereby expressing the transgene within a host cell.
  • the polynucleotide, expression cassette or vector described herein may also contain a ribosome binding site for translation initiation, a transcription terminator, and/or polynucleotide sequences for amplifying expression.
  • Illustrative Expression Cassettes [0133] The disclosure provides expression cassettes comprising a polynucleotide comprising a 5’ to 3’ arrangement of elements.
  • the elements comprise one or more promoters; optionally one or more enhancers; optionally one or more introns; one or more transgenes; optionally one or more WPRE sequences; and optionally one or more polyadenylation sequences (p(A)).
  • the 5’ to 3’ arrangement of elements is selected from: [0134] 5’-promoter-transgene-WPRE-p(A)-3’; [0135] 5’-promoter-intron-transgene-WPRE-p(A)-3’; [0136] 5’-promoter-transgene-WPRE-p(A)-promoter-transgene-WPRE-p(A); [0137] 5’-enhancer-promoter-transgene-WPRE-p(A)-3’; [0138] 5’-enhancer-promoter-intron-transgene-WPRE-p(A)-3’; [0139] 5’-enhancer-enhancer-promoter-transgene-WPRE-p(A)-3’; [0140] 5’-enhancer-enhancer-promoter-intron-transgene-WPRE-p(A)-3’; [0141] 5’-en
  • the WPRE element is replaced by any other post-transcriptional regulatory element known in the art.
  • the expression cassettes provided herein comprise any post-transcriptional regulatory element known in the art.
  • the expression cassettes provided herein do not comprise a post-transcriptional regulatory element (e.g., do not comprise the WPRE element).
  • the expression cassettes provided herein comprise WPRE.
  • the orientation of the promoter, enhancer, transgene and poly(A) elements can be forward or reverse (e.g., in cases where there are more than one promoters, one promoter, optionally enhancer, and operably linked transgene can be oriented in a forward direction, and another promoter, optionally enhancer, and operably linked transgene can be oriented in a reverse direction).
  • an expression cassette described herein is flanked by ITRs as described herein.
  • regulatory element refers those non-translated regions of the vector (e.g., origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence) introns, a polyadenylation sequence, 5 ⁇ and 3 ⁇ untranslated regions) which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.
  • the transcriptional regulatory element may be functional in either a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., bacterial or archaeal cell).
  • a polynucleotide sequence encoding a BAG3 protein is operably linked to multiple control elements that allow expression of the polynucleotide in both prokaryotic and eukaryotic cells.
  • transcription start site refers to the first base pair transcribed by an RNA polymerase when the RNA polymerase initiates transcription. A TSS is different from the start codon (canonically, ATG), which must be downstream of the TSS in the transcribed region of the polynucleotide. The location of a transcription start site can be determined experimentally or by prediction using any of various prediction algorithms.
  • TSSs are available from the Eukaryotic Promoter Database and the UCSC Genome Browser. Multiple TSSs for TNNT2 are identified in the UCSC Genome Browser.
  • the TSS for TNNT2 is defined to be the sequence identified by the C at the 5 ⁇ end of the motif identified by dbTSS: CTCCATC.
  • the vectors described herein comprise a transcription termination signal. Elements directing the efficient termination and polyadenylation of the heterologous nucleic acid transcripts increases heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal.
  • vectors comprise a polyadenylation sequence 3 ⁇ of a polynucleotide encoding a polypeptide to be expressed.
  • Promoters refers to a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range of cells, tissues and species or cell-type specific, tissue-specific, or species specific. Examples of ubiquitous promoters include the CAG promoter and CMB promoter (Yue et al. BioTechniques 33:672-678 (2002)).
  • Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors.
  • enhancer sequences are included in the nucleic acid constructs or vectors of the invention. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5 ⁇ or 3 ⁇ regions of the native gene.
  • the expression cassette comprises a single promoter. In some embodiments, the expression cassette comprises at least one promoter. In some embodiments, the expression cassette comprises two promoters. In some embodiments, the expression cassette comprises a ubiquitous promoter. In some embodiments, the expression cassette comprises an inducible promoter.
  • the expression cassette comprises a cell-type specific promoter.
  • the promoter specifically promotes expression of the polynucleotide encoding a polypeptide, or functional variant thereof, in a cardiac cell (e.g., a cardiomyocyte).
  • the polynucleotides, expression cassettes and vectors described herein comprise a muscle-specific promoter.
  • the polynucleotides, expression cassettes and vectors described herein comprise a cardiac-specific promoter.
  • the polynucleotides, expression cassettes and vectors described herein comprise a cardiomyocyte-specific promoter.
  • the polynucleotides, expression cassettes and vectors described herein designed to express a cardioprotective gene product, such as BAG3 or BAG3-C151R comprise a cardiomyocyte-specific promoter.
  • a “cardiomyocyte-specific promoter”, as used herein, specifies a promoter whose activity in cardiomyocytes is at least 2-fold higher than in any other non-cardiac cell type or cardiac cell which is not a cardiomyocyte.
  • a cardiomyocyte-specific promoter suitable for being used in the vector of the present disclosure has an activity in cardiomyocytes which is at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher compared to its activity in a non-cardiac cell type or a cardiac cell type which is not a cardiomyocyte.
  • the cardiac-specific or cardiomyocyte-specific promoter is a human promoter.
  • cardiac-specific or cardiomyocyte-specific promoter examples include, but are not limited to, the alpha myosin heavy chain promoter, the myosin light chain 2v promoter, the alpha myosin heavy chain promoter, the alpha-cardiac actin promoter, the alpha- tropomyosin promoter, the cardiac troponin C promoter, the cardiac troponin I promoter, the cardiac myosin-binding protein C promoter, and the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) promoter (e.g. isoform 2 of SERCA2).
  • SERCA sarco/endoplasmic reticulum Ca 2+ ATPase
  • the cardiac-specific promoter is the cardiac troponin T promoter (TNNT2) promoter.
  • the polynucleotides, expression cassettes and vectors described herein comprise a TNNT2 promoter, such as any of the TNNT2 promoters described herein.
  • the TNNT2 promoter is a human TNNT2 promoter.
  • the TNNT2 promoter is a chicken TNNT2 promoter.
  • the cardiac TNNT2 promoter is modified, e.g., by the deletion, insertion, or substitution of polynucleotides.
  • TNNT2 promoter is a modified human TNNT2 promoter (e.g., a truncated human TNNT2 promoter).
  • Illustrative polynucleotide sequences of the cardiac TNNT2 promoter are shown in Table 2 below.
  • the transcription start site (TSS) of the TNNT2 promoters are bolded and underlined. Table 2.
  • Illustrative TNNT2 promoters herein comprise any promoter, e.g., any TNNT2 promoter, described in WO2021/163357A2 and/or U.S. Patent No.11,129,908, both of which are incorporated by reference herein in their entirety.
  • the polynucleotides, expression cassettes and vectors described herein comprise any promoter, e.g., any TNNT2 promoter, described in WO2023/283649, which is incorporated by reference herein in its entirety.
  • the polynucleotides and expression cassettes described herein comprise a cardiac troponin T promoter, comprising a polynucleotide having between 300 bp and 500 bp, or between 350 bp and 450 bp.
  • the promoter comprises a sequence that shares at least 80%, at least 90%, or at least 100% identity to any one of SEQ ID NOs: 1-4.
  • the promoter comprises a sequence that shares at least 80%, at least 90%, or at least 100% identity to SEQ ID NO: 1. In some embodiments, the promoter comprises a sequence that shares at least 80%, at least 90%, or at least 100% identity to SEQ ID NO: 3. In some embodiments, the promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with a genomic polynucleotide sequence upstream of and including the transcription start site of a troponin T gene.
  • the promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with a genomic polynucleotide sequence -450 bp to +1 bp relative to the transcription start site of a troponin T gene. In some embodiments, the promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with a genomic polynucleotide sequence -350 bp to +1 bp relative to the transcription start site of a troponin T gene.
  • the promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with a genomic polynucleotide sequence -250 bp to +1 bp relative to the transcription start site of a troponin T gene. In some embodiments, the promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with a genomic polynucleotide sequence -450 bp to +50 bp relative to the transcription start site of a troponin T gene.
  • the promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with a genomic polynucleotide sequence -350 bp to +50 bp relative to the transcription start site of a troponin T gene. In some embodiments, the promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with a genomic polynucleotide sequence -250 bp to +50 bp relative to the transcription start site of a troponin T gene. In some embodiments, the troponin T gene is a human troponin T gene. [0163] In some embodiments, the promoter is a muscle-specific promoter.
  • the promoter is a cardiac cell-specific promoter. In some embodiments, the promoter is a cardiomyocyte-specific promoter. In some embodiments, the promoter has the same cell-type specificity as a native troponin T promoter of about 600 bp. In some embodiments, the promoter described herein has the same cell-type specificity as a reference promoter comprising SEQ ID NO: 1. In some embodiments, the promoter expresses a gene product operatively linked thereto at least about 10%, at least about 20%, at least about 30% more than a native troponin T promoter.
  • the promoter described herein expresses a gene product operatively linked thereto at least about 10%, at least about 20%, at least about 30% more than a reference promoter comprising SEQ ID NO: 1.
  • modified cardiac TNNT2 promoter refers to a promoter that comprises a polynucleotide sequence of at least 200 base pairs that comprises one or more continuous or discontinuous polynucleotide segments each sharing 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a corresponding segment of the TNNT2p-600 segment provided in Table 2 as SEQ ID NO: 1.
  • a modified cardiac TNNT2 promoter must be capable of promoting initiation of transcription by an RNA polymerase in a host or target cell at or near a TSS within the promoter (i.e. at or near the TTS of TNNT2 as defined herein) or, if the endogenous TSS of TNNT2 is not present in the modified cardiac TNNT2 promoter then at a heterologous TSS at most 100 base pairs downstream (3 ⁇ on the sense strand) to the downstream (3 ⁇ ) end of the modified cardiac TNNT2 promoter.
  • a modified cardiac TNNT2 promoter may comprise only sequences upstream of the TSS of TNNT2 or more comprise the TSS of TNNT2.
  • the cardiac TNNT2 promoter is modified to comprise a polynucleotide sequence of between about 200 and 500 base pairs, between about 250 and 500 base pairs, between about 300 to 500 base pairs, between about 350 to 500 base pairs, between about 350 to 450 bp, between about 400 to 500 base pairs, between about 450 to 500 base pairs, between about 200 and 450 base pairs, between about 200 and 400 base pairs, between about 200 and 350 base pairs, between about 200 and 300 base pairs, and between about 200 and 250 base pairs in length.
  • the modified cardiac TNNT2 promoter comprises a polynucleotide sequence of between about 350 base pairs to about 450 base pairs, between about 375 base pairs to about 425 base pairs, between about 375 base pairs to about 400 base pairs, between about 375 base pairs to about 425 base pairs, between about 400 base pairs to about 425 base pairs, or between about 400 base pairs to about 450 base pairs.
  • the cardiac TNNT2 promoter comprises a polynucleotide sequence of about 400 base pairs.
  • the modified cardiac troponin T promoter comprises between 300 bp and 500 bp, or 350 bp to 450 bp, of SEQ ID NO: 1.
  • the modified cardiac troponin T promoter may comprise, consist essentially of, or consist of SEQ ID NO: 3.
  • the 300 bp-500 bp sequence may be linked to further polynucleotide sequences but may not be linked to additional sequences derived from SEQ ID NO: 1.
  • the modified cardiac troponin T promoter may include no more than 500 bp of SEQ ID NO: 1 but may include additional unrelated polynucleotide sequences.
  • the modified cardiac troponin T promoter may include SEQ ID NO: 3, no additional sequences derived from SEQ ID NO: 1, but may include additional unrelated polynucleotide sequences.
  • the cardiac TNNT2 promoter is modified by the deletion of polynucleotides.
  • a modification may include one, two, three or more internal deletions.
  • Each deletion may be a deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having about 600 base pairs.
  • the TNNT2 promoter is modified by the deletion of polynucleotides from the upstream end of the promoter with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having about 600 base pairs.
  • a modification may include the deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs from the upstream end of the promoter with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having about 600 base pairs.
  • the modification is a 200 base pair deletion from the upstream end of the promoter with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having about 600 base pairs.
  • the cardiac TNNT2 promoter is modified by the deletion of polynucleotides from the downstream end of the promoter with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having about 600 base pairs.
  • a modification may include the deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs from the downstream end of the promoter with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) having about 600 base pairs.
  • the cardiac TNNT2 promoter is modified by an internal deletion of polynucleotides.
  • a modification may include the internal deletion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1).
  • the cardiac TNNT2 promoter is modified by the insertion of polynucleotides.
  • a modification may include the insertion of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 10 base pairs, 15 base pairs, 20 base pairs, 25 base pairs, 30 base pairs, 35 base pairs, 40 base pairs, 45 base pairs, 50 base pairs, 55 base pairs, 60 base pairs, 65 base pairs, 70 base pairs, 75 base pairs, 80, base pairs, 85 base pairs, 90 base pairs, 100 base pairs, 125 base pairs, 150 base pairs, 175 base pairs, 200 base pairs, 225 base pairs, 250 base pairs, 275 base pairs, or 300 base pairs with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1) .
  • the cardiac TNNT2 promoter is modified by the substitution of polynucleotides.
  • a modification may include the substitution of 1 base pair, 2 base pairs, 3 base pairs, 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, or 10 base pairs with respect to a reference cardiac TNNT2 promoter (SEQ ID NO: 1).
  • the polynucleotide sequence of the TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the polynucleotide sequence -450 base pairs to +1 base pairs relative to the transcription start site of the human TNNT2 gene.
  • the polynucleotide sequence of the TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with the polynucleotide sequence -350 base pairs to +1 base pairs relative to the transcription start site of the human TNNT2 gene. In some embodiments, the polynucleotide sequence of the TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with the polynucleotide sequence -250 base pairs to +1 base pairs relative to the transcription start site of the human TNNT2 gene.
  • the polynucleotide sequence of the cardiac TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with the polynucleotide sequence -450 base pairs to +50 base pairs relative to the transcription start site of the TNNT2 gene. In some embodiments, the polynucleotide sequence of the cardiac TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with the polynucleotide sequence -350 base pairs to +50 base pairs relative to the transcription start site of the TNNT2 gene.
  • the polynucleotide sequence of the cardiac TNNT2 promoter shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% sequence identity with the polynucleotide sequence -250 base pairs to +5 base pairs relative to the transcription start site of the TNNT2 gene.
  • the cardiac TNNT2 promoter comprises a polynucleotide comprising a sequence that shares at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or and 100% identity to any one of SEQ ID NOs: 1-4.
  • the polynucleotide comprises a sequence that shares at least 80% identity to any one of SEQ ID NOS: 1-4. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to any one of SEQ ID NOS: 1-4. In some embodiments, the polynucleotide comprises a sequence that shares at least 100% identity to any one of SEQ ID NOS: 1-4. In some embodiments, the polynucleotide comprises a sequence that shares at least 80% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to SEQ ID NO: 1.
  • the polynucleotide comprises a sequence that shares at least 100% identity to SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a sequence that shares at least 80% identity to SEQ ID NO: 3. In some embodiments, the polynucleotide comprises a sequence that shares at least 90% identity to SEQ ID NO: 3. In some embodiments, the polynucleotide comprises a sequence that shares at least 100% identity to SEQ ID NO: 3.
  • cytomegalovirus (CMV) immediate early herpes simplex virus (HSV) thymidine kinase
  • SV40 viral simian virus 40
  • SFFV spleen focus forming virus
  • LTRs long terminal repeats from retrovirus
  • MoMLV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • the promoter is any promoter suitable for expression in mammalian cells.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is a CMV promoter.
  • the promoter is a SV40 promoter.
  • Enhancers [0178] The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and/or other enhancer elements.
  • the term “enhancer” further refers to a DNA sequence that directs the binding of transcriptional regulatory proteins (e.g., transcriptional machinery) and RNA polymerase, and thereby promotes RNA synthesis.
  • An enhancer may overlap with a promoter or be upstream or downstream of the promoter.
  • the expression cassette can include one or more enhancers.
  • the enhancer can be operably linked to a promoter and modulate the expression of a transgene operably linked to a promoter.
  • the presence of an enhancer can modulate transgene expression by, for example, increasing expression or decreasing expression.
  • An enhancer can modulate transgene expression by, for example, increasing expression levels in a desired cell type, for example, a cardiac cell.
  • an enhancer can modulate transgene expression by, for example, decreasing expression levels in an “off-target” cell type, or a cell type in which expression is not desired.
  • the expression cassette comprises a single enhancer. In some embodiments, the expression cassette comprises at least one enhancer. In some embodiments, the expression cassette comprises two enhancers. In some embodiments, the expression cassette comprises three enhancers. In some embodiments, the expression cassette comprises four enhancers. In some embodiments, the expression cassette comprises an enhancer that is operably linked to a promoter. For example, a ACTC1 cardiac enhancer can be linked to a human cTnT promoter. In some embodiments, the expression cassette described herein comprises one or more enhancers.
  • the expression cassette described herein comprises no enhancer.
  • the expression cassette comprises an enhancer that is operably linked to another enhancer.
  • a ACTC1 cardiac enhancer can be operably linked to an ⁇ MHC enhancer.
  • the expression cassette comprises an enhancer that is operably linked to a promoter and operably linked to another enhancer.
  • the enhancer comprises an ACTC1 cardiac enhancer (ACTC1e).
  • the ACTC1 cardiac enhancer shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5.
  • the ACTC1 cardiac enhancer comprises SEQ ID NO: 5.
  • the enhancer comprises an ⁇ MHC enhancer ( ⁇ MHCe).
  • ⁇ MHCe ⁇ MHC enhancer
  • the ⁇ MHC enhancer comprises SEQ ID NO: 6.
  • the expression cassette can include an intron sequence, for example, a synthetic or chimeric intron sequence. The intron sequence can be used to adjust the length (i.e., size) of the expression cassette for improving recombinant AAV packaging.
  • the intron sequence can be used to improve the efficiency of transgene expression (i.e., mRNA production or transcription) in a host cell containing the expression cassette.
  • the expression cassette comprises an intron.
  • the intron comprises the CMV intron (CMVint).
  • the CMV intron shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 7.
  • the CMV intron comprises SEQ ID NO: 7.
  • the intron comprises a chimeric intron.
  • the chimeric intron shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 8.
  • the chimeric intron comprises SEQ ID NO: 8. Table 4. Illustrative Intron Sequences posttranscriptional regulatory element(s).
  • the expression cassette comprises no WPRE.
  • the expression cassette comprises a woodchuck hepatitis virus post-transcriptional element (WPRE). The WPRE sequence can be inserted, for example, proximal to on the 3’ end of a transgene in a viral vector to, for example, optimize gene expression in a viral vector (Lee et al. Exp Physiol. 90:33-37 (2005)).
  • the WPRE comprises a polynucleotide sequence that shares at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9. In some embodiments, the WPRE comprises SEQ ID NO: 9. Table 5: Illustrative WPRE Sequence Polyadenylation Sequences [0185]
  • the expression cassette comprises a poly(A) signal sequence.
  • the poly(A) sequence can be any poly(A) sequence known in the art or described herein. [0186] .
  • expression cassettes comprise a polyadenylation sequence 3 ⁇ of a polynucleotide encoding a polypeptide to be expressed.
  • polyA site or “polyA sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II.
  • Polyadenylation sequences can promote mRNA stability by addition of a polyA tail to the 3 ⁇ end of the coding sequence and thus, contribute to increased translational efficiency.
  • Cleavage and polyadenylation is directed by a poly(A) sequence in the RNA.
  • the core poly(A) sequence for mammalian pre-mRNAs has two recognition elements flanking a cleavage-polyadenylation site.
  • an almost invariant AAUAAA hexamer lies 20-50 nucleotides upstream of a more variable element rich in U or GU residues. Cleavage of the nascent transcript occurs between these two elements and is coupled to the addition of up to 250 adenosines to the 5 ⁇ cleavage product.
  • the core poly(A) sequence is an ideal polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA).
  • the poly(A) sequence is an SV40 polyA sequence, a bovine growth hormone polyA sequence (BGHpA), a rabbit ⁇ -globin polyA sequence (r ⁇ gpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art.
  • the polyA sequence comprises, consists essentially of, or consists of a sequence that shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10.
  • the polyA sequence is SEQ ID NO: 10.
  • the poly(A) sequence is a BGH poly(A) sequence.
  • the BGH poly(A) sequence comprises, consists essentially of, or consists of a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11.
  • the polyA sequence is SEQ ID NO: 11.
  • the poly(A) sequence is an SV40 poly(A) sequence.
  • the SV40 poly(A) sequence comprises, consists essentially of, or consists of a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12.
  • the polyA sequence is SEQ ID NO: 12.
  • Table 6 Illustrative Poly(A) Sequences Inverted Terminal Repeat Sequences
  • the expression cassette is flanked by AAV inverted terminal repeats (ITRs).
  • ITRs AAV inverted terminal repeats
  • the expression cassette is flanked by AAV ITRs at the 5’ and 3’ ends.
  • ITRs Any suitable ITRs described herein or known in the art may be used.
  • the ITRs function as recognition sites for replication. ITRs serve as markers used for viral packaging of the expression cassette (Clark et al. Hum Gene Ther. 6:1329-41 (1995)). ITRs form T-shaped secondary structures by two adjacent inverted repeats separated by a sing unpaired nucleotide.
  • ITRs are required for packaging the expression cassette into an rAAV virion, which provide the function of expressing the transgene after a host cell is targeted by the rAAV virion.
  • the ITRs contain tetranucleotide repeat motifs called Rep-binding elements (RBE) that act as contact points for the Rep68/78 proteins encoded by the rep gene.
  • RBE Rep-binding elements
  • the ITRs also contain a packaging signal for genome encapsidation, which directs 3’ genomic transport into preassembled capsids by Rep proteins (Wilmott et al. Hum Gene Ther Methods. 30:206-213 (2019)). Any naturally occurring or synthetically derived ITRs described herein or known in the art can be used.
  • the ITRs flanking the transgene expression cassette are ITRs of the same AAV serotype as the Rep protein used in making the virions described herein.
  • the transgene expression cassette used in the expression system comprises ITRs from AAV9 as well.
  • the transgene expression cassette used in the expression system comprises ITRs from AAV2 as well.
  • the transgene expression cassette used in the expression system comprises ITRs from AAV5 as well.
  • the ITRs may be of the same or different serotype as the capsid protein used in packaging the virion described herein.
  • the ITRs comprise the polynucleotide sequence that shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13 and/or SEQ ID NO: 14.
  • Table 7 Illustrative ITR Sequences
  • rAAV genome comprises an expression cassette that is flanked by one or both of a 5 ⁇ inverted terminal repeat (ITR) and a 3 ⁇ ITR.
  • the 5 ⁇ ITR comprises a sequence that shares at least 90%, 95%, 98% or 100% identity to SEQ ID NO: 13.
  • the 3 ⁇ ITR comprises a sequence that shares at least 90%, 95%, 98% or 100% identity to SEQ ID NO: 14.
  • the disclosure provides vectors comprising the expression cassettes provided herein.
  • the vector can be any viral vector or any non-viral vector known in the art or described herein.
  • the vector is a viral vector.
  • the viral vector is an adeno-associated virus vector (AAV), an adenoviral vector (AV), a lentiviral vector (LV), a retroviral vector (RV), a herpes simplex virus vector (HSV), or a poxvirus vector.
  • an AAV comprising any expression cassette described herein.
  • an AV comprising any expression cassette described herein.
  • an LV comprising any expression cassette described herein.
  • an RV comprising any expression cassette described herein.
  • an HSV comprising any expression cassette described herein.
  • a poxvirus-based vector comprising any expression cassette described herein.
  • the viral vector is a retroviral vector, e.g., a lentiviral vector.
  • retrovirus refers an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Retrovirus vectors are a common tool for gene delivery (Miller, Nature. 357: 455-460 (2000)). Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoded by the virus. In some embodiments, a retroviral vector is altered so that it does not integrate into the host cell genome.
  • Illustrative retroviruses include, but are not limited to: (1) genus gammaretrovirus, such as, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV), (2) genus spumavirus, such as, simian foamy virus, (3) genus lentivirus, such as, human immunodeficiency virus-1 and simian immunodeficiency virus.
  • M-MuLV Moloney murine leukemia virus
  • MoMSV Moloney murine sarcoma virus
  • MoMTV murine mammary tumor virus
  • GaLV gibbon ape leukemia virus
  • FLV feline leukemia virus
  • genus spumavirus such as, simian foamy virus
  • genus lentivirus such as, human immunodeficiency
  • lentiviral refers to a group (or genus) of complex retroviruses.
  • Illustrative lentiviruses include but are not limited to HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2; visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).
  • the viral vector is an adenoviral vector.
  • the viral vector is an adeno-associated viral (AVV) vector, such as an AAV vector selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 or chimeric AAV derived thereof.
  • AAV expression vector is pseudotyped to enhance targeting. A pseudotyping strategy can promote gene transfer and sustain expression in a target cell type.
  • the AAV2 genome can be packaged into the capsid of another AAV serotype such as AAV5, AAV7, or AAV8, producing pseudotyped vectors such as AAV2/5, AAV2/7, and AAV2/8 respectively, as described in Balaji et al. J Surg Res. Sep; 184(1): 691–698 (2013).
  • an AAV9 may be used to target expression in myofibroblast-like lineages, as described in Piras et al. Gene Therapy 23:469–478 (2016).
  • AAV1, AAV6, or AAV9 is used, and in some embodiments, the AAV is engineered, as described in Asokari et al. Hum Gene Ther.
  • the viral vector is AAV engineered to increase target cell infectivity as described in US20180066285A1.
  • the vector is an AAV9 vector.
  • the vector is a non-viral vector.
  • the non- viral vector is a naked DNA (e.g., a DNA plasmid). In some embodiments, the non-viral vector is a plasmid. In some embodiments, the non-viral vector is a liposome or lipid vector comprising plasmid DNA and a lipid solution.
  • the vectors are recombinant vectors.
  • the vectors described herein comprise an expression cassette comprising a polynucleotide encoding BAG3 protein or a variant thereof (e.g., C151R) as described herein.
  • a vector is used to deliver the expression cassettes described herein to cardiac cells of a subject, e.g., to treat cardiomyopathy.
  • the disclosure provides a viral vector comprising an expression cassette comprising a polynucleotide encoding BAG3 protein or a variant thereof (e.g., C151R) operatively linked to a promoter and a pharmaceutically acceptable carrier.
  • the disclosure provides a virion comprising a capsid and an expression cassette comprising a polynucleotide encoding BAG3 protein or a variant thereof (e.g., C151R) operatively linked to a promoter and a pharmaceutically acceptable carrier.
  • the disclosure provides a plasmid comprising an expression cassette comprising a polynucleotide encoding BAG3 protein or a variant thereof (e.g., C151R) operatively linked to a promoter and a pharmaceutically acceptable carrier.
  • the viral vectors described herein are replication incompetent, in that it cannot independently further replicate and package its genome.
  • the viral vectors described herein are replication-competent.
  • the vectors described herein are capable of being delivered to both dividing and non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to non-dividing cells. In some embodiments, the vectors described herein are capable of being delivered to dividing cells.
  • the vectors comprising the expression cassettes described herein lead to cardiac cell-specific expression of a transgene (such as BAG3 or BAG3-C151R). In some embodiments, the vectors comprising the expression cassettes described herein lead to cardiomyocyte-specific expression of a transgene. In some embodiments, the vectors comprising the expression cassettes described herein allow high expression of a transgene in a cardiac cell (e.g., a cardiomyocyte) and low or no expression in other cells (e.g., low or no expression in liver cells, low or no expression in muscle cells except for muscle cells of the heart, low or no expression in cardiac fibroblasts).
  • a transgene such as BAG3 or BAG3-C151R
  • the vectors comprising the expression cassettes described herein lead to cardiomyocyte-specific expression of a transgene.
  • the vectors comprising the expression cassettes described herein allow high expression of a transgene in a cardiac cell (e.g., a cardiomyocyte) and low
  • the vectors comprising the expression cassettes described herein allow high expression of a transgene in heart tissue of a subject (e.g., in human heart). In some embodiments, the vectors comprising the expression cassettes described herein allow no or low expression of a transgene in tissues of a subject other than the heart (e.g., in liver or in muscles except those of the heart).
  • the expression of a transgene in cardiac cells (e.g., cardiomyocytes) and/or heart tissue can be at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 50 fold, 100 fold, 150 fold, or 200 fold higher than its expression in other cells and tissues (e.g., liver, muscle except for the heart).
  • the vector genome has a size of less than 6 kilobases. In some embodiments, the vector genome has a size of less than 5.6 kilobases.
  • the vector genome has a size of about, at most or less than 4.0 kilobases, 4.5 kilobases, 4.6 kilobases, 4.7 kilobases, 4.8 kilobases, 4.9 kilobases, 5 kilobases, 5.1 kilobases, 5.2 kilobases, 5.3 kilobases, 5.4 kilobases, or 5.5 kilobases.
  • the vector genome has a size of 4 kilobases to 5.2 kilobases.
  • the vector genome has a size of 4 kilobases to 5 kilobases.
  • the vector genome has a size of 4 kilobases to 4.8 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.9 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.8 kilobases. In some embodiments, the vector genome has a size of equal to or less than 4.7 kilobases. [0213] In some of these embodiments, the vector is an AAV vector or a variant thereof. In some of these embodiments, the vector is an AAV9 vector or a variant thereof. In some of these embodiments, the vector is an AAV5 vector or a variant thereof.
  • the vector is an AAV2 vector or a variant thereof.
  • Methods of introducing polynucleotides into a host cell are known in the art, and any known method can be used to introduce the polynucleotides described herein into a cell. Suitable methods include e.g., viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery, microfluidics delivery methods, and the like.
  • PEI polyethyleneimine
  • an rAAV virion is used to deliver the expression cassettes described herein to cardiac cells.
  • an adeno-associated virus (AAV) referenced herein is any AAV known in the art or described herein.
  • an AAV is an AAV selected from the group consisting of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, or a chimeric AAV derived therefrom.
  • AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, or a variant thereof.
  • the disclosure provides an rAAV virion comprising an AAV capsid (e.g., AAV9 capsid) and an expression cassette comprising a polynucleotide encoding a transgene described herein (such as BAG3 or BAG3-C151R) operatively linked to a promoter (e.g., a TNNT2 promoter).
  • AAV capsid e.g., AAV9 capsid
  • an expression cassette comprising a polynucleotide encoding a transgene described herein (such as BAG3 or BAG3-C151R) operatively linked to a promoter (e.g., a TNNT2 promoter).
  • the disclosure provides an rAAV virion comprising a modified AAV capsid (e.g., a modified AAV9 capsid comprising one or more substitutions or insertions) and an expression cassette comprising a polynucleotide encoding a transgene described herein (such as BAG3 or BAG3-C151R) operatively linked to a promoter (e.g., a TNNT2 promoter).
  • the rAAV virions of the disclosure comprise a capsid protein.
  • Capsid proteins are structural proteins that make up the assembled icosahedral packaging of the rAAV virion that contains the expression cassette. Capsid proteins are classified by the serotype.
  • Wild type capsid serotypes in rAAV virions can be, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 (Naso et al. BioDrugs 31:317– 334 (2017)).
  • Engineered capsid types include chimeric capsids and mosaic capsids (Choi et al. Curr Gene Ther. 5: 299–310 (2005)). Capsids are selected for rAAV virions based on their ability to transduce specific tissue or cell types (Liu et al. Curr Pharm Des.21:3248-56 (2015)).
  • capsid protein that can facilitate rAAV virion transduction into cardiac cells for delivery of a transgene, as described herein, can be used.
  • Capsid proteins used in rAAV virions for transgene delivery to cardiac cells that result in high expression can be, for example, AAV4, AAV6, AAV7, AAV8, and AAV9 (Zincarelli et al. Mol. Ther.16:P1073-1080 (2008)).
  • the AAV capsid protein described herein is a wild type AAV capsid protein from AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or a variant thereof.
  • the AAV is AAV9 or a variant thereof.
  • the AAV is AAV5 or a variant thereof. In some embodiments, the AAV is AAV2 or a variant thereof [0220]
  • Artificial capsids such as chimeric capsids generated through combinatorial libraries, can also be used for transgene delivery to cardiac cells that results in high expression. Other capsid proteins with various features can also be used in the rAAV virions of the disclosure.
  • AAV vectors and capsids are provided in U.S. Pat. Pub. Nos.
  • the rAAV virions of the disclosure comprise a wild type AAV9 capsid protein.
  • the AAV9 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 22, as shown below.
  • the AAV9 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 21.
  • the wild type AAV9 VP1 has the amino acid sequence of SEQ ID NO:22.
  • the wild type AAV9 VP3 has the amino acid sequence of SEQ ID NO: 21.
  • Engineered capsid proteins can be derived from a parental, e.g. wild type, capsid and include, for example, variant polypeptide sequence with respect to a parental capsid sequence at one or more sites.
  • variant sites of the parental capsid can occur at the VR-IV site, VR-V site, VR-VII site and/or VR-VIII site (see, e.g. Büning and Srivastava. Mol Ther Methods Clin Dev.12:248-265 (2019)).
  • the variant has a substitution or insertion in the VR-IV region of the capsid protein (e.g., of AAV9).
  • the variant has a substitution or insertion in the VR-V region of the capsid protein (e.g., of AAV9). In some embodiments, the variant has a substitution or insertion in the VR-VII region of the capsid protein (e.g., of AAV9). In some embodiments, the variant has a substitution or insertion in the VR-VIII region of the capsid protein (e.g., of AAV9). In some embodiments, the variant has a substitution or insertion in the VR-IV region and the VR-VIII region of the capsid protein (e.g., of AAV9). [0225] In some embodiments, the capsid protein is an AAV5/AAV9 chimeric capsid protein.
  • the chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide segments that are derived from AAV5 capsid protein. In some embodiments, the chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide segments that are derived from AAV9 capsid protein. In some embodiments, at least one polypeptide segment is derived from the AAV5 capsid protein and at least one polypeptide segment is derived from the AAV9 capsid protein. [0226] In some embodiments, the capsid protein is a combinatory capsid proteins.
  • “combinatory capsid protein” refers to a AAV5/AAV9 chimeric capsid protein, which further comprises amino acid variations with respect to the chimeric parental sequence at one or more sites.
  • the one or more sites of the chimeric parental sequence are selected from those equivalent to the VR-IV site, the VR-V site, the VR-VII site and the VR-VIII site of the AAV9 capsid protein.
  • the rAAV virions described herein comprise any capsid protein or variant capsid protein, e.g., any AAV9 variant capsid protein (e.g., comprising one or more substitutions or insertions), described in WO2021/163357A2 and/or U.S. Patent No. 11,129,908, both of which are incorporated by reference herein in their entirety.
  • the rAAV virions described herein comprise any capsid protein or variant capsid protein, e.g., any AAV9 variant capsid protein (e.g., comprising one or more substitutions or insertions), described in WO 2021/216456, which is incorporated by reference herein in its entirety.
  • the rAAV virions described herein comprise any capsid protein or variant capsid protein, e.g., any AAV9 variant capsid protein (e.g., comprising one or more substitutions or insertions), described in Int’l Pat. Pub. No. WO2023201207A1 (the entire disclosure of which forms part of this disclosure).
  • a recombinant adeno-associated virus (rAAV) capsid protein shares at least 80%, at least 85%, at least 90%, or at least 95% polypeptide sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 21, and wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, one or more modifications (such as any of the modifications described in Int’l Pat. Pub. No. WO2023201207A1).
  • the capsid protein comprises one, two, three, four or more substitutions in the VR-VIII site.
  • the capsid protein comprises one, two, three, four or more insertions in the VR-VIII site.
  • the capsid protein comprises, relative to reference SEQ ID NO:22, one, two, three, four or more substitutions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four or more substitutions at positions from 585 to 590 in the VR-VIII site. In some embodiments, the capsid protein comprises, relative to reference SEQ ID NO:22, one, two, three, four or more insertions at positions from 584 to 590 in the VR-VIII site, or one, two, three, four or more insertions at positions from 585 to 590 in the VR-VIII site. [0232] In some embodiments, the capsid protein comprises at least two, three, four, five or more substitutions in the VR-VIII site.
  • the capsid protein comprises at least two, three, four or more insertions in the VR-VIII site. In some embodiments, the capsid protein comprises, relative to reference SEQ ID NO:22, at least two, three, four, five or more substitutions at positions from 584 to 590 in the VR-VIII site, or at least two, three, four, five or more substitutions at positions from 585 to 590 in the VR-VIII site. In some embodiments, the capsid protein comprises, relative to reference SEQ ID NO:22, at least two, three, four or more insertions at positions from 584 to 590 in the VR-VIII site, or at least two, three, four or more insertions at positions from 585 to 590 in the VR-VIII site.
  • the capsid protein (i) is cardiotrophic, (ii) exhibits increased transduction efficiency in cardiac cells compared to the parental sequence, (iii) exhibits decreased transduction efficiency in liver cells compared to the parental sequence, and/or (iv) exhibits increased selectivity for the cardiac cells over liver cells compared to the parental sequence.
  • iPSC-derived cardiac cells or cardiomyocytes e.g., iPSC-derived cardiac cells or cardiomyocytes
  • the capsid protein may comprise an amino acid insertion at position 584 (relative to reference sequence SEQ ID NO:22) comprising one or more of an asparagine (N), a threonine (T), a tyrosine (Y), phenylalanine (F), and an alanine (A).
  • the capsid protein may comprise an amino acid insertion at position 585 (relative to reference sequence SEQ ID NO:22) comprising one or more of a histidine (H) and a methionine (M).
  • the capsid protein may comprise an amino acid insertion at position 586 (relative to reference sequence SEQ ID NO:22) comprising one or more of a histidine (H), a tyrosine (Y), a valine (V), a threonine (T), an alanine (A), an isoleucine (I), a tryptophan (W), a methionine (M), and a leucine.
  • the capsid protein may comprise an amino acid insertion at position 587 (relative to reference sequence SEQ ID NO:22) comprising one or more of an isoleucine (I) and a proline (P).
  • the capsid protein may comprise an amino acid insertion at position 588 (relative to reference sequence SEQ ID NO:22) comprising one or more of an isoleucine (I), a threonine (T), and a proline (P).
  • the capsid protein may comprise one or more amino acid substitutions selected from the group consisting of N452K, N452A, N452V, G453A, G453N, S454T, S454D, G455N, Q456L, Q456K, N457L, N457V, Q458I, and Q458H (relative to reference sequence SEQ ID NO:22).
  • the capsid protein may comprise one or more amino acid substitutions selected from the group consisting of T582D, T582L, T582E, T582A, T582F, T582R, T582P, N583V, N583T, H584R, H584Q, H584K, H584V, H584Y, H584M, H584T, H584W, H584E, H584D, Q585T, Q585C, Q585V, Q585L, Q585N, Q585S, Q585P, Q585A, Q585M, Q585E, Q585Y, Q585G, Q585H, Q585I, S586D, S586T, S586G, S586K, S586M, S586N, S586I, S586Q, S586L, S586P, S586F, S586R,
  • a recombinant adeno-associated virus (rAAV) capsid protein shares, or comprises a sequence sharing, at least 80% amino acid sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 21, and wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: an amino acid insertion between position 583 and 584 comprising one or more of an asparagine (N), a threonine (T), a tyrosine (Y), phenylalanine (F), and an alanine (A); an amino acid insertion between position 584 and 585 comprising one or more of a histidine (H) and a methionine (M); an amino acid insertion between position 585 and 586 comprising one or more of a histidine (H), a tyrosine (Y), a valine (V), a threonine (T), an alanine (A), an isoleucine
  • the capsid protein may comprise an amino acid insertion at position 584 (relative to reference sequence SEQ ID NO:22) consisting of a TY, FN, or AT. [0243] In some embodiments, the capsid protein may comprise an amino acid insertion at position 585 (relative to reference sequence SEQ ID NO:22) consisting of MH. [0244] In some embodiments, the capsid protein may comprise an amino acid insertion at position 586 (relative to reference sequence SEQ ID NO:22) consisting of HY, VT, AI, WM, or ML.
  • the capsid protein may comprise an amino acid insertion at position 587 (relative to reference sequence SEQ ID NO:22) consisting of PI. [0246] In some embodiments, the capsid protein may comprise an amino acid insertion at position 588 (relative to reference sequence SEQ ID NO:22) consisting of IT or PT.
  • the capsid protein may comprise one or more amino acid substitutions selected from the group consisting of T582D, T582E, N583V, H584Q, S586K, A587P, A587S, Q588G, Q588M, A589S, A591I, G594Q, and G594D (relative to reference sequence SEQ ID NO:22).
  • the capsid protein may comprise one or more amino acid substitutions selected from the group consisting of T582L, T582A, T582F, T582R, T582P, H584R, H584K, H584V, H584Y, H584M, H584Q, H584W, H584E, H584D, Q585T, Q585N, Q585M, Q585E, Q585V, Q585H, S586T, S586G, S586Q, S586I, S586L, S586F, S586D, S586R, S586M, A587F, A587I, A587H, A587M, A587N, A587W, Q588Y, Q588S, Q588T, and Q588R (relative to reference sequence SEQ ID NO:22).
  • the capsid protein may comprise one or more amino acid substitutions selected from the group consisting of Q585C, Q585S, and S586I (relative to reference sequence SEQ ID NO:22). [0250] In some embodiments, the capsid protein may comprise one or more amino acid substitutions selected from the group consisting of Q585C, Q585S, S586I, A587V and A587G (relative to reference sequence SEQ ID NO:22).
  • the capsid protein may comprise one or more amino acid substitutions selected from the group consisting of Q585V, Q585T, Q585L, Q585C, Q585N, Q585S, Q585M, Q585E, Q585P, Q585A, Q585G, Q585H, Q585I, S586D, S586G, S586T, S586M, S586N, S586L, S586R, S586I, S586K, A587S, A587T, A587N, A587L, A587V, A587K, A587I, A587F, A587P, A587R, A587D, Q588L, Q588S, Q588F, Q588N, Q588R, Q588I, Q588V, Q588T, Q588H, Q588Y, Q588M, Q588K,
  • the capsid protein may comprise one or more amino acid substitutions selected from the group consisting of A587V and A587G (relative to reference sequence SEQ ID NO:22). [0253] In some embodiments, the capsid protein may comprise the amino acid sequence ANYG at positions 586-589 or at about positions 586-589 (relative to reference sequence SEQ ID NO:22).
  • the capsid protein may comprise two or more amino acid substitutions selected from the group consisting of N452K, N452A, N452V, G453A, G453N, S454T, S454D, G455N, Q456L, Q456K, N457L, N457V, Q458I, and Q458H (relative to reference sequence SEQ ID NO:22).
  • the capsid protein may comprise the amino acid substitution N452K, N452A, or N452V (relative to reference sequence SEQ ID NO:22).
  • the capsid protein may comprise the amino acid substitution N452K (relative to reference sequence SEQ ID NO: 22). [0257] In some embodiments, the capsid protein may comprise the amino acid substitution G453A or G453N (relative to reference sequence SEQ ID NO:22). [0258] In some embodiments, the capsid protein may comprise the amino acid substitution S454T or S454D (relative to reference sequence SEQ ID NO:22). [0259] In some embodiments, the capsid protein may comprise the amino acid substitution G455N (relative to reference sequence SEQ ID NO:22).
  • the capsid protein may comprise the amino acid substitution Q456L or Q456K (relative to reference sequence SEQ ID NO:22). [0261] In some embodiments, the capsid protein may comprise the amino acid substitution N457L or N457V (relative to reference sequence SEQ ID NO:22). [0262] In some embodiments, the capsid protein may comprise the amino acid substitution Q458I or Q458H (relative to reference sequence SEQ ID NO:22). [0263] In some embodiments, the capsid protein comprises relative to reference sequence SEQ ID NO: 22, at position 452 an amino acid selected from the group consisting of: K and N.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, an amino acid substitution N452K.
  • a recombinant adeno-associated virus (rAAV) capsid protein shares, or comprises a sequence sharing, at least 80% amino acid sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 21, and wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid substitution N452K.
  • N452K is the only substitution in the capsid protein relative to the parental or wild-type AAV9. In some embodiments, N452K is not the only substitution in the capsid protein relative to the parental or wild-type AAV9.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 585 an amino acid selected from: E, N, G, M, C, V, T and Q; at position 586 an amino acid selected from: N, T, M, G, D, and S; at position 587 an amino acid selected from: T, L, I, K, S, N, V and A; at position 588 an amino acid selected from: V, F, Y, L, T, S, I, R and Q; at position 589 an amino acid selected from: S, N, L, T, I, R and A; and/or at position 590 an amino acid selected from: I, S, G, H, R and Q.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 585 an amino acid selected from: E, N, G, M, C, V, T and Q; at position 586 an amino acid selected from: N, T, M, G, D, and S; at position 587 an amino acid selected from: T, L, I, K, S, N, V and A; at position 588 an amino acid selected from: V, F, Y, L, T, S, I, R and Q; at position 589 an amino acid selected from: S, N, L, T, I, R and A; and at position 590 an amino acid selected from: I, S, G, H, R and Q.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 585 an amino acid selected from: E, N, G, M, C, V and T; at position 586 an amino acid selected from: N, T, M, G, and D; at position 587 an amino acid selected from: T, L, I, K, S, N and V; at position 588 an amino acid selected from: V, F, Y, L, T, S, I and R; at position 589 an amino acid selected from: S, N, L, T, I and R; and/or at position 590 an amino acid selected from: I, S, G, H and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 585 an amino acid selected from: E, N, G, M, C, V and T; at position 586 an amino acid selected from: N, T, M, G, and D; at position 587 an amino acid selected from: T, L, I, K, S, N and V; at position 588 an amino acid selected from: V, F, Y, L, T, S, I and R; at position 589 an amino acid selected from: S, N, L, T, I and R; and at position 590 an amino acid selected from: I, S, G, H and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 584 an amino acid selected from the group consisting of: R and H; at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, L and Q; at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, I and S; at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, P and A; at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, G and Q; at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, Y and A; and/or at position 590 an amino acid selected from the group consisting of: G, R,
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; at position 584 an amino acid selected from the group consisting of: R and H; at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, L and Q; at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, I and S; at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, P and A; at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, G and Q; at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, Y and A; and at position 452 an amino acid selected from the
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 584 amino acid R; at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H and, L; at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, and I; at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, and P; at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, and G; at position 589 an amino acid selected from the group consisting of: L, I, R, S, G, N, T, V, Q, F, E, and Y; and/or at position 590 an amino acid selected from the group consisting of: G, R, S, I, H, N, Y, L, and M;
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four, five, six, seven or all eight of any of the following: (i) at position 452 amino acid K; (ii) at position 584 amino acid R; (iii) at position 585 an amino acid selected from the group consisting of: N, M, C, E, G, S, V, A, T, H, and L; (iv) at position 586 an amino acid selected from the group consisting of: M, D, N, G, A, T, R, and I; (v) at position 587 an amino acid selected from the group consisting of: T, N, V, L, I, S, R, and P; (vi) at position 588 an amino acid selected from the group consisting of: Y, T, S, I, V, F, L, R, N, D, and G; (vii) at position 589 an amino acid selected from the group consisting of: L, I, R, S, G,
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V, T and Q; at position 586 an amino acid selected from the group consisting of: N, T, M, G, D, and S; at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N, V and A; at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I, R and Q; at position 589 an amino acid selected from the group consisting of: S, N, L, T, I, R and A; and/or at position 590 an amino acid selected from the group consisting of: I, S, G, H, R and Q; and optionally at position 452 an amino acid selected from the group consisting of: N and K.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V, T and Q; at position 586 an amino acid selected from the group consisting of: N, T, M, G, D, and S; at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N, V and A; at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I, R and Q; at position 589 an amino acid selected from the group consisting of: S, N, L, T, I, R and A; and at position 590 an amino acid selected from the group consisting of: I, S, G, H, R and Q.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V and T; at position 586 an amino acid selected from the group consisting of: N, T, M, G, and D; at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N and V; at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I and R; at position 589 an amino acid selected from the group consisting of: S, N, L, T, I and R; and/or at position 590 an amino acid selected from the group consisting of: I, S, G, H and R; and optionally at position 452 an amino acid selected from the group consisting of: N and K.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four, five, six or all seven of any of the following: (i) at position 452 amino acid K; (ii) at position 585 an amino acid selected from the group consisting of: E, N, G, M, C, V and T; (iii) at position 586 an amino acid selected from the group consisting of: N, T, M, G, and D; (iv) at position 587 an amino acid selected from the group consisting of: T, L, I, K, S, N and V; (v) at position 588 an amino acid selected from the group consisting of: V, F, Y, L, T, S, I and R; (vi) at position 589 an amino acid selected from the group consisting of: S, N, L, T, I and R; and (vii) at position 590 an amino acid selected from the group consisting of: I, S, G, H and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 585 an amino acid selected from the group consisting of: E, N, M, C, and Q; at position 586 an amino acid selected from the group consisting of: A, M, G, D, N and S; at position 587 an amino acid selected from the group consisting of: T, N, V and A; at position 588 an amino acid selected from the group consisting of: V, Y, T, S, I and Q; at position 589 an amino acid selected from the group consisting of: S, G, L, I, R and A; and/or at position 590 an amino acid selected from the group consisting of: I, S, G, R and Q; and optionally at position 452 an amino acid selected from the group consisting of: N and K.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; at position 585 an amino acid selected from the group consisting of: E, N, M, C, and Q; at position 586 an amino acid selected from the group consisting of: A, M, G, D, N and S; at position 587 an amino acid selected from the group consisting of: T, N, V and A; at position 588 an amino acid selected from the group consisting of: V, Y, T, S, I and Q; at position 589 an amino acid selected from the group consisting of: S, G, L, I, R and A; and at position 590 an amino acid selected from the group consisting of: I, S, G, R and Q.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 585 an amino acid selected from the group consisting of: E, N, M, and C; at position 586 an amino acid selected from the group consisting of: A, M, G, D, and N; at position 587 an amino acid selected from the group consisting of: T, N, and V; at position 588 an amino acid selected from the group consisting of: V, Y, T, S, and I; at position 589 an amino acid selected from the group consisting of: S, G, L, I and R; and/or at position 590 an amino acid selected from the group consisting of: I, S, G, and R; and optionally at position 452 an amino acid selected from the group consisting of: N and K.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four, five, six or all seven of any of the following: (i) at position 452 amino acid K; (ii) at position 585 an amino acid selected from the group consisting of: E, N, M, and C; (iii) at position 586 an amino acid selected from the group consisting of: A, M, G, D, and N; (iv) at position 587 an amino acid selected from the group consisting of: T, N, and V; (v) at position 588 an amino acid selected from the group consisting of: V, Y, T, S, and I; (vi) at position 589 an amino acid selected from the group consisting of: S, G, L, I and R; and (vii) at position 590 an amino acid selected from the group consisting of: I, S, G, and R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; and at position 587 amino acid substitution A587T. [0282] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; and amino acid N or R at one, two or more positions selected from the group consisting of: 584, 585, 586, 588, 589, and 590.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; and amino acid S at two or more positions selected from the group consisting of: 585, 586, 587, 588, 589 and 590.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; and at three, four or more positions in the region 585-590 of the VR-VIII site, an amino acid selected from the group consisting of: N, S, T, R and I.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at three, four or more positions in the region 585-590 of the VR-VIII site, an amino acid selected from the group consisting of: N, S, T, and R. [0286] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; and at three, four or more positions in the region 585-590 of the VR-VIII site, amino acids selected from the group consisting of: N, S, T, R and I (such as any combination and number of each of these amino acids).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at three, four or more positions in the region 585-590 of the VR-VIII site, amino acids selected from the group consisting of: N, S, T, and R (such as any combination and number of each of these amino acids).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at position 452 an amino acid selected from the group consisting of: K and N; and at four, five or more positions in the region 585-590 of the VR-VIII site, amino acids selected from the group consisting of: N, S, T, R and I (such as any combination and number of each of these amino acids).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22: at four, five or more positions in the region 585-590 of the VR-VIII site, amino acids selected from the group consisting of: N, S, T, and R (such as any combination and number of each of these amino acids).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions Q585E, S586N, A587T, Q588V, A589S, Q590I, and/or N452K (or any combination of these substitutions).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585E, S586N, A587T, Q588V, A589S, Q590I, and N452K. [0291] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions S586T, A587L, Q588F, A589N, Q590S, and/or N452K (or any combination of these substitutions).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions S586T, A587L, Q588F, A589N, Q590S, and N452K. [0292] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions Q585N, A587T, Q588Y, A589L, Q590G, and/or N452K (or any combination of these substitutions).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585N, A587T, Q588Y, A589L, Q590G, and N452K. [0293] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions Q585G, A587I, Q588L, A589T, Q590H, and/or 452K (or any combination of these substitutions).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585G, A587I, Q588L, A589T, Q590H, and N452K. [0294] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions Q585M, S586M, A587T, Q588T, and/or Q590R (or any combination of these substitutions). In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585M, S586M, A587T, Q588T, and Q590R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585M, S586M, A587T, Q588T, and Q590R; and amino acid N at position 452. [0295] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions Q585N, A587T, Q588Y, A589L, and/or Q590G (or any combination of these substitutions). In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585N, A587T, Q588Y, A589L, and Q590G.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585N, A587T, Q588Y, A589L, and Q590G; and amino acid N at position 452. [0296] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions Q585C, A587T, Q588S, A589I, and/or Q590R (or any combination of these substitutions). In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585C, A587T, Q588S, A589I, and Q590R.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585C, A587T, Q588S, A589I, and Q590R; and amino acid N at position 452. [0297] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, and/or Q590S (or any combination of these substitutions).
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, and Q590S. In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, and Q590S; and amino acid N at position 452.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, Q590S, and/or N452K (or any combination of these substitutions). In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions Q585E, S586D, A587N, Q588I, A589R, Q590S, and N452K. [0299] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, amino acid S586G and/or Q588Y.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions S586G and Q588Y; and amino acid N at position 452. [0300] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 22, at least two, three, four or more of the amino acid substitutions S586A, A587N, Q588Y, A589G, and/or N452K (or any combination of these substitutions). In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO: 1, amino acid substitutions S586A, A587N, Q588Y, A589G, and N452K.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO:22, amino acids ATN at positions 581-583, and amino acids AQTG at positions 591- 594. [0302] In some embodiments, the capsid protein comprises, relative to reference sequence SEQ ID NO:22, amino acids ATNH at positions 581-584, and amino acids AQTG at positions 591- 594.
  • the capsid protein comprises, relative to reference sequence SEQ ID NO:22, any one of the following: (i) amino acid sequence ATNHENTVSIAQTG (SEQ ID NO: 35) at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452; (ii) amino acid sequence ATNHQTLFNSAQTG (SEQ ID NO: 36) at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452; (iii) amino acid sequence ATNHNSTYLGAQTG (SEQ ID NO: 37) at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452; (iv) amino acid sequence ATNHGSILTHAQTG (SEQ ID NO: 38) at the VR-VIII positions 581-594, and amino acid K at the VR-IV position 452; (v) amino acid sequence ATNHMMTTARAQTG (SEQ ID NO: 39) at the VR-VIII positions 581-594,
  • the capsid protein comprises a variant polypeptide sequence at the VR-VIII site, wherein the VR-VIII site (e.g., the entire VR-VIII site) comprises, consists essentially of, or consists of, a sequence having at least about 60%, 65%, 70%, 71%, 74%, 75%, 78%, 78.5%, 79%, 80%, 83%, 85%, 86%, 90%, 92%, 93% or 100% identity to any one of the following sequences (e.g., with at most 1, 2, or 3 amino acid substitutions relative to any one of the following sequences): [0305] In some embodiments, the capsid protein comprises any substitution and/or insertion motif described herein.
  • the capsid protein comprises a substitution motif having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any substitution motif described in Int’l Pat. Pub. No. WO2023201207A1.
  • the capsid protein comprises an insertion motif having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any insertion motif described in Int’l Pat. Pub. No. WO2023201207A1.
  • the capsid protein shares, or comprises a sequence sharing, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% amino acid sequence identity to an AAV9 VP3 sequence according to SEQ ID NO: 21, except for the specified modifications.
  • the capsid protein shares, or comprises a sequence sharing, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% amino acid sequence identity to AAV9 VP2 sequence, except for the specified modifications.
  • the capsid protein shares, or comprises a sequence sharing, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% amino acid sequence identity to an AAV9 VP1 sequence according to SEQ ID NO:22, except for the specified modifications.
  • the capsid protein comprises, consists essentially of, or consists of an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the modified capsid protein sequences disclosed herein (e.g., VP1, VP2, or VP3), or a functional fragment thereof.
  • the capsid protein comprises, consists essentially of, or consists of a polypeptide sequence of any one of the modified capsid protein sequences disclosed herein (e.g., VP1, VP2, or VP3).
  • the capsid protein comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from the group consisting of the following sequences provided in Int’l Pat. Pub. No.
  • WO2023201207A1 SEQ ID NOs: 488, 499, 504, 505, 506, 510, 512, 513, 516, 518, 521, 522, 533, 536, 539, 558, 562, 566, 571, 576, 578, 579, 580, 581, 585, 588, 589, 705, 706, 707, 708, 710, 772, and 774, or a functional fragment thereof.
  • the capsid protein comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any capsid protein sequence provided in Int’l Pat. Pub. No. WO2023201207A1 (e.g., any capsid protein sequence in any one of the tables and/or sequences provided herein).
  • the capsid protein comprises, consists essentially of, or consists of a polypeptide sequence of any one selected from the group consisting of the following sequences provided in Int’l Pat. Pub. No.
  • WO2023201207A1 SEQ ID NOs: 488, 499, 504, 505, 506, 510, 512, 513, 516, 518, 521, 522, 533, 536, 539, 558, 562, 566, 571, 576, 578, 579, 580, 581, 585, 588, 589, 705, 706, 707, 708, 710, 772, and 774.
  • the capsid protein is any capsid protein described in any one of the tables and/or sequences provided in Int’l Pat. Pub. No. WO2023201207A1.
  • the capsid protein comprises, consists essentially of, or consists of an amino acid sequence of any capsid protein described in any one of the tables and/or sequences provided in Int’l Pat. Pub. No. WO2023201207A1.
  • the capsid is not a chimeric capsid and/or not a combinatory capsid.
  • a recombinant adeno-associated virus (rAAV) virion comprises a capsid protein (such as any described herein) and a vector genome.
  • the vector genome may comprise an expression cassette flanked by inverted terminal repeats (ITRs), wherein the expression cassette is any one described herein for expression of BAG3 or a variant thereof.
  • ITRs inverted terminal repeats
  • the rAAV virion specifically transduces heart cells.
  • the rAAV virion specifically transduces cardiomyocytes.
  • the rAAV virion traffics to the heart.
  • the rAAV virion traffics to at least one organ other than the liver.
  • the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22.
  • administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22.
  • administration of the rAAV virion to a subject leads to a lower liver viral load in a primate or as assessed in a primate, than administration of an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22.
  • administration of the rAAV virion to a subject leads to at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower liver viral load than administration of an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22 (e.g., in a primate or as assessed in a primate).
  • the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22.
  • the rAAV virion exhibits a heart-to-liver transduction ratio which is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22.
  • the rAAV virion exhibits a higher transduction efficiency than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22, assessed in a primate.
  • the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22 (e.g., as assessed in a primate).
  • the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22, assessed in a primate.
  • the rAAV virion exhibits at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 22 (e.g., as assessed in a primate).
  • the rAAV virions comprise an AAVrh.74 capsid protein or a variant thereof. AAVrh.74 capsid proteins are known in the art.
  • the rAAV virions comprise an AAVrh.10 capsid protein or a variant thereof. AAVrh.10 capsid proteins are known in the art.
  • the rAAV virions comprise an AAV- SLB101 capsid protein or a variant thereof as known in the art or described in, e.g., WO 2021/072197, which is incorporated by reference herein in its entirety.
  • the rAAV virions comprise an AAVmod capsid protein or a variant thereof as known in the art or described in, e.g., WO 2022/173847 or in Olivieri et al. (2021) 24 th Annual Meeting of the American Society of Gene & Cell Therapy available at https://www.affiniatx.com/pdf/asgct_2021_olivieri.pdf, both of which are incorporated by reference herein in their entirety.
  • the rAAV virions comprise the AAV mut1dec1 , AAV deco1 , and/or AAV mut1 capsid protein or a variant thereof as known in the art or described in, e.g., WO 2022/173847.
  • the rAAV virions comprise an AAVcc.47 capsid protein or a variant thereof as known in the art or described in, e.g., Gonzalez et al. Nature Communications 13:5947 (2022), which is incorporated by reference herein in its entirety.
  • the rAAV virions comprise an AAVHSC16 capsid protein or a variant thereof as known in the art or described in, e.g., Smith et al. Molecular Therapy Methods & Clinical Development 26:224-238 (2022), which is incorporated by reference herein in its entirety.
  • the rAAV virions comprise a MyoAAV capsid protein or variant thereof as known in the art or described in, e.g., Tabebordbar et al. Cell 184(19):4919-4938. (2021), which is incorporated by reference herein in its entirety.
  • the rAAV virions comprise the MyoAAV-4E, MyoAAV-3F, MyoAAV-4A, or MyoAAV-4D capsid protein or variant thereof as known in the art or described in, e.g., Tabebordbar et al.
  • the rAAV virions comprise the 4D-C102 or C102 capsid protein or a variant thereof as known in the art or described in, e.g., US2021/0380643. Exemplary sequences of some of these capsid proteins are provided below.
  • the rAAV is replication defective, in that the rAAV virion cannot independently further replicate and package its genome.
  • rAAV virions of the present disclosure encapsulating the expression cassettes as described herein, can be produced using helper-free production.
  • rAAVs are replication-deficient viruses and normally require components from a live helper virus, such as adenovirus, in a host cell for packaging of infectious rAAV virions.
  • rAAV helper-free production systems allow the production of infectious rAAV virions without the use of a live helper virus.
  • a host packaging cell line is co-transfected with three plasmids.
  • a first plasmid may contain adenovirus gene products (e.g. E2A, E4, and VA RNA genes) needed for the packaging of rAAV virions.
  • a second plasmid may contain required AAV genes (e.g., REP and CAP genes).
  • a third plasmid contains the polynucleotide sequence encoding the transgene of interest and a promoter flanked by ITRs.
  • a host packaging cell line can be, for example, AAV-293 host cells. Suitable host cells contain additional components required for packaging infectious rAAV virions that are not supplied by the plasmids.
  • the CAP genes can encode, for example, AAV capsid proteins as described herein.
  • administration of the vectors described herein causes specific expression of BAG3 (or a cardioprotective mutant or variant thereof), in cardiomyocytes and/or the heart of the subject.
  • administration of the vectors described herein causes specific expression of BAG3-C151R in cardiomyocytes and/or the heart of the subject.
  • the specific expression is at least or more than 1.25 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 old, 7 fold, 8 fold, 9 fold or 10 fold (or any range between these any of these values) over the wild-type level of expression of BAG3 or the level of expression of BAG3 before administration of the vectors.
  • administration of the vectors described herein leads to low or undetectable overexpression of BAG3 (or a mutant thereof) or BAG3-C151R in non-cardiac cells and/or the skeletal tissue, brain, and/or liver of the subject (e.g., overexpression by less than that in cardiac cells, e.g., less than 1.5 fold, or less than 1.25 fold over the wild-type level of expression of these proteins or the level of expression of these proteins before administration of the vectors).
  • the expression differential between cardiac and non-cardiac cells and/or tissues can be at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 50 fold, 100 fold, 150 fold, or 200 fold or higher.
  • the compositions and methods described herein can induce detectable expression of BAG3 or a variant thereof to modulate contractile function of the myocardial tissue in a subject in need thereof.
  • the amount, concentration, and volume of the composition that modulates contractile function in myocardial tissue administered to a subject can be controlled and/or optimized to substantially improve the functional parameters of the heart while mitigating adverse side effects.
  • the administration of the therapies described herein to a subject restores or improves cardiac function in the subject.
  • the administration of the therapies described herein to a subject restores or improves contractile function of the heart in the subject.
  • the administration of the therapies described herein to a subject improves ejection fraction in the subject. In some embodiments, the administration of the therapies described herein to a subject increases ejection fraction in the subject. [0335] In some embodiments, the administration of the therapies described herein to a subject reduces left ventricular hypertrophy, left ventricular mass and/or left ventricular wall thickness in the subject. In some embodiments, the administration of the therapies described herein to a subject improves left ventricular relaxation and/or left ventricular filling pressure in the subject. [0336] In some embodiments, the administration of the therapies described herein to a subject restores or improves sarcomere function in a cardiac cell, e.g., cardiomyocyte, in the subject.
  • a cardiac cell e.g., cardiomyocyte
  • the administration of the therapies described herein to a subject restores or improves sarcomere organization in a cardiac cell, e.g., cardiomyocyte, in the subject.
  • a cardiac cell e.g., cardiomyocyte
  • the improvement in sarcomere function or organization can be demonstrated by an increase in sarcomere number (sarcomere count), increase in sarcomere length, increase in length uniformity (length variation), and/or increase in orientation uniformity (angle variation).
  • the improvement in sarcomere function or organization is as assessed by any one, two, three, four or more of these parameters.
  • Cells and Cell Therapies [0337] Also provided herein is an isolated cell or population of cells comprising any vector described herein.
  • an isolated cell or population of cells comprising two or more vectors described herein.
  • the cell is a cardiac cell.
  • cardiac cell refers to any cell present in the heart that provides a cardiac function, such as heart contraction or blood supply, or otherwise serves to maintain the structure of the heart.
  • Cardiac cells as used herein encompass cells that exist in the epicardium, myocardium or endocardium of the heart. Cardiac cells also include, for example, cardiac muscle cells or cardiomyocytes, and cells of the cardiac vasculatures, such as cells of a coronary artery or vein.
  • cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem or progenitor cells, cardiac conducting cells and cardiac pacemaking cells that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure.
  • Cardiac cells may be derived from stem cells, including, for example, embryonic stem cells or induced pluripotent stem cells.
  • the cell is a cardiomyocyte.
  • the cell is an induced pluripotent stem cell (iPSC).
  • a cell is an iPSC-derived cardiomyocyte.
  • the disclosure provides methods of manipulating polypeptide expression in a cell comprising contacting the cell with any vector or virion (e.g., rAAV virion) described herein.
  • the cell is a cardiac cell.
  • the cell is a cardiomyocyte.
  • the contacting is in vitro.
  • the contacting is in vivo.
  • the polypeptide is any polypeptide for use in treating or preventing a heart disease.
  • the polypeptide is any polypeptide described herein.
  • the polypeptide is encoded by any transgene described herein.
  • the disclosure provides methods of manipulating polypeptide expression in a tissue comprising contacting the tissue with any vector or virion (e.g., rAAV virion) described herein.
  • the tissue is cardiac tissue.
  • the contacting is in vitro.
  • the contacting is in vivo.
  • the disclosure provides methods of manipulating polypeptide expression in an organ comprising contacting the organ with any vector or virion (e.g., rAAV virion) described herein.
  • the organ is a heart.
  • the heart is diseased or at risk of disease.
  • the heart has borderline or reduced ejection fraction.
  • the heart has a normal ejection fraction.
  • the heart comprises a genetic mutation associated with a heart disease.
  • the genetic mutation is a BAG3 mutation.
  • the genetic mutation is a deleterious or loss of function mutation in BAG3 (e.g., such as any of the mutations known in the art or described herein, e.g., E455K BAG3 mutation).
  • the contacting is in vitro. In some embodiments, the contacting is in vivo.
  • the method may comprise, for example, transducing a target cell with the rAAV virions, rAAV vector genomes, or expression cassettes described herein.
  • a target cell can be, for example and without limitation, a cardiac cell, a muscle cell, an induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM), a cardiomyocyte, a BAG3 +/- cell or BAG3 +/- .
  • iPSC-CM induced pluripotent stem cell-derived cardiomyocyte
  • a cardiomyocyte a BAG3 +/- cell or BAG3 +/- .
  • Pharmaceutical Compositions and Kits [0347] Also provided herein are pharmaceutical compositions comprising at least one vector described herein. [0348]
  • the present disclosure provides pharmaceutical compositions for treating and/or preventing heart disease (e.g., heart disease associated with BAG3 gene variants).
  • the present disclosure provides pharmaceutical compositions comprising a vector (e.g., an rAAV vector genome or rAAV virion) described herein, and one or more pharmaceutically acceptable carriers, diluents or excipients for parenteral delivery.
  • the vector genome comprises a gene encoding BAG3 or BAG3-C151R.
  • the pharmaceutical compositions described herein contain one or more pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients can include vehicles (e.g., carriers, diluents and excipients) that are pharmaceutically acceptable for a formulation capable of being injected.
  • injectable solutions may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • saline solutions monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts
  • dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • Illustrative pharmaceutical forms suitable for injectable use include, e.g., sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the pharmaceutical compositions of the disclosure comprise about 1 ⁇ 10 8 genome copies per milliliter (GC/mL), about 5 ⁇ 10 8 GC/mL, about 1 ⁇ 10 9 GC/mL, about 5 ⁇ 10 9 GC/mL, about 1 ⁇ 10 10 GC/mL, about 5 ⁇ 10 10 GC/mL, about 1 ⁇ 10 11 GC/mL, about 5 ⁇ 10 11 GC/mL, about 1 ⁇ 10 12 GC/mL, about 5 ⁇ 10 12 GC/mL, about 5 ⁇ 10 13 GC/mL, about 1 ⁇ 10 14 GC/mL, or about 5 ⁇ 10 14 GC/mL of the viral vector (e.g. rAAV virion).
  • the viral vector e.g. rAAV virion
  • the pharmaceutical compositions of the disclosure comprise about 1 ⁇ 10 8 viral genomes per milliliter (vg/mL), about 5 ⁇ 10 8 vg/mL, about 1 ⁇ 10 9 vg/mL, about 5 ⁇ 10 9 vg/mL, about 1 ⁇ 10 10 vg/mL, about 5 ⁇ 10 10 vg/mL, about 1 ⁇ 10 11 vg/mL, about 5 ⁇ 10 11 vg/mL, about 1 ⁇ 10 12 vg/mL, about 5 ⁇ 10 12 vg/mL, about 5 ⁇ 10 13 vg/mL, about 1 ⁇ 10 14 vg/mL, or about 5 ⁇ 10 14 vg/mL of the viral vector (e.g.
  • the pharmaceutical compositions of the disclosure comprise less than about 1 ⁇ 10 15 viral genomes per milliliter (vg/mL), less than about 5 ⁇ 10 14 vg/mL, less than about 1 ⁇ 10 14 vg/mL, less than about 5 ⁇ 10 13 vg/mL, less than about 1 ⁇ 10 13 vg/mL, less than about 5 ⁇ 10 12 vg/mL, less than about 1 ⁇ 10 12 vg/mL, less than about 5 ⁇ 10 11 vg/mL, or less than about 1 ⁇ 10 11 vg/mL of the viral vector (e.g. rAAV virion).
  • the viral vector e.g. rAAV virion
  • the pharmaceutical compositions of the disclosure comprise less than about 1 ⁇ 10 14 viral genomes per milliliter (vg/mL) or less than about 1 ⁇ 10 13 vg/mL of the viral vector (e.g. rAAV virion).
  • the pharmaceutical compositions of the disclosure comprise from about 1 ⁇ 10 11 viral genomes per milliliter (vg/mL) to about 1 ⁇ 10 15 vg/mL of the viral vector (e.g. rAAV virion).
  • the pharmaceutical compositions of the disclosure comprise from about 1 ⁇ 10 11 viral genomes per milliliter (vg/mL) to about 1 ⁇ 10 14 vg/mL of the viral vector (e.g.
  • the pharmaceutical compositions of the disclosure comprise from about 1 ⁇ 10 12 viral genomes per milliliter (vg/mL) to about 1 ⁇ 10 14 vg/mL of the viral vector (e.g. rAAV virion). In some embodiments, the pharmaceutical compositions of the disclosure comprise from about 1 ⁇ 10 12 viral genomes per milliliter (vg/mL) to about 1 ⁇ 10 13 vg/mL of the viral vector (e.g. rAAV virion). In some embodiments, the pharmaceutical compositions of the disclosure comprise from about 1 ⁇ 10 12 viral genomes per milliliter (vg/mL) to about 6 ⁇ 10 13 vg/mL of the viral vector (e.g. rAAV virion).
  • the pharmaceutical compositions of the disclosure comprise any amount or concentration range of the viral vectors or rAAV virions of the disclosure between the values referenced herein.
  • the pharmaceutical compositions of the disclosure are administered in a total volume of about 1 mL, 5 mL, 10 mL, about 20 mL, about 25mL, about 30 mL, about 35 mL, about 40 mL, about 45 mL, about 50 mL, about 55 mL, about 60 mL, 65 mL, about 70 mL, about 75 mL, about 80 mL, about 85 mL, about 90 mL, about 95 mL, about 100 mL, about 105 mL, about 110 mL, about 115 mL, about 120 mL, about 125 mL, about 130 mL, about 135 mL, about 140 mL, about 145 mL, about 150 mL, about 155
  • the present disclosure provides a kit comprising a container housing a pharmaceutical composition as described herein.
  • Patient Populations Subjects who are suitable for treatment using the compositions and methods of the present disclosure include individuals (e.g., mammalian subjects, such as humans, non-human primates, domestic mammals, experimental non-human mammalian subjects such as mice, rats, etc.) having a cardiac condition.
  • the subject to be treated in accordance with the methods described herein is a mammal.
  • the subject to be treated in accordance with the methods described herein is a human.
  • the subject to be treated has a mutation in BAG3.
  • the subject to be treated has any mutation in BAG3 known in the art or described herein.
  • the subject to be treated has a deleterious mutation in, loss of function (LOF) mutation in, or deletion of BAG3.
  • the subject to be treated has a E455K mutation in BAG3.
  • the deleterious or LOF mutation is any mutation known in the art or described herein.
  • the BAG3 mutation is a mutation provided in Table 8 corresponding to NCBI Reference Sequence: NM_004281.4.
  • the BAG3 mutation is a BAG3 protein mutation provided in Table 8 (corresponding to the position in the polypeptide translated from NCBI Reference Sequence NM_004281.4). Table 8. BAG3 Mutations [0363] In some embodiments, the BAG3 mutation relative to NCBI Reference Sequence NM_004281.4 is selected from the group consisting of: 29_35del, 394C>T, 481C>T, 1034_1038del, 1257del, 1306G>T, 607dup, 72dup, 367C>T, 626C>T, 1363G>A, 262C>T, 403C>T, 765G>A, 699C>A, 1267_1276del, 268C>T, 568_577del, 652del, 670dup, 751dup, 901_902del, 1418dup, 165del, 358C>T, 434_437del, 448_449del
  • the BAG3 mutation is a BAG3 protein mutation selected from the group consisting of: Met10fs, Gln132Ter, Gln161Ter, Glu345fs, Pro420fs, Glu436Ter, Arg203fs, Gly25fs, Arg123Ter, Pro209Leu, Glu455Lys, Gln88Ter, Gln135Ter, Trp255Te, Tyr233Ter, Leu423fs, Arg90Ter, Ser190fs, Arg218fs, Ser224fs, Gln251fs, Arg301fs, Ala474fs, Ser56fs, Gln120Ter, Thr145fs, Gln150fs, Gln172Ter, Gln200Ter, Pro209Gln, Gln214Ter, Gln251fs, Glu256Ter, Ile339fs, Lys3
  • the subject to be treated does not have a deleterious mutation in, loss of function mutation in, or deletion of BAG3. In some embodiments, the subject to be treated does not have E455K mutation in BAG3. [0366] In some embodiments, the subject to be treated has a haploinsufficiency of BAG3. In some embodiments, the subject to be treated has a reduced level of BAG3 proteins, e.g., in cardiac cells of the subject. [0367] In some embodiments, the subject to be treated has a heart disease. In some embodiments, the subject to be treated is at risk of heart disease. [0368] In some embodiments, the subject to be treated has a BAG3 LOF (loss of function)- associated heart disease.
  • BAG3 LOF loss of function
  • the subject to be treated is at risk of BAG3 LOF-associated heart disease.
  • the subject to be treated has cardiomyopathy, e.g., DCM (dilated cardiomyopathy).
  • the subject to be treated is at risk of cardiomyopathy, e.g., DCM.
  • the subject to be treated has hypertrophic cardiomyopathy (HCM) or is at risk of HCM.
  • HCM hypertrophic cardiomyopathy
  • the subject to be treated has a BAG3 LOF-associated cardiomyopathy, e.g., BAG3 LOF-associated DCM.
  • the subject to be treated is at risk of BAG3 LOF-associated cardiomyopathy, e.g., BAG3 LOF-associated DCM. In some embodiments, the subject to be treated is at risk of BAG3 LOF-associated HCM. [0371] In some embodiments, the subject to be treated has an idiopathic DCM. In some embodiments, the subject to be treated is at risk of an idiopathic DCM. [0372] In some embodiments, the subject to be treated has a BAG3 LOF-associated idiopathic DCM. In some embodiments, the subject to be treated is at risk of a BAG3 LOF-associated idiopathic DCM.
  • the subject to be treated has a heart failure. In some embodiments, the subject to be treated is at risk of heart failure. [0374] In some embodiments, the subject to be treated has a BAG3 LOF-associated heart failure. In some embodiments, the subject to be treated is at risk of BAG3 LOF-associated heart failure. In some embodiments, the heart failure is a heart failure with reduced ejection fraction. In some embodiments, the heart failure is an ischemic heart failure. [0375] In some embodiments, the subject to be treated has a systolic dysfunction, e.g., left ventricular systolic dysfunction.
  • the subject to be treated is at risk of a systolic dysfunction, e.g., left ventricular systolic dysfunction.
  • the subject to be treated has a BAG3 LOF-associated systolic dysfunction, e.g., left ventricular systolic dysfunction.
  • the subject to be treated is at risk of BAG3 LOF-associated systolic dysfunction, e.g., left ventricular systolic dysfunction.
  • the subject to be treated has an arrhythmia, e.g., atrial and/or ventricular arrhythmia, and/or malignant ventricular arrhythmia.
  • the subject to be treated is at risk of an arrhythmia, e.g., atrial and/or ventricular arrhythmia, and/or malignant ventricular arrhythmia.
  • the subject to be treated has a BAG3 LOF-associated an arrhythmia, e.g., atrial and/or ventricular arrhythmia, and/or malignant ventricular arrhythmia.
  • the subject to be treated is at risk of BAG3 LOF-associated an arrhythmia, e.g., atrial and/or ventricular arrhythmia, and/or malignant ventricular arrhythmia.
  • compositions and methods of the present disclosure include, but are not limited to, individuals having a congenital heart defect, individuals suffering from a degenerative muscle disease, individuals suffering from a condition that results in ischemic heart tissue (e.g., individuals with coronary artery disease), and the like.
  • a method is useful to treat a degenerative muscle disease or condition (e.g., familial cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, or coronary artery disease with resultant ischemic cardiomyopathy).
  • a subject method is useful to treat individuals having a cardiac or cardiovascular disease or disorder, for example, cardiovascular disease, angina, arrhythmia, congenital heart disease, congestive heart failure, myocarditis, valve disease coronary, artery disease dilated, diastolic dysfunction, cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, coronary artery disease with resultant ischemic cardiomyopathy, mitral valve prolapse, or myocardial infarction (heart attack).
  • cardiovascular disease angina, arrhythmia, congenital heart disease, congestive heart failure, myocarditis, valve disease coronary, artery disease dilated, diastolic dysfunction, cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, coronary artery disease with resultant ischemic cardiomyopathy, mitral valve prolapse, or myocardial infarction (heart attack).
  • cardiovascular disease for example, cardiovascular disease, angina, arrhythmia, congenital heart disease, congestive heart failure, myocardi
  • compositions and methods described herein can be used to treat cardiomyopathies affiliated with mutations in BAG3 gene.
  • the mutations in BAG3 gene is any pathogenic mutation in the BAG3 gene.
  • Methods of Treatment Heart diseases to be treated [0381] In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is any heart disease. [0382] In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is a BAG3 LOF-associated heart disease.
  • the heart disease to be treated or prevented in accordance with the methods described herein is associated with a mutation in BAG3, e.g., any mutation in BAG3 known in the art or described herein. In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is associated with a deleterious or loss of function mutation of BAG3. In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is associated with any of the mutations known in the art or described herein (e.g., E455K). [0384] In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is cardiomyopathy, e.g., DCM.
  • cardiomyopathy e.g., DCM.
  • the heart disease to be treated or prevented in accordance with the methods described herein is an idiopathic DCM.
  • the heart disease to be treated or prevented in accordance with the methods described herein is HCM.
  • the heart disease to be treated or prevented in accordance with the methods described herein is BAG3 LOF-associated cardiomyopathy, e.g., BAG3 LOF- associated DCM or BAG3 LOF-associated HCM.
  • the heart disease to be treated or prevented in accordance with the methods described herein is a BAG3 LOF- associated idiopathic DCM.
  • the heart disease to be treated or prevented in accordance with the methods described herein is a heart failure. In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is a BAG3 LOF - associated heart failure. [0387] In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is a heart failure with reduced ejection fraction. In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is a BAG3 LOF -associated heart failure with reduced ejection fraction. [0388] In some embodiments, the heart disease to be treated or prevented in accordance with the methods described herein is an ischemic heart failure.
  • the heart disease to be treated or prevented in accordance with the methods described herein is a BAG3 LOF -associated ischemic heart failure.
  • the heart disease is selected from cardiomyopathy, systolic dysfunction, and arrhythmia, e.g., BAG3 LOF -associated cardiomyopathy, systolic dysfunction, and arrhythmia.
  • the heart disease is selected from dilated cardiomyopathy, left ventricular systolic dysfunction, atrial and/or ventricular arrhythmia, and malignant ventricular arrhythmia, e.g., when these conditions are associated with a BAG3 mutation.
  • the heart disease to be treated or prevented in accordance with the methods described herein is a systolic dysfunction, e.g., left ventricular systolic dysfunction.
  • the heart disease to be treated or prevented in accordance with the methods described herein is a BAG3 LOF -associated systolic dysfunction, e.g., left ventricular systolic dysfunction.
  • the heart disease to be treated or prevented in accordance with the methods described herein is an arrhythmia, e.g., atrial and/or ventricular arrhythmia, and/or malignant ventricular arrhythmia.
  • the heart disease to be treated or prevented in accordance with the methods described herein is a BAG3 LOF -associated an arrhythmia, e.g., atrial and/or ventricular arrhythmia, and/or malignant ventricular arrhythmia.
  • the cardiomyopathy treated or prevented by the compositions and methods described herein can also include cardiomyopathies associated with a pulmonary embolus, a venous thrombosis, a myocardial infarction, a transient ischemic attack, a peripheral vascular disorder, atherosclerosis, ischemic cardiac disease and/or other myocardial injury or vascular disease.
  • the cardiomyopathies treated by the compositions and methods described herein can include cardiac diseases associated with myocardial tissue hypercontractility, such as heart failure related to left ventricular hypercontractility.
  • Methods of Treating Heart Disease [0393]
  • the compositions that are described herein can be employed in a method of treating a subject with a cardiac disease or condition. “Treating” or “treatment of a condition or subject in need thereof” refers to (1) taking steps to obtain beneficial or desired results, including clinical results such as the reduction of symptoms; (2) inhibiting the disease, for example, arresting or reducing the development of the disease or its clinical symptoms; (3) relieving the disease, for example, causing regression of the disease or its clinical symptoms; and/or (4) delaying the disease.
  • beneficial or desired clinical results include, but are not limited to, promoting cardiac sarcomere contraction.
  • the compositions and methods described herein can induce detectable expression of a therapeutic protein or nucleic acid (e.g., BAG3 or BAG3-C151R protein), or a mutant, variant, or fragment thereof, to modulate sarcomere architecture and/or contractile function of the myocardial tissue in a subject in need thereof.
  • a therapeutic protein or nucleic acid e.g., BAG3 or BAG3-C151R protein
  • the vectors and virions (e.g., AAV) of the present disclosure can be administered to a subject in need thereof by systemic application (such as parenteral application), e.g., by intravenous, intra-arterial or intraperitoneal delivery of a vector in analogy to what has been shown in animal models (Katz et al., 2012, Gene Ther. 19:659-669).
  • systemic application such as parenteral application
  • the vectors and virions (e.g., AAV) of the present disclosure are to be administered parenterally.
  • the vectors and virions (e.g., AAV) of the present disclosure are to be administered intravenously (e.g., by IV infusion).
  • the vectors and virions (e.g., AAV) of the present disclosure can be delivered by direct administration to the heart tissue.
  • the vectors and virions (e.g., AAV) of the present disclosure can be delivered by intracoronary administration.
  • the administration is by antegrade epicardial coronary artery infusion, e.g., a single infusion over a 10-minute period in a cardiac catheterization laboratory after angiography (percutaneous intracoronary delivery without vessel balloon occlusion) with the use of standard 5F or 6F guide or diagnostic catheters (Jaski et al., 2009, J Card Fail.15: 171-181).
  • the vectors and virions (e.g., AAV) of the present disclosure can be delivered by direct injection into the heart or cardiac catheterization.
  • the vectors and virions (e.g., AAV) of the present disclosure can be delivered by intracardiac catheter delivery via retrograde coronary sinus infusion (RCSI).
  • RCSI retrograde coronary sinus infusion
  • direct injection it may be performed either by open-heart surgery or by minimally invasive surgery.
  • the vectors and virions (e.g., AAV) can be delivered to the pericardial space by injection or infusion.
  • the amount, concentration, and volume of the composition that modulates contractile function in myocardial tissue administered to a subject can be controlled and/or optimized to substantially improve the functional parameters of the heart while mitigating adverse side effects.
  • the amount of the composition that modulates contractile function administered to myocardial tissue can also be an amount required to result in the detectable expression of a therapeutic protein or nucleic acid (e.g., BAG3 or BAG3-C151R protein) or a mutant, variant, or fragment thereof in the heart; preserve and/or improve contractile function; delay the emergence of cardiomyopathy or reverse the pathological course of the disease; increase myocyte viability; improve myofilament function; inhibit left ventricular hypertrophy; cardiac hypertrophy regression, normalize systolic and diastolic function in heart; and restore normal cross-bridge behavior at the myofilament level.
  • a therapeutic protein or nucleic acid e.g., BAG3 or BAG3-C151R protein
  • compositions and methods disclosed herein result in detectable expression (or detectable overexpression, over the wild-type level of expression) of BAG3 protein, or a mutant, variant, or fragment thereof (in particular, BAG3-C151R), in a cardiac cell of the subject being treated.
  • administration of the compositions and methods described herein e.g., an rAAV vector genome or rAAV virion
  • Detectable expression typically refers to expression at least 5%, 10%, 15%, 20% or more compared to a control subject or tissue not treated with the vector.
  • detectable expression means expression at 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater than a no-vector control.
  • Expression can be assess by Western blot, as described in the example that follows, or enzyme-linked immunosorbent assay (ELISA), or other methods known in the art. In some cases, expression is measured quantitatively using a standard curve. Standard curves can be generated using purified protein by methods described in the examples or known in the art. Alternatively, expression of the therapeutic gene product can be assessed by quantification of the corresponding mRNA.
  • the detectable expression of the therapeutic gene product in heart tissue occurs at doses, in vector genomes (vg) per kilogram weight of subject (kg), of 3 ⁇ 10 14 vg/kg or less, 2 ⁇ 10 14 vg/kg or less, 1 ⁇ 10 14 vg/kg or less, 9 ⁇ 10 13 vg/kg or less, 8 ⁇ 10 13 vg/kg or less, 7 ⁇ 10 13 vg/kg or less, 6 ⁇ 10 13 vg/kg or less, 5 ⁇ 10 13 vg/kg or less, 4 ⁇ 10 13 vg/kg or less, 3 ⁇ 10 13 vg/kg or less, 2 ⁇ 10 13 vg/kg or less, or 1 ⁇ 10 13 vg/kg or less.
  • vg vector genomes
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose of about 1 ⁇ 10 8 genome copies per milliliter (GC/mL), about 5 ⁇ 10 8 GC/mL, about 1 ⁇ 10 9 GC/mL, about 5 ⁇ 10 9 GC/mL, about 1 ⁇ 10 10 GC/mL, about 5 ⁇ 10 10 GC/mL, about 1 ⁇ 10 11 GC/mL, about 5 ⁇ 10 11 GC/mL, about 1 ⁇ 10 12 GC/mL, about 5 ⁇ 10 12 GC/mL, about 5 ⁇ 10 13 GC/mL, about 1 ⁇ 10 14 GC/mL, or about 5 ⁇ 10 14 GC/mL of the rAAV virion.
  • the methods of the disclosure comprise intravenously administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose of about 3 ⁇ 10 12 GC/mL, about 3 ⁇ 10 13 GC/mL, about 1 ⁇ 10 14 GC/mL, or about 3 ⁇ 10 14 GC/mL of the rAAV virion.
  • an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose of about 3 ⁇ 10 12 GC/mL, about 3 ⁇ 10 13 GC/mL, about 1 ⁇ 10 14 GC/mL, or about 3 ⁇ 10 14 GC/mL of the rAAV virion.
  • the methods of the disclosure comprise administering, by localized delivery to the heart, an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose of about 3 ⁇ 10 11 GC/mL, about 3 ⁇ 10 12 GC/mL, about 1 ⁇ 10 13 GC/mL, or about 3 ⁇ 10 13 GC/mL of the rAAV virion.
  • an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose of about 3 ⁇ 10 11 GC/mL, about 3 ⁇ 10 12 GC/mL, about 1 ⁇ 10 13 GC/mL, or about 3 ⁇ 10 13 GC/mL of the rAAV virion.
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose of about 1 ⁇ 10 8 viral genomes per milliliter (vg/mL), about 5 ⁇ 10 8 vg/mL, about 1 ⁇ 10 9 vg/mL, about 5 ⁇ 10 9 vg/mL, about 1 ⁇ 10 10 vg/mL, about 5 ⁇ 10 10 vg/mL, about 1 ⁇ 10 11 vg/mL, about 5 ⁇ 10 11 vg/mL, about 1 ⁇ 10 12 vg/mL, about 5 ⁇ 10 12 vg/mL, about 5 ⁇ 10 13 vg/mL, about 1 ⁇ 10 14 vg/mL, or about 5 ⁇ 10 14 vg/mL of the rAAV virion.
  • vg/mL viral genomes per milliliter
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose of less than about 1 ⁇ 10 15 viral genomes per milliliter (vg/mL), less than about 5 ⁇ 10 14 vg/mL, less than about less than about 5 ⁇ 10 13 vg/mL, less than about 1 ⁇ 10 13 vg/mL, less than about less than about 1 ⁇ 10 12 vg/mL, less than about 5 ⁇ 10 11 vg/mL, or less than about of the viral vector (e.g. rAAV virion).
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose of less than about 1 ⁇ 10 14 viral genomes per milliliter (vg/mL) or less than about 1 ⁇ 10 13 vg/mL of the viral vector (e.g. rAAV virion).
  • an rAAV virion encoding BAG3 or a variant thereof e.g., BAG3-C151R
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose from about 1 ⁇ 10 11 viral genomes per milliliter (vg/mL) to about 1 ⁇ 10 15 vg/mL of the viral vector (e.g. rAAV virion).
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose from about 1 ⁇ 10 11 viral genomes per milliliter (vg/mL) to about 1 ⁇ 10 14 vg/mL of the viral vector (e.g.
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose from about 1 ⁇ 10 12 viral genomes per milliliter (vg/mL) to about 1 ⁇ 10 14 vg/mL of the viral vector (e.g. rAAV virion).
  • an rAAV virion encoding BAG3 or a variant thereof e.g., BAG3-C151R
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose from about 1 ⁇ 10 12 viral genomes per milliliter (vg/mL) to about 1 ⁇ 10 13 vg/mL of the viral vector (e.g. rAAV virion).
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at a dose from about 1 ⁇ 10 12 viral genomes per milliliter (vg/mL) to about 6 ⁇ 10 13 vg/mL of the viral vector (e.g.
  • the methods of the disclosure comprise administering an rAAV virion encoding BAG3 or a variant thereof (e.g., BAG3-C151R) at any dose or dose range of the disclosure between the values referenced herein.
  • the methods of the disclosure comprise intravenously administering an rAAV virion encoding BAG3 or BAG3-C151R at a dose of about 1 ⁇ 10 12 vg/mL, about 3 ⁇ 10 12 vg/mL, about 6 ⁇ 10 12 vg/mL, or about 9 ⁇ 10 12 vg/mL of the rAAV virion.
  • the methods of the disclosure comprise administering, by localized delivery to the heart, an rAAV virion encoding BAG3 or BAG3-C151R at a dose of about 1 ⁇ 10 12 vg/mL, about 3 ⁇ 10 12 vg/mL, about 6 ⁇ 10 12 vg/mL, or about 9 ⁇ 10 12 vg/mL of the rAAV virion.
  • Genome copies per milliliter can be determined by quantitative polymerase change reaction (qPCR) using a standard curve generated with a reference sample having a known concentration of the polynucleotide genome of the virus.
  • the reference sample used is often the transfer plasmid used in generation of the rAAV virion but other reference samples may be used.
  • the concentration of a viral vector can be determined by measuring the titer of the vector on a cell line. Viral titer is typically expressed as viral particles (vp) per unit volume (e.g., vp/mL).
  • the pharmaceutical compositions of the disclosure comprise about 1 ⁇ 10 8 viral particles per milliliter (vp/mL), about 5 ⁇ 10 8 vp/mL, about 1 ⁇ 10 9 vp/mL, about 5 ⁇ 10 9 vp/mL, about 1 ⁇ 10 10 vp/mL, about 5 ⁇ 10 10 vp/mL, about 1 ⁇ 10 11 vp/mL, about 5 ⁇ 10 11 vp/mL, about 1 ⁇ 10 12 vp/mL, about 5 ⁇ 10 12 vp/mL, about 5 ⁇ 10 13 vp/mL, or about 1 ⁇ 10 14 vp/mL, or about 5 ⁇ 10 14 of the viral vector (e.g., rAAV virion).
  • the viral vector e.g., rAAV virion
  • the viral vector administered to the subject can be traced by a variety of methods.
  • recombinant viruses labeled with or expressing a marker such as green fluorescent protein, or beta-galactosidase
  • the recombinant viruses may be engineered to cause the target cell to express a marker protein, such as a surface-expressed protein or a fluorescent protein.
  • the infection of target cells with recombinant viruses can be detected by their expression of a cell marker that is not expressed by the animal employed for testing (for example, a human-specific antigen when injecting cells into an experimental animal).
  • the presence and phenotype of the target cells can be assessed by fluorescence microscopy (e.g., for green fluorescent protein, or beta-galactosidase), by immunohistochemistry (e.g., using an antibody against a human antigen), by ELISA (using an antibody against a human antigen), or by RT-PCR analysis using primers and hybridization conditions that cause amplification to be specific for RNA indicative of a cardiac phenotype.
  • a single administration of a vector or virion described herein is effective in maintaining or improving heart function in the treated subject for at least 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 10 weeks, 12 weeks, or more.
  • a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 2 weeks. In some embodiments, a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 3 weeks. In some embodiments, a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 4 weeks. In some embodiments, a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 5 weeks.
  • a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 6 weeks. In some embodiments, a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 7 weeks. In some embodiments, a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 8 weeks. In some embodiments, a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 9 weeks.
  • a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 10 weeks. In some embodiments, a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 11 weeks. In some embodiments, a single administration of a vector or virion (e.g., rAAV) described herein is effective in maintaining or improving heart function in the treated subject for at least 12 weeks.
  • Cardiomyocytes were treated with siRNA to inhibit BAG3 and subsequently treated with AAVs comprising a codon optimized wild type or C151R mutant BAG3 coding sequence (where codon optimized C151R BAG3 nucleic acid sequence of SEQ ID NO:19 was used).
  • the AAV9-based vector used comprised a 5’ ITR, a 600bp Troponin T2 promoter (SEQ ID NO: 1), the BAG3 transgene, a WPRE, a bGH Poly A tail, and a 3’ ITR.
  • iCell Cardiomyocytes (‘CDI cells’) were thawed and seeded on 384-well pre- coated plates at a density of 10k/well. Cells were allowed to recover in plating media for 4 hours and switched to maintenance media for 7 days, with media changes every other day. After 7 days, cells were treated with one of five conditions: a) a scrambled siRNA control (SCR), siBAG3, siBAG3 and AAV-BAG3 wild-type , siBAG3 and AAV-BAG3 C151R , siBAG3 and AAV-GFP.7 days after initial siRNA treatment cells were fixed with 4% PFA and stained with a nuclear stain and florescence antibodies for ACTN2 and cMYBPC.
  • SCR scrambled siRNA control
  • siBAG3, siBAG3 and AAV-BAG3 wild-type siBAG3 and AAV-BAG3 C151R
  • siBAG3 and AAV-GFP 7 days after initial siRNA
  • sarcomere parameters including count, length, angle, and fit-score were identified using a custom which employs a characteristic 3-element ‘wavelet’ pattern to identify and place sarcomeres within the image. Output of images were averaged for each well, and wells were averaged for each condition. Results [0422] Treatment with siRNA of BAG3 led to a decrease in sarcomere count normalized (ns) to the number of nuclei within each cardiomyocyte (SCR had a mean normalized sarcomere count (nsc) of 4,553 vs. siBAG3 mean nsc with 3,735, p ⁇ 0.001).
  • mice By 5-months of age, the mice showed increased left ventricular internal diameter, decreased ejection fraction, and worse survival relative to wild-type animals (FIG. 2).
  • Another pathogenic BAG3 mutant mouse model with a pathogenic human point mutation BAG3-E455K crossed with the Bag3 cardiac knockout, was also evaluated and showed increased left ventricular internal diameter, decreased ejection fraction, and severe early mortality (FIG.2).
  • BAG3 could rescue heart function in the BAG3 knock-out mouse model codon optimized BAG3 expression vectors were administered to the mice.
  • AAV9 viral particles used comprised BAG3 expression cassette with 5’ ITR, a 600bp Troponin T2 promoter (SEQ ID NO: 1), a BAG3 transgene encoding a wild-type BAG3, a WPRE, a bGH Poly A tail, and a 3’ ITR.
  • Bag3-cKO animals were administered a single 6e13 vg/kg dose of vehicle, hAAV9-BAG3 comprising a codon optimized human BAG3 (SEQ ID NO: 15), or mAAV9-Bag3 comprising a codon optimized mouse Bag3 (SEQ ID NO: 16). Wild-type mice were administered Hanks Balanced Salt Solution (HBSS) as a control.
  • HBSS Hanks Balanced Salt Solution
  • hAAV9-BAG3 and mAAV9-Bag3 injected animals maintained their baseline ejection fraction (p ⁇ 0.001) (FIG. 3A). Additionally, hAAV9-BAG3 and mAAV9- Bag3 treated animals had a statistically significant effect in maintaining LV internal diameters (LVID) at systole (LVIDs) and diastole (LVIDd) showing early efficacy of BAG3 AAV gene therapy (FIG. 3B and 3C).
  • LVID LV internal diameters
  • LVIDd diastole

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Environmental Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des cassettes d'expression, des vecteurs, des virions, des compositions et des cellules comprenant un acide nucléique codant pour un polypeptide BAG3 ou un variant cardioprotecteur de celui-ci, tel que BAG3-C151R. La présente invention concerne également la prévention et le traitement des maladies cardiaques (par exemple, les cardiomyopathies, les insuffisances cardiaques ou les troubles apparentés) par l'utilisation de telles cassettes d'expression, de vecteurs, de virions, de compositions et de cellules.
PCT/US2024/023699 2023-04-10 2024-04-09 Thérapies cardioprotectrices par bag3 Ceased WO2024215655A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363495293P 2023-04-10 2023-04-10
US63/495,293 2023-04-10

Publications (1)

Publication Number Publication Date
WO2024215655A1 true WO2024215655A1 (fr) 2024-10-17

Family

ID=90829158

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/023699 Ceased WO2024215655A1 (fr) 2023-04-10 2024-04-09 Thérapies cardioprotectrices par bag3

Country Status (1)

Country Link
WO (1) WO2024215655A1 (fr)

Citations (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
WO1998011244A2 (fr) 1996-09-11 1998-03-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vecteur de vaa4 et ses utilisations
WO1999061601A2 (fr) 1998-05-28 1999-12-02 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vecteurs d'aav5 et leurs utilisation
WO2000028061A2 (fr) 1998-11-05 2000-05-18 The Trustees Of The University Of Pennsylvania Sequences d'acide nucleique du serotype i du virus associe aux adenovirus, vecteurs et cellules hotes contenant ces derniers
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
WO2001011034A2 (fr) 1999-08-09 2001-02-15 Targeted Genetics Corporation Vecteurs viraux de recombinaison actives metaboliquement et procede de preparation et d'utilisation de ceux-ci
WO2001092551A2 (fr) 2000-06-01 2001-12-06 University Of North Carolina At Chapel Hill Vecteurs de parvovirus dupliques
US6962815B2 (en) 2001-01-05 2005-11-08 Children's Hopital Inc. AAV2 vectors and methods
US6984517B1 (en) 1998-05-28 2006-01-10 The United States Of America As Represented By The Department Of Health And Human Services AAV5 vector and uses thereof
US7105345B2 (en) 1998-11-05 2006-09-12 The University Of Pennsylvania Adeno-associated virus serotype 1 nucleic acid sequences, vectors and host cells containing same
US7198951B2 (en) 2001-12-17 2007-04-03 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 9 sequences, vectors containing same, and uses therefor
US7259151B2 (en) 2003-06-19 2007-08-21 Genzyme Corporation AAV virions with decreased immunoreactivity and uses therefor
WO2008124724A1 (fr) 2007-04-09 2008-10-16 University Of Florida Research Foundation, Inc. Compositions à base de vecteurs raav comprenant des protéines de capside modifiées par la tyrosine et procédés d'utilisation correspondants
US7790449B2 (en) 2001-12-17 2010-09-07 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing the same, and uses therefor
WO2010129021A1 (fr) 2009-05-02 2010-11-11 Genzyme Corporation Thérapie génique pour les maladies neurodégénératives
US7892809B2 (en) 2004-12-15 2011-02-22 The University Of North Carolina At Chapel Hill Chimeric vectors
US7906111B2 (en) 2003-09-30 2011-03-15 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor
WO2012145601A2 (fr) 2011-04-22 2012-10-26 The Regents Of The University Of California Virions de virus adéno-associé avec capside variant et procédés d'utilisation de ceux-ci
US8632764B2 (en) 2008-04-30 2014-01-21 University Of North Carolina At Chapel Hill Directed evolution and in vivo panning of virus vectors
US8889641B2 (en) 2009-02-11 2014-11-18 The University Of North Carolina At Chapel Hill Modified virus vectors and methods of making and using the same
US8906675B2 (en) 2001-11-13 2014-12-09 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) sequences and isolating novel sequences identified thereby
US9233131B2 (en) 2003-06-30 2016-01-12 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
WO2016054554A1 (fr) 2014-10-03 2016-04-07 University Of Massachusetts Virus adéno-associés greffés au moyen de peptides de ciblage hétérologues
US9434928B2 (en) 2011-11-23 2016-09-06 Nationwide Children's Hospital, Inc. Recombinant adeno-associated virus delivery of alpha-sarcoglycan polynucleotides
US20160340393A1 (en) 2003-06-30 2016-11-24 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
WO2017100671A1 (fr) 2015-12-11 2017-06-15 California Institute Of Technology Peptides de ciblage pour diriger des virus adéno-associés (aav)
WO2017106236A1 (fr) 2015-12-14 2017-06-22 The University Of North Carolina At Chapel Hill Protéines capsidiques modifiées pour la libération améliorée de vecteurs parvoviraux
WO2017192750A1 (fr) 2016-05-04 2017-11-09 Oregon Health & Science University Vecteurs viraux adéno-associés recombinés
WO2017191274A2 (fr) * 2016-05-04 2017-11-09 Curevac Ag Arn codant pour une protéine thérapeutique
WO2017212019A1 (fr) 2016-06-09 2017-12-14 Centre National De La Recherche Scientifique (Cnrs) Raav à capside chimiquement modifiée
US20180066285A1 (en) 2015-03-24 2018-03-08 The Regents Of The University Of California Adeno-associated virus variants and methods of use thereof
US10011640B2 (en) 2012-05-15 2018-07-03 University Of Florida Research Foundation, Incorporated Capsid-modified rAAV vector compositions and methods therefor
US10036016B2 (en) 2013-09-12 2018-07-31 President And Fellows Of Harvard College Methods for inducing glucose uptake
WO2018152333A1 (fr) 2017-02-15 2018-08-23 The University Of North Carolina At Chapel Hill Méthodes et compositions pour le transfert de gènes à travers le système vasculaire
WO2018222503A1 (fr) 2017-05-31 2018-12-06 The Regents Of The University Of California Virus adéno-associé à capside variante et ses méthodes d'utilisation
WO2019028306A2 (fr) 2017-08-03 2019-02-07 Voyager Therapeutics, Inc. Compositions et procédés permettant l'administration de virus adéno-associés
US10480011B2 (en) 2014-10-21 2019-11-19 University Of Massachusetts Recombinant AAV variants and uses thereof
WO2020117898A1 (fr) 2018-12-05 2020-06-11 Abeona Therapeutics Inc. Vecteur viral adéno-associé recombiné pour l'insertion de gènes
WO2020191300A1 (fr) 2019-03-21 2020-09-24 Stridebio, Inc. Vecteurs viraux adéno-associés recombinants
WO2020198737A1 (fr) 2019-03-28 2020-10-01 The General Hospital Corporation Vecteurs de virus adéno-associés (aav) modifiés pour l'expression transgénique
US10894949B2 (en) 2014-02-19 2021-01-19 University Of Massachusetts Recombinant AAVS having useful transcytosis properties
WO2021072197A1 (fr) 2019-10-10 2021-04-15 Solid Biosciences Inc. Capsides de vaa modifiées et leurs utilisations
WO2021163357A2 (fr) 2020-02-13 2021-08-19 Tenaya Therapeutics, Inc. Vecteurs de thérapie génique de traitement de maladie cardiaque
US11129908B2 (en) 2020-02-13 2021-09-28 Tenaya Therapeutics, Inc. Gene therapy vectors for treating heart disease
WO2021216456A2 (fr) 2020-04-20 2021-10-28 Tenaya Therapeutics, Inc. Virus adéno-associé comprenant une capside modifiée
US20210380643A1 (en) 2017-09-20 2021-12-09 4D Molecular Therapeutics Inc. Adeno-associated virus variant capsids and methods of use thereof
WO2022173847A2 (fr) 2021-02-09 2022-08-18 Affinia Therapeutics Inc. Vaa recombinants ayant un tropisme et une spécificité améliorés
WO2023283649A1 (fr) 2021-07-08 2023-01-12 Tenaya Therapeutics, Inc. Cassettes d'expression optimisées pour thérapie génique
WO2023108159A1 (fr) * 2021-12-10 2023-06-15 University Of Florida Research Foundation, Incorporated Méthodes et compositions pour traiter une cardiomyopathie liée à bag-3 avec un vecteur viral
WO2023201207A1 (fr) 2022-04-11 2023-10-19 Tenaya Therapeutics, Inc. Virus adéno-associé comprenant une capside modifiée
WO2023200742A2 (fr) * 2022-04-11 2023-10-19 Tenaya Therapeutics, Inc. Capsides pour la thérapie génique à plakophilline-2
WO2023205767A2 (fr) * 2022-04-22 2023-10-26 Spacecraft Seven, Llc Thérapie génique de l'anthanogène 3 associé au lymphome b 2 (bag3) faisant intervenir un vecteur de vaa

Patent Citations (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
WO1998011244A2 (fr) 1996-09-11 1998-03-19 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Vecteur de vaa4 et ses utilisations
US7718424B2 (en) 1996-09-11 2010-05-18 The United States Of America As Represented By The Department Of Health And Human Services AAV4 vector and uses thereof
US6156303A (en) 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
US6984517B1 (en) 1998-05-28 2006-01-10 The United States Of America As Represented By The Department Of Health And Human Services AAV5 vector and uses thereof
WO1999061601A2 (fr) 1998-05-28 1999-12-02 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vecteurs d'aav5 et leurs utilisation
WO2000028061A2 (fr) 1998-11-05 2000-05-18 The Trustees Of The University Of Pennsylvania Sequences d'acide nucleique du serotype i du virus associe aux adenovirus, vecteurs et cellules hotes contenant ces derniers
US7105345B2 (en) 1998-11-05 2006-09-12 The University Of Pennsylvania Adeno-associated virus serotype 1 nucleic acid sequences, vectors and host cells containing same
WO2001011034A2 (fr) 1999-08-09 2001-02-15 Targeted Genetics Corporation Vecteurs viraux de recombinaison actives metaboliquement et procede de preparation et d'utilisation de ceux-ci
WO2001092551A2 (fr) 2000-06-01 2001-12-06 University Of North Carolina At Chapel Hill Vecteurs de parvovirus dupliques
US6962815B2 (en) 2001-01-05 2005-11-08 Children's Hopital Inc. AAV2 vectors and methods
US8906675B2 (en) 2001-11-13 2014-12-09 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) sequences and isolating novel sequences identified thereby
US9790472B2 (en) 2001-11-13 2017-10-17 The Trustees Of The University Of Pennsylvania Method of detecting and/or identifying adeno-associated virus (AAV) sequences and isolating novel sequences identified thereby
US7198951B2 (en) 2001-12-17 2007-04-03 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 9 sequences, vectors containing same, and uses therefor
US7790449B2 (en) 2001-12-17 2010-09-07 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing the same, and uses therefor
US7259151B2 (en) 2003-06-19 2007-08-21 Genzyme Corporation AAV virions with decreased immunoreactivity and uses therefor
US9233131B2 (en) 2003-06-30 2016-01-12 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
US20160340393A1 (en) 2003-06-30 2016-11-24 The Regents Of The University Of California Mutant adeno-associated virus virions and methods of use thereof
US7906111B2 (en) 2003-09-30 2011-03-15 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor
US9737618B2 (en) 2003-09-30 2017-08-22 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) glades, sequences, vectors containing same, and uses therefor
US7892809B2 (en) 2004-12-15 2011-02-22 The University Of North Carolina At Chapel Hill Chimeric vectors
WO2008124724A1 (fr) 2007-04-09 2008-10-16 University Of Florida Research Foundation, Inc. Compositions à base de vecteurs raav comprenant des protéines de capside modifiées par la tyrosine et procédés d'utilisation correspondants
US8632764B2 (en) 2008-04-30 2014-01-21 University Of North Carolina At Chapel Hill Directed evolution and in vivo panning of virus vectors
US8889641B2 (en) 2009-02-11 2014-11-18 The University Of North Carolina At Chapel Hill Modified virus vectors and methods of making and using the same
US9475845B2 (en) 2009-02-11 2016-10-25 The University Of North Carolina At Chapel Hill Modified virus vectors and methods of making and using the same
WO2010129021A1 (fr) 2009-05-02 2010-11-11 Genzyme Corporation Thérapie génique pour les maladies neurodégénératives
WO2012145601A2 (fr) 2011-04-22 2012-10-26 The Regents Of The University Of California Virions de virus adéno-associé avec capside variant et procédés d'utilisation de ceux-ci
US9434928B2 (en) 2011-11-23 2016-09-06 Nationwide Children's Hospital, Inc. Recombinant adeno-associated virus delivery of alpha-sarcoglycan polynucleotides
US10011640B2 (en) 2012-05-15 2018-07-03 University Of Florida Research Foundation, Incorporated Capsid-modified rAAV vector compositions and methods therefor
US10036016B2 (en) 2013-09-12 2018-07-31 President And Fellows Of Harvard College Methods for inducing glucose uptake
US10894949B2 (en) 2014-02-19 2021-01-19 University Of Massachusetts Recombinant AAVS having useful transcytosis properties
WO2016054554A1 (fr) 2014-10-03 2016-04-07 University Of Massachusetts Virus adéno-associés greffés au moyen de peptides de ciblage hétérologues
US10480011B2 (en) 2014-10-21 2019-11-19 University Of Massachusetts Recombinant AAV variants and uses thereof
US20180066285A1 (en) 2015-03-24 2018-03-08 The Regents Of The University Of California Adeno-associated virus variants and methods of use thereof
WO2017100671A1 (fr) 2015-12-11 2017-06-15 California Institute Of Technology Peptides de ciblage pour diriger des virus adéno-associés (aav)
WO2017106236A1 (fr) 2015-12-14 2017-06-22 The University Of North Carolina At Chapel Hill Protéines capsidiques modifiées pour la libération améliorée de vecteurs parvoviraux
WO2017191274A2 (fr) * 2016-05-04 2017-11-09 Curevac Ag Arn codant pour une protéine thérapeutique
WO2017192750A1 (fr) 2016-05-04 2017-11-09 Oregon Health & Science University Vecteurs viraux adéno-associés recombinés
WO2017212019A1 (fr) 2016-06-09 2017-12-14 Centre National De La Recherche Scientifique (Cnrs) Raav à capside chimiquement modifiée
WO2018152333A1 (fr) 2017-02-15 2018-08-23 The University Of North Carolina At Chapel Hill Méthodes et compositions pour le transfert de gènes à travers le système vasculaire
WO2018222503A1 (fr) 2017-05-31 2018-12-06 The Regents Of The University Of California Virus adéno-associé à capside variante et ses méthodes d'utilisation
WO2019028306A2 (fr) 2017-08-03 2019-02-07 Voyager Therapeutics, Inc. Compositions et procédés permettant l'administration de virus adéno-associés
US20210380643A1 (en) 2017-09-20 2021-12-09 4D Molecular Therapeutics Inc. Adeno-associated virus variant capsids and methods of use thereof
WO2020117898A1 (fr) 2018-12-05 2020-06-11 Abeona Therapeutics Inc. Vecteur viral adéno-associé recombiné pour l'insertion de gènes
WO2020191300A1 (fr) 2019-03-21 2020-09-24 Stridebio, Inc. Vecteurs viraux adéno-associés recombinants
WO2020198737A1 (fr) 2019-03-28 2020-10-01 The General Hospital Corporation Vecteurs de virus adéno-associés (aav) modifiés pour l'expression transgénique
WO2021072197A1 (fr) 2019-10-10 2021-04-15 Solid Biosciences Inc. Capsides de vaa modifiées et leurs utilisations
US11129908B2 (en) 2020-02-13 2021-09-28 Tenaya Therapeutics, Inc. Gene therapy vectors for treating heart disease
WO2021163357A2 (fr) 2020-02-13 2021-08-19 Tenaya Therapeutics, Inc. Vecteurs de thérapie génique de traitement de maladie cardiaque
US20220160896A1 (en) * 2020-02-13 2022-05-26 Tenaya Therapeutics, Inc. Gene therapy vectors for treating heart disease
WO2021216456A2 (fr) 2020-04-20 2021-10-28 Tenaya Therapeutics, Inc. Virus adéno-associé comprenant une capside modifiée
WO2022173847A2 (fr) 2021-02-09 2022-08-18 Affinia Therapeutics Inc. Vaa recombinants ayant un tropisme et une spécificité améliorés
WO2023283649A1 (fr) 2021-07-08 2023-01-12 Tenaya Therapeutics, Inc. Cassettes d'expression optimisées pour thérapie génique
WO2023108159A1 (fr) * 2021-12-10 2023-06-15 University Of Florida Research Foundation, Incorporated Méthodes et compositions pour traiter une cardiomyopathie liée à bag-3 avec un vecteur viral
WO2023201207A1 (fr) 2022-04-11 2023-10-19 Tenaya Therapeutics, Inc. Virus adéno-associé comprenant une capside modifiée
WO2023200742A2 (fr) * 2022-04-11 2023-10-19 Tenaya Therapeutics, Inc. Capsides pour la thérapie génique à plakophilline-2
WO2023205767A2 (fr) * 2022-04-22 2023-10-26 Spacecraft Seven, Llc Thérapie génique de l'anthanogène 3 associé au lymphome b 2 (bag3) faisant intervenir un vecteur de vaa

Non-Patent Citations (62)

* Cited by examiner, † Cited by third party
Title
"GenBank", Database accession no. NC 006261
"Gene Transfer and Expression in Mammalian Cells", 2003
"Immobilized Cells and Enzymes", 1986, IRL PRESS
"Immunochemical Methods in Cell and Molecular Biology", 1987, COLD SPRING HARBOR LABORATORY
"NCBI", Database accession no. NM_004281.4
"Weir's Handbook of Experimental Immunology", 1996
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUC. ACIDS RES., vol. 25, 1977, pages 3389 - 3402
ANDERSON, NUCLEIC ACID HYBRIDIZATION, 1999
ASOKARI ET AL., HUM GENE THER., vol. 24, no. 11, 2013, pages 906 - 913
BALAJI ET AL., J SURG RES., vol. 184, no. 1, 2013, pages 691 - 698
BANTEL-SCHAAL ET AL., J. VIROL., vol. 73, 1999, pages 3994
BRENT ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, 2003
BRIELER ET AL., AM FAM PHYSICIAN, vol. 96, 2017, pages 640 - 646
BULCHA ET AL., SIGNAL TRANSDUCTION AND TARGETED TTTERAPY, 2020
BUNINGSRIVASTAVA, MOL THER METHODS CLIN DEV., vol. 12, 2019, pages 248 - 265
CHIORINI, J. VIROL., vol. 71, 1998, pages 6823
CHOI ET AL., CURR GENE THER., vol. 5, 2005, pages 299 - 310
CHU ET AL., GENE, vol. 13, 1981, pages 197
CLARK ET AL., HUM GENE THEN, vol. 6, 1995, pages 1329 - 41
CLARK ET AL., HUM GENE THER., vol. 6, 1995, pages 1329 - 41
DAVIS ET AL., BASIC METHODS IN MOLECULAR BIOLOGY, 1986
FANG ET AL., JCI, vol. 127, no. 8, 2017, pages 3189 - 3200
GAO ET AL., PROC. NAT. ACAD. SCI. USA, vol. 99, 2002, pages 11854
GHOSH ET AL., APPLIED BIOSAFETY: JOURNAL OF ABSA INTERNATIONAL, vol. 25, no. 1, 2020, pages 7 - 18
GONZALEZ ET AL., NATURE COMMUNICATIONS, vol. 13, 2022, pages 5947
GRAHAM ET AL., VIROLOGY, vol. 52, 1973, pages 456
GRUNHAUS ET AL., SEMINAR IN VIROLOGY, vol. 200, no. 2, 1992, pages 535 - 546
HARDEE ET AL., GENES, vol. 8, 2017, pages 65
IRIONS ET AL., NATURE BIOTECHNOLOGY, vol. 25, 2007, pages 1477 - 1482
JASKI ET AL., J CARD FAIL, vol. 15, 2009, pages 171 - 181
KATZ ET AL., GENE THER., vol. 19, 2012, pages 659 - 669
KOTTERMAN, M.A.D.V. SCHAFFER: "Engineering Adeno-Associated Viruses for Clinical Gene Therapy", NATURE REVIEWS GENETICS, vol. 15, 2014, pages 445 - 451, XP055317129, DOI: 10.1038/nrg3742
LEE ET AL., EXP PHYSIOL., vol. 90, 2005, pages 33 - 37
LIU ET AL., CURR PHARM DES., vol. 21, 2015, pages 3248 - 56
MACPHERSON ET AL.: "PCR 1: A Practical Approach", 1991, ACADEMIC PRESS, INC.
MALI, INDIAN JOURNAL OF HUMAN GENETICS, vol. 19, no. 1, 2013, pages 3 - 8
MCCARTY D., MO. THER., vol. 16, 2008, pages 1648 - 1656
MCKENNA ET AL., CIRC RES., vol. 121, 2017, pages 722 - 730
MILLER, NATURE, vol. 357, 2000, pages 455 - 460
MORIS ET AL., VIROLOGY, vol. 33, 2004, pages 375 - 383
MURAMATSU ET AL., VIROL., vol. 221, 1996, pages 208
NASO ET AL., BIODRUGS, vol. 31, 2017, pages 317 - 334
NEEDLEMANWUNSCH, J.MOL. BIOL., vol. 48, 1970, pages 443
OLIVIERI ET AL., 24TH ANNUAL MEETING OF THE AMERICAN SOCIETY OF GENE CELL THERAPY, 2021, Retrieved from the Internet <URL:https://www.affiniatx.com/pdf/asgct_2021_olivieri.pdf>
PEARSONLIPMAN, PROC. NAT'I. ACTID. SCI. USA, vol. 85, 1988, pages 2444
PERBAL: "A Practical Guide to Molecular Cloning", 1984
PEREZ-BERMEJO JUAN A ET AL: "Functional analysis of a common BAG3 allele associated with protection from heart failure", BIORXIV, 6 October 2021 (2021-10-06), XP093182522, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2021.10.06.463213v1.full> [retrieved on 20240703], DOI: 10.1101/2021.10.06.463213 *
PIRAS ET AL., GENE THERAPY, vol. 23, 2016, pages 469 - 478
POZSGAI ET AL., MOL THER., vol. 25, no. 4, 2017, pages 855 - 869
QU HUI-QI ET AL: "Genetics of BAG3: A Paradigm for Developing Precision Therapies for Dilated Cardiomyopathies", JOURNAL OF THE AMERICAN HEART ASSOCIATION, vol. 11, no. 23, 6 December 2022 (2022-12-06), XP093024288, ISSN: 2047-9980, Retrieved from the Internet <URL:http://dx.doi.org/10.1161/JAHA.122.027373> [retrieved on 20240708], DOI: 10.1161/JAHA.122.027373 *
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001
SHADE ET AL., J. VIROL., vol. 58, 1986, pages 921
SMITH ET AL., MOLECULAR THERAPY METHODS & CLINICAL DEVELOPMENT, vol. 26, 2022, pages 224 - 238
SOHAIL: "Gene Silencing by RNA Interference: Technology and Application", 2004, CRC PRESS
SRIVISTAVA ET AL., J. VIROL., vol. 45, 1983, pages 555
SUNGKIM, BIOMATERIALS RESEARCH, vol. 23, 2019, pages 8
TABEBORDBAR ET AL., CELL, vol. 184, no. 19, 2021, pages 4919 - 4938
WILMOTT ET AL., HUM GENE THER METHODS, vol. 30, 2019, pages 206 - 213
WU ET AL., MOL THER., vol. 18, 2010, pages 80
YUC ET AL., BIOTECHNIQUES, vol. 33, 2002, pages 672 - 678
ZINCARELLI ET AL., MOL THER., vol. 16, 2008, pages 1073 - 1080

Similar Documents

Publication Publication Date Title
EP4103214B1 (fr) Vecteurs de thérapie génique de traitement de maladie cardiaque
US20230220421A1 (en) Gene-therapy vectors for treating cardiomyopathy
US12151001B2 (en) Optimized expression cassettes for gene therapy
US12564647B2 (en) Optimized expression cassettes for gene therapy
WO2021163357A2 (fr) Vecteurs de thérapie génique de traitement de maladie cardiaque
CN116194154A (zh) 使用aav载体的plakophilin-2(pkp2)基因疗法
US20250332288A1 (en) Cardioprotective heart disease therapies
JP2022522196A (ja) ラミノパシーを処置するための組成物および方法
CN119233831A (zh) 用于与肌营养不良蛋白有关的心肌病的基因疗法组合物和治疗
JP2004519222A (ja) Ｃａｒｐ遺伝子の上流配列、それらを含むベクターとその使用
US20260027236A1 (en) Guide rnas, vectors, and virions for targeting mutations in the pln gene
WO2025122739A1 (fr) Compositions et méthodes de traitement d&#39;une maladie cardiaque
WO2025129079A1 (fr) Promoteurs cardiaques spécifiques pour thérapie génique
WO2026080685A1 (fr) Arn guides, vecteurs et virions pour cibler des mutations dans le gène pln
EA051433B1 (ru) Векторы для генной терапии для лечения заболевания сердца
WO2025155918A1 (fr) Compositions cardioprotectrices et procédés de fibrillation auriculaire
WO2025129095A1 (fr) Compositions et procédés d&#39;édition de gène myh7 spécifique d&#39;un allèle

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24721472

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 24721472

Country of ref document: EP

Kind code of ref document: A1