WO2023122804A1 - Compositions et méthodes comprenant un promoteur spécifique du coeur - Google Patents

Compositions et méthodes comprenant un promoteur spécifique du coeur Download PDF

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WO2023122804A1
WO2023122804A1 PCT/US2022/082384 US2022082384W WO2023122804A1 WO 2023122804 A1 WO2023122804 A1 WO 2023122804A1 US 2022082384 W US2022082384 W US 2022082384W WO 2023122804 A1 WO2023122804 A1 WO 2023122804A1
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promoter
aav
cardiac
sequence
raav
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Christian HINDERER
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The Trustees Of The University Of Pennsylvania
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    • 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
    • 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
    • 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
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • 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
    • 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
    • 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

  • Adeno-associated viral (AAV) vectors are safe and effective gene transfer vehicles used for several clinical indications.
  • Recombinant AAV vectors have a vector genome lacking AAV coding sequences packaged in an AAV capsid.
  • Treatment approaches based on AAV vectors have been approved by the US Food and Drug Administration and other worldwide regulatory authorities for the treatment of Leber congenital amaurosis, lipoprotein lipase deficiency, and spinal muscular atrophy.
  • a central challenge for gene therapy is the difficulty of modulating and targeting expression of the transgene in vivo, more specifically targeting a particular tissue.
  • Virtually all pre-clinical and clinical applications of gene therapy have used vectors that express the transgene from a constitutive and/or ubiquitous promoters.
  • many diseases that are amenable to gene therapy may need to have expression of the transgene regulated and/or targeted to a specific tissue.
  • a hybrid cardiac promoter is provided herein which comprises a CMV IE enhancer, a spacer sequence, and a chicken cardiac troponin T (cTnT) promoter.
  • the promoter has a nucleic acid sequence of SEQ ID NO: 3 or a sequence at least about 99% identical to SEQ ID NO: 3.
  • a recombinant adeno-associated virus comprising an adeno-associated virus (AAV) capsid and a vector genome packaged in the AAV capsid
  • the vector genome comprises an AAV 5’ inverted terminal repeat (ITR), an expression cassette, and an AAV 3’ ITR
  • the expression cassette comprises an engineered open reading frame (ORF) for transgene coding sequence which encodes for protein of interest
  • the ORF is operably linked to regulatory control sequences which direct expression of the protein of interest in a cardiac cell
  • the regulatory control sequences comprise the hybrid cardiac promoter.
  • the hybrid promoter comprises a shortened cTnT promoter.
  • the AAV capsid is a Clade F AAV, e.g., AAVhu68, AAVhu95, AAVhu96, AAV9 or an AAV9 mutant.
  • the rAAV is useful in the treatment of cardiac disorders.
  • a composition is provided which comprises a stock of rAAV in an aqueous suspension media.
  • the suspension is formulated for intravenous (IV) injection.
  • the expression cassette comprises an engineered open reading frame (ORF) encoding a protein of interest and/or a RNA sequence of interest, wherein the ORF is operably linked to hybrid cardiac promoter and other regulatory sequences.
  • ORF engineered open reading frame
  • the expression cassette is in a viral vector selected from a recombinant parvovirus, a recombinant lentivirus, a recombinant retrovirus, or a recombinant adenovirus; or a non-viral vector selected from naked DNA, naked RNA.
  • the vector is non-viral, e.g., the expression cassette is encapsulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • a recombinant nucleic acid molecule comprising the hybrid cardiac promoter.
  • a packaging host cell comprising the nucleic acid molecule is provided.
  • an rAAV production system is provided which comprised the nucleic acid molecule.
  • a method of treating a cardiac disorder or disease in a human subject comprises administering to the subject a suspension of a rAAV comprising the hybrid cardiac promoter and an ORF comprising a therapeutic gene or sequence in a formulation buffer.
  • FIG. 1A shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP of NHP heart tissue (septum) collected on day 21 post administration with AAVhu68-eGFP comprising CB7 promoter.
  • IHC immunohistochemical
  • FIG. IB shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP of NHP heart tissue (left ventricle) collected on day 21 post administration with AAVhu68-eGFP comprising CB7 promoter.
  • IHC immunohistochemical
  • FIG. 1C shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP of NHP heart tissue (septum) collected on day 21 post administration with AAVhu68-eGFP comprising CMV IE enhancer and chicken TnT promoter.
  • IHC immunohistochemical
  • FIG. ID shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP of NHP heart tissue (left ventricle) collected on day 21 post administration with AAVhu68-eGFP comprising CMV IE enhancer and chicken TnT promoter.
  • IHC immunohistochemical
  • FIG. 2A shows results of the cardiac promoter evaluation in mice, plotted as pg of GFP over pg protein as examined from heart tissue collected at 14 days post treatment with vehicle control, or with rAAVhu68.eGFP vectors comprising CB7, MH-MH (rat a-myosin heavy enhancer with rat a-myosin heavy promoter), TnT/MH- MH (chicken TnT enhancer with rat a-myosin heavy promoter), CMV-MH (CMV IE enhancer with rat a-myosin heavy promoter), TnT (chicken TnT promoter), MH/TnT- TnT (rat a-myosin heavy enhancer with chicken TnT promoter), or CMV-TnT promoter (CMV IE enhancer with chicken TnT promoter).
  • MH-MH rat a-myosin heavy enhancer with rat a-myosin heavy promoter
  • FIG. 2B shows results of the cardiac promoter evaluation in mice, plotted as pg of GFP over pg protein as examined from liver tissue collected at 14 days post treatment with vehicle control, or with rAAVhu68.eGFP vectors comprising CB7, MH-MH (rat a-myosin heavy enhancer with rat a-myosin heavy promoter), TnT/MH- MH (chicken TnT enhancer with rat a-myosin heavy promoter), CMV-MH (CMV IE enhancer with rat a-myosin heavy promoter), TnT (chicken TnT promoter), MH/TnT- TnT (rat a-myosin heavy enhancer with chicken TnT promoter), or CMV-TnT promoter (CMV IE enhancer with chicken TnT promoter).
  • MH-MH rat a-myosin heavy enhancer with rat a-myosin heavy promoter
  • FIG. 3A shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-TPl of FRG mouse liver tissue, following administration at newborn stage with AAVhu68.TTl intravenously at a dose of 5 x IO 10 GC (approximately 3 x 10 13 GC/kg).
  • IHC immunohistochemical
  • FIG. 3B shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-TPl of mouse heart tissue, following administration at newborn stage with vehicle control.
  • IHC immunohistochemical
  • FIG. 3C shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-TPl of mouse heart tissue, following administration at newborn stage with AAVhu68.TTl intravenously at a dose of 5 x IO 10 GC (approximately 3 x 10 13 GC/kg).
  • IHC immunohistochemical
  • compositions, kits and methods which utilize a recombinant adeno-associated virus (rAAV) comprising regulatory control sequences comprising a hybrid cardiac promoter which are designed to produce cardiac-specific expression of a transgene.
  • rAAV recombinant adeno-associated virus
  • Use of a compositions and methods which utilize rAAV comprising cardiac tissue targeting regulatory control sequences are also provided herein.
  • the compositions and methods of the invention are well suited for use with protein replacement therapy. However, other applications will be apparent to one of skill in the art.
  • a novel regulatory control sequence comprising a hybrid cardiac promoter comprising a cytomegalovirus immediate early (CMV IE) enhancer and a chicken cardiac troponin T (chicken cTnT or chTnT or chicken TnT) promoter and the nucleic acid sequence thereof, which are engineered for a targeted expression of a transgene in a cardiac cell following systemic administration.
  • CMV IE enhancer comprises a nucleic acid sequence of SEQ ID NO: 1 or a sequence at least 99% identical to SEQ ID NO: 1.
  • the chicken cardiac troponin T promoter comprises nucleic acid sequence of SEQ ID NO: 2 or a sequence at least 99% identical to SEQ ID NO: 2.
  • the chTnT promoter is a shortened chTnT promoter.
  • the regulatory control sequences comprise a short non-coding spacer sequence between the CMV IE enhancer and chicken troponin T promoter.
  • the noncoding spacer sequence as described herein includes variations in length and identity.
  • the spacer sequence comprises at least two (2) to at least ten (10) nucleotides.
  • the spacer sequence is at least nine (9) nucleotides.
  • the spacer comprises nucleic acid sequence “CAATAGCTT”.
  • the spacer sequence comprises nucleic acid sequence “CA”.
  • a “spacer” is any selected nucleic acid sequence, e.g., of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length which is located in the hybrid cardiac promoter between the enhancer and promoter sequences.
  • the spacer is 1 to 8 nucleotides in length, 2 to 7 nucleotides in length, 3 to 6 nucleotides in length, four nucleotides in length, 4 to 9 nucleotides, 3 to 7 nucleotides, or values which are longer.
  • a spacer is a non-coding sequence.
  • the spacer may be of four (4) nucleotides.
  • the spacer is GGAT.
  • the spacer is six (6) nucleotides.
  • the spacer is CACGTG or GCATGC.
  • the hybrid cardiac promoter comprises a CMV IE enhancer. A spacer sequence, and a chTnT promoter. In certain embodiments, the hybrid cardiac promoter comprises nucleic acid sequence of SEQ ID NO: 3. In certain embodiment, the hybrid cardiac promoter comprises nucleic acid sequence at least 99% identical to SEQ ID NO: 3. The variations in the nucleic acid sequence of the hybrid cardiac promoter includes substitutions of nucleotides in the spacer sequence, and optionally include insertion and deletion of nucleotides in spacer sequence.
  • a “nucleic acid”, as described herein, can be RNA, DNA, or a modification thereof, and can be single or double stranded, and can be selected, for example, from a group including: nucleic acid encoding a protein of interest, oligonucleotides, nucleic acid analogues, for example peptide-nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), locked nucleic acid (LNA) etc.
  • PNA peptide-nucleic acid
  • pc-PNA pseudocomplementary PNA
  • LNA locked nucleic acid
  • nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins, for example that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example but are not limited to RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.
  • sequence identity refers to the residues in the two sequences which are the same when aligned for correspondence.
  • the length of sequence identity comparison may be over the full-length of the genome, the full-length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides, is desired. However, identity among smaller fragments, e.g., of at least about nine nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides, may also be desired.
  • Percent identity may be readily determined for amino acid sequences over the full-length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or a peptide fragment thereof or the corresponding nucleic acid sequence coding sequences.
  • a suitable amino acid fragment may be at least about 8 ammo acids in length, and may be up to about 700 amino acids.
  • identity”, “homology”, or “similarity” is determined in reference to “aligned” sequences. “Aligned” sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
  • Sequence alignment programs are available for amino acid sequences, e.g., the “Clustal X”, “Clustal Omega” “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensive comparison of multiple sequence alignments”, 27(13):2682-2690 (1999).
  • nucleic acid sequences are also available for nucleic acid sequences. Examples of such programs include, “Clustal W”, “Clustal Omega”, “CAP Sequence Assembly”, “BLAST”, “MAP”, and “MEME”, which are accessible through Web Servers on the internet. Other sources for such programs are known to those of skill in the art. Alternatively, Vector NTI utilities are also used. There are also a number of algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the programs described above. As another example, polynucleotide sequences can be compared using FastaTM, a program in GCG Version 6.1. FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using FastaTM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
  • FastaTM provides alignments and percent sequence identity of the regions
  • Nucleic acid sequences described herein can be cloned using routine molecular biology techniques, or generated de novo by DNA synthesis, which can be performed using routine procedures by service companies having business in the field of DNA synthesis and/or molecular cloning (e.g., GeneArt, GenScript, Life Technologies, Eurofins).
  • nucleic acid sequences encoding the miRNA or modified snRNA described herein are assembled and placed into any suitable genetic element, e.g., naked DNA, phage, transposon, cosmid, episome, etc., which transfers the sequences carried thereon to a host cell, e.g., for generating non-viral delivery systems (e.g., RNA-based systems, naked DNA, or the like), or for generating viral vectors in a packaging host cell, and/or for delivery to a host cells in a subject.
  • the genetic element is a vector.
  • the genetic element is a plasmid.
  • engineered constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).
  • compositions in the regulatory control sequences comprising hybrid cardiac promoter described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.
  • a novel regulatory control sequence comprising a hybrid cardiac promoter comprising a cytomegalovirus immediate early (CMV IE enhancer), a spacer sequence, and a chicken cardiac troponin T (chicken cTnT or chTnT) and expression cassette or vector genome thereof, which are engineered for a targeted expression of a transgene in a cardiac cell.
  • CMV IE enhancer cytomegalovirus immediate early
  • spacer sequence a spacer sequence
  • a chicken cardiac troponin T chicken cTnT or chTnT
  • expression cassette or vector genome are engineered for targeted expression of a transgene in a cardiac cell following systemic administration.
  • an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.
  • a biologically useful nucleic acid sequence e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.
  • regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.
  • “operably linked” sequences include both regulatory sequences that are contiguous or non-contiguous with the nucleic acid sequence and regulatory sequences that act in trans or cis nucleic acid sequence.
  • Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal.
  • the expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region (3’ UTR) comprising a polyadenylation site, among other elements.
  • the regulatory sequences are operably linked to the nucleic acid sequence of a gene product, wherein the regulatory sequences are separated from nucleic acid sequence of a gene product by an intervening nucleic acid sequences, i.e., 5 ’-untranslated regions (5 ’UTR).
  • the expression cassette comprises nucleic acid sequence of one or more of gene products.
  • the expression cassette can be a monocistronic or a bicistronic expression cassette.
  • the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell.
  • such an expression cassette can be used for generating a viral vector and contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.
  • a vector genome may contain two or more expression cassettes.
  • exogenous nucleic acid sequence or protein means that the nucleic acid or protein does not naturally occur in the position in which it exists in a chromosome, or host cell.
  • An exogenous nucleic acid sequence also refers to a sequence derived from and inserted into the same host cell or subject, but which is present in a non-natural state, e.g., a different copy number, or under the control of different regulatory elements.
  • the expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a hybrid promoter, a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region (3’ UTR) comprising a polyadenylation (poly A) site, among other elements.
  • regulatory sequences upstream (5’ to) of the gene sequence e.g., one or more of a hybrid promoter, a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region (3’ UTR) comprising a polyadenylation (poly A) site, among other elements.
  • the regulatory sequences comprise an enhancer and a promoter, and one or more of a transcription factor, a transcription terminator, an efficient RNA processing signals such as splicing and polyadenylation signals (poly A), a sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE), and sequences that enhance translation efficiency (i.e., Kozak consensus sequence).
  • the term “hybrid cardiac promoter” comprises a CMV IE enhancer sequence, a spacer sequence, a chicken cardiac troponin T promoter.
  • the promoter is a tissue- or cell specific-promoter.
  • the promoter is cardiac specific promoter, e.g., cardiac troponin T (cTNT), desmin (DES), alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2) promoters. See also, Pacak, C.A., et al., Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice, Genetic Vaccines and Therapy 2008, 6:13.
  • the expression cassette comprises a promoter which is a chicken cardiac Troponin T promoter (also referred to as chicken TnT or chTnT).
  • the chTnT promoter comprises nucleic acid sequence of SEQ ID NO: 2 or a sequence at least 99% identical to SEQ ID NO: 2.
  • the expression cassette comprises one or more expression enhancers.
  • the expression cassette contains two or more expression enhancers. These enhancers may be the same or may differ from one another.
  • the enhancer is a cytomegalovirus (CMV) immediate early enhancer (CMV IE enhancer).
  • CMV IE enhancer comprises nucleic acid of SEQ ID NO: 1 or a sequence at least 99% identical to SEQ ID NO: 1.
  • the enhancer is a cardiac enhancer.
  • the cardiac enhancer is chicken troponin T enhancer.
  • the enhancer is a rat a-myosin heavy enhancer.
  • the dual copies of the enhancer may be separated by one or more sequences.
  • the enhancer(s) is selected from one or more of an APB enhancer, an ABPS enhancer, an alpha mic/bik enhancer, a TTR enhancer, an en34 enhancer, an ApoE enhancer, a CMV enhancer, or an RSV enhancer.
  • the regulatory elements comprise an intron.
  • the intron is selected from chicken beta actin intron (CBA), human beta globin, IVS2, SV40 (Promega), bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53. See, e.g., WO 2011/126808.
  • the regulatory elements comprise a poly A.
  • the polyA is a synthetic polyA or from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit P-globin (RBG), or modified RBG (mRBG).
  • bGH bovine growth hormone
  • hGH human growth hormone
  • SV40 rabbit P-globin
  • RBG modified RBG
  • a modified WPRE sequence which may be engineered upstream of the polyA sequence and downstream of the coding sequence [see, e.g., MA Zanta-Boussif, et al, Gene Therapy (2009) 16: 605-619.
  • the expression cassettes may include one or more expression enhancers such as post-transcriptional regulatory element from hepatitis viruses of woodchuck (WPRE), human (HPRE), ground squirrel (GPRE) or arctic ground squirrel (AGSPRE); or a synthetic post-transcriptional regulatory element.
  • WPRE woodchuck
  • HPRE human
  • GPRE ground squirrel
  • AGSPRE arctic ground squirrel
  • the expressions cassettes provided include a regulator sequence that is a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or a variant thereof. Suitable WPRE sequences are provided in the vector genomes described herein and are known in the art (e.g., such as those are described in US Patent Nos.
  • the WPRE is a variant that has been mutated to eliminate expression of the woodchuck hepatitis B virus X (WHX) protein, including, for example, mutations in the start codon of the WHX gene. See also, Kingsman S.M., Mitrophanous K., & Olsen J.C. (2005), Potential Oncogene Activity of the Woodchuck Hepatitis Post- Transcriptional Regulatory Element (Wpre)." Gene Ther.
  • WHX woodchuck hepatitis B virus X
  • enhancers are selected from a non-viral source. In certain embodiments, no WPRE sequence is present.
  • the expression cassette comprises regulatory control sequences comprising a hybrid cardiac promoter comprising a cardiac promoter and an enhancer sequence. In certain embodiments, the expression cassette comprises regulatory control sequences comprising a hybrid cardiac promoter comprising a cardiac troponin T (cTnT) promoter. In certain embodiments, the expression cassette comprises regulatory control sequences comprising a hybrid cardiac promoter comprising a shorted (or truncated) cardiac troponin T (cTnT) promoter. In certain embodiments, the expression cassette comprises regulatory control sequences comprising a hybrid cardiac promoter comprising chicken cardiac Troponin T promoter (chTnT) with CMV IE enhancer.
  • the expression cassette comprises a hybrid cardiac promoter comprising chTnT comprising nucleic acid sequence of SEQ ID NO: 2 with CMV IE enhancer comprising nucleic acid sequence of SEQ ID NO: 1.
  • the expression cassette comprises regulatory control sequences comprising a hybrid cardiac promoter having nucleic acid sequence of SEQ ID NO: 3 comprising CMV IE enhancer, short spacer sequence, and a chicken cardiac troponin T promoter.
  • the expression cassette comprises regulatory control sequences comprising a hybrid cardiac promoter having nucleic acid sequence of at least 99% identical to SEQ ID NO: 3.
  • the regulatory control sequences further comprise polyA sequence which is a rabbit beta globin polyA sequence.
  • the expression cassette comprises an engineered open reading frame (ORF) for transgene coding sequence which encodes for protein of interest, wherein the ORF is operably linked to regulatory control sequences which direct expression of the protein of interest in a cardiac cell, and wherein the regulatory control sequences comprise a hybrid cardiac promoter and a polyadenylation (polyA) sequence, wherein the hybrid cardiac promoter comprises an enhancer which is a CMV IE enhancer (SEQ ID NO: 1), a spacer sequence, and a cardiac specific promoter which is a chicken cardiac TnT promoter (SEQ ID NO: 2).
  • ORF engineered open reading frame
  • the regulatory control sequences comprise a hybrid cardiac promoter and a polyadenylation (polyA) sequence
  • the hybrid cardiac promoter comprises an enhancer which is a CMV IE enhancer (SEQ ID NO: 1), a spacer sequence, and a cardiac specific promoter which is a chicken cardiac TnT promoter (SEQ ID NO: 2).
  • the expression cassette comprises an engineered open reading frame (ORF) for transgene coding sequence which encodes for protein of interest, wherein the ORF is operably linked to regulatory control sequences which direct expression of the protein of interest in a cardiac cell, and wherein the regulatory control sequences comprise a hybrid cardiac promoter comprising nucleic acid sequence of SEQ ID NO: 3 or at least 99% identical to SEQ ID NO: 3.
  • ORF engineered open reading frame
  • the vector genome comprises a recombinant nucleic acid molecule comprising, 5’ to 3’, AAV5’ ITR - hybrid cardiac promoter - transgene coding sequence - polyA - AAV3’ ITR.
  • the vector genome comprises a nucleic acid molecule comprising, 5’ to 3’, AAV5’ ITR - CMV IE promoter - spacer sequence - chTnT promoter - transgene coding sequence - polyA - AAV3’ ITR.
  • the vector genome comprises a nucleic acid molecule comprising, 5’ to 3’, AAV5’ ITR - CMV IE promoter - spacer sequence - chTnT promoter - transgene coding sequence - rabbit beta globin polyA - AAV3’ ITR.
  • a vector genome comprises an AAV 5’ inverted terminal repeat (ITR), an expression cassette, and an AAV3’ ITR
  • the expression comprises an engineered open reading frame (ORF) for transgene coding sequence which encodes for protein of interest
  • ORF engineered open reading frame
  • the regulatory control sequences comprise a hybrid cardiac promoter comprising an enhancer, a spacer sequence, a cardiac specific promoter, and a polyadenylation (poly A) sequence
  • the enhancer is a CMV IE enhancer (SEQ ID NO: 1)
  • the cardiac specific promoter is a chicken cardiac TnT promoter (SEQ ID NO: 2).
  • a vector genome comprises an AAV 5’ inverted terminal repeat (ITR), an expression cassette, and an AAV3’ ITR, wherein the expression comprises an engineered open reading frame (ORF) for transgene coding sequence which encodes for protein of interest, wherein the ORF is operably linked to regulatory control sequences which direct expression of the protein of interest in a cardiac cell, and wherein the regulatory control sequences comprise a hybrid cardiac promoter comprising a nucleic acid sequence of SEQ ID NO: 3 or a sequence at least 99% identical to SEQ ID NO: 3.
  • ITR inverted terminal repeat
  • ORF engineered open reading frame
  • a “cardiac cell” refers to general cardiac tissue cells including but not limited to heart cells, cardiac muscle cells (cardiomyocyte), conduction cells, fibroblasts, endothelial cells, smooth muscle cells and peri-vascular cells.
  • a “vector genome” refers to the nucleic acid sequence packaged inside a parvovirus (e.g., rAAV) capsid which forms a viral particle.
  • a nucleic acid sequence contains AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • a vector genome contains, at a minimum, from 5’ to 3’, an AAV 5’ ITR (also referred to as 5’ ITR), coding sequence(s) (i.e., transgene(s)), and an AAV 3’ ITR (also referred to as 3’ ITR). ITRs from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected.
  • the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV.
  • ITRs e.g., self- complementary (scAAV) ITRs
  • scAAV self- complementary
  • Both single-stranded AAV and self- complementary (sc) AAV are encompassed with the rAAV.
  • the transgene is a nucleic acid coding sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
  • a “vector genome” contains, at a minimum, from 5’ to 3’, a vector-specific sequence, a nucleic acid sequence encoding protein of interest operably linked to regulatory control sequences (which direct their expression in a target cell), where the vector-specific sequence may be a terminal repeat sequence which specifically packages the vector genome into a viral vector capsid or envelope protein.
  • AAV inverted terminal repeats are utilized for packaging into AAV and certain other parvovirus capsids.
  • the vector genome is an expression cassette having inverted terminal repeat (ITR) sequences necessary for packaging the vector genome into the AAV capsid at the extreme 5’ and 3’ end and containing therebetween a transgene as described herein operably linked to sequences which direct expression thereof.
  • a vector genome may comprise at a minimum from 5’ to 3’, an AAV 5’ ITR, coding sequence(s), and an AAV 3’ ITR.
  • the ITRs are from AAV2, a different source AAV than the capsid, or other than full-length ITRs may be selected.
  • the ITRs are from the same AAV source as the AAV which provides the rep function during production or a transcomplementing AAV. Further, other ITRs may be used.
  • the AAV sequences of the vector typically comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)).
  • the ITR sequences are about 145 bp in length.
  • substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible.
  • the ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning.
  • ITRs are from an AAV different than that supplying a capsid.
  • the ITR sequences from AAV2. However, ITRs from other AAV sources may be selected.
  • a shortened version of the 5’ ITR termed AITR
  • the vector genome includes a shortened AAV2 ITR of 130 base pairs, wherein the external A elements is deleted.
  • the shortened ITR reverts back to the wild-type length of 145 base pairs during vector DNA amplification using the internal (A’) element as a template.
  • full-length AAV 5’ and 3’ ITRs are used.
  • the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped.
  • other configurations of these elements may be suitable.
  • compositions in the expression cassette and vector genomes described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.
  • a vector comprising an engineered open reading frame (ORF) for transgene coding sequence which encodes for protein of interest, wherein the ORF is operably linked to regulatory control sequences which direct expression of the protein of interest in a cell, and wherein the regulatory control sequences comprise a hybrid cardiac promoter and a polyadenylation (poly A) sequence, wherein the hybrid cardiac promoter comprises an enhancer which is a CMV IE enhancer, a spacer sequence, and a cardiac specific promoter which is a chicken cardiac TnT promoter.
  • the vector comprises regulatory control sequences wherein the CMV IE enhancer comprises nucleic acid of SEQ ID NO: 1 or a sequence at least 99% identical to SEQ ID NO: 1.
  • the vector comprises regulatory control sequences wherein the chicken TnT promoter comprises nucleic acid of SEQ ID NO: 2 or a sequence at least 99% identical to SEQ ID NO: 2.
  • the vector comprises regulatory control sequences comprising a hybrid cardiac promoter comprising nucleic acid sequence of SEQ ID NO: 3 or a sequence at least 99% identical to SEQ ID NO: 3.
  • a “vector” as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate target cell for replication or expression of said nucleic acid sequence.
  • a vector includes but not limited to a recombinant virus, a plasmid, Lipoplexes, a Polymersome, Polyplexes, a dendrimer, a cell penetrating peptide (CPP) conjugate, a magnetic particle, or a nanoparticle.
  • a vector is a nucleic acid molecule into which an exogenous or heterologous or engineered nucleic acid encoding a functional SGSH may be inserted, which can then be introduced into an appropriate target cell.
  • Such vectors preferably have one or more origin of replication, and one or more site into which the recombinant DNA can be inserted.
  • Vectors often have means by which cells with vectors can be selected from those without, e.g., they encode drug resistance genes.
  • Common vectors include plasmids, viral genomes, and "artificial chromosomes". Conventional methods of generation, production, characterization or quantification of the vectors are available to one of skill in the art.
  • the vector is a non-viral plasmid that comprises an expression cassette described thereof, e.g., “naked DNA”, “naked plasmid DNA”, naked RNA, and mRNA; coupled with various compositions and nano particles, including, e.g., micelles, liposomes, cationic lipid - nucleic acid compositions, poly- glycan compositions and other polymers, lipid and/or cholesterol-based - nucleic acid conjugates, and other constructs such as are described herein. See, e.g., X. Su, et al, Mol. Pharmaceutics, 2011, 8 (3), pp 774-787; web publication: March 21, 2011; WO2013/182683, WO 2010/053572 and WO 2012/170930, all of which are incorporated herein by reference.
  • an expression cassette described thereof e.g., “naked DNA”, “naked plasmid DNA”, naked RNA, and mRNA
  • various compositions and nano particles including, e
  • the vector or a recombinant nucleic acid molecule thereof is encapsulated in a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • the phrase "lipid nanoparticle” or “nanoparticle” refers to a transfer vehicle comprising one or more lipids (e.g., cationic lipids, non- cationic lipids, and PEG-modified lipids).
  • the lipid nanoparticles are formulated to deliver one or more nucleic acid sequences to one or more target cells (e.g., liver and/or muscle).
  • lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). Also contemplated is the use of polymers as transfer vehicles, whether alone or in combination with other transfer vehicles.
  • phosphatidyl compounds e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • polymers as transfer vehicles, whether alone or in combination with other transfer vehicles.
  • Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide- polyglycolide copolymers, poly caprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, dendrimers and polyethylenimine.
  • the transfer vehicle is selected based upon its ability to facilitate the transfection of a nucleic acid sequence encapsulated therein to a target cell.
  • Useful lipid nanoparticles for nucleic acid sequence comprise a cationic lipid to encapsulate and/or enhance the delivery of such nucleic acid sequence into the target cell that will act as a depot for protein production.
  • cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.
  • the contemplated lipid nanoparticles may be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and PEG- modified lipids.
  • Several cationic lipids have been described in the literature, many of which are commercially available. See, e.g., WO2014/089486, US 2018/0353616A1, and US 8,853,377B2, which are incorporated by reference.
  • LNP formulation is performed using routine procedures comprising cholesterol, ionizable lipid, helper lipid, PEG-lipid and polymer forming a lipid bilayer around encapsulated nucleic acid sequence (Kowalski et al., 2019, Mol. Ther. 27(4):710- 728).
  • LNP comprises a cationic lipids (i.e. N-[l-(2,3- di oleoyloxy )propyl]-N,N,N-trimethylammonium chloride (DOTMA), or 1,2-dioleoyl- 3-trimethylammonium-propane (DOTAP)) with helper lipid DOPE.
  • DOTMA 1,2-dioleoyl- 3-trimethylammonium-propane
  • LNP comprises an ionizable lipid Dlin-MC3-DMA ionizable lipids, or diketopiperazine-based ionizable lipids (cKK-E12).
  • polymer comprises a polyethyleneimine (PEI), or a poly(P-amino)esters (PBAEs). See, e.g., WO2014/089486, US 2018/0353616A1, US2013/0037977A1, WO2015/074085 Al, US9670152B2, and US 8,853,377B2, which are incorporated by reference.
  • the vector described herein is a “replication-defective virus” or a “viral vector” which refers to a synthetic or artificial viral particle in which an expression cassette containing a nucleic acid sequence of a transgene is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
  • the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the nucleic acid sequence of a transgene flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.
  • the vector as described herein is for use in viral delivery of gene-editing nucleases. In certain embodiments, the vector as described herein is for use in non-viral delivery (LNP) of gene-editing nucleases. See also, US Provisional Patent Application No. 63/273,373, filed October 29, 2021.
  • a recombinant virus vector is an adeno-associated virus (AAV), an adenovirus, a bocavirus, a hybrid AAV/bocavirus, a herpes simplex virus or a lenti virus.
  • AAV adeno-associated virus
  • adenovirus an adenovirus
  • a bocavirus a bocavirus
  • a hybrid AAV/bocavirus a herpes simplex virus or a lenti virus.
  • the term “host cell” may refer to the packaging cell line in which a vector (e.g., a recombinant AAV) is produced.
  • a host cell may be a prokaryotic or eukaryotic cell (e.g., human, insect, or yeast) that contains exogenous or heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • host cells may include, but are not limited to an isolated cell, a cell culture, an Escherichia coli cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a non-mammahan cell, an insect cell, an HEK-293 cell, a liver cell, a kidney cell, a cell of the central nervous system, a heart cell, or a stem cell.
  • compositions in the vector described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.
  • rAAV Recombinant Adeno-associated Virus
  • a recombinant adeno-associated virus useful for treating cardiac diseases such as cardiomyopathies including long QT syndrome, dilated cardiomyopathy, hypertrophic cardiomyopathy, and mitochondrial cardiomyopathy associated with Barth Syndrome.
  • the rAAV comprises (a) an AAV capsid; and (b) a vector genome packaged in the AAV capsid of (a).
  • the AAV capsid selected targets the cells to be treated.
  • the capsid is from Clade F.
  • another AAV capsid source may be selected, i.e., Clade A.
  • the AAV capsid is AAVhu68 capsid.
  • the AAV capsid is AAVhu95 capsid. In certain embodiments, the AAV capsid is AAVhu96 capsid.
  • the vector genome comprises an AAV 5’ inverted terminal repeat (ITR), a nucleic acid sequence of a transgene, a regulatory sequence which direct expression of the transgene in a cardiac cell, and an AAV 3’ ITR, and wherein the regulatory control sequences comprise a hybrid cardiac promoter comprising a nucleic acid sequence of SEQ ID NO: 3 or a sequence at least 99% identical to SEQ ID NO: 3 comprising an enhancer which is a CMV IE enhancer, a spacer sequence, and a cardiac promoter which is a chicken TnT promoter.
  • ITR inverted terminal repeat
  • the rAAV is for use in the treatment of cardiomyopathies.
  • the rAAV is for the use in treatment of cardiomyopathy wherein the rAAV is delivered systemically (e.g., intravenous administration).
  • the rAAV is for the use in treatment of long QT syndrome cardiomyopathy.
  • the rAAV is for the use in treatment of dilated cardiomyopathy.
  • the rAAV is for the use in treatment of hypertrophic cardiomyopathy.
  • the rAAV is for the use in treatment of mitochondrial cardiomyopathy associated with Barth Syndrome.
  • the rAAV is for the use in treatment of cardiomyopathy or cardiac symptoms associated with Freidreich’s ataxia. See also, US Provisional Patent Application No. 62/950,834, filed December 19, 2019, and International Patent Application No. PCT/US20/66167, filed December 18, 2020, which is now published as WO/2021/127533, US Provisional Application No. 63/136,059, filed January 11, 2021, and US Provisional Patent Application No. 63/232,927, filed August 13, 2021, which are incorporated herein by reference it its entirety.
  • the rAAV is for use in viral delivery of gene-editing nucleases. See also, US Provisional Patent Application No. 63/273,373, filed October 29, 2021.
  • the rAAV comprises a vector genome comprising 5’ AAV ITR, an expression cassette, and 3’ AAV ITR, wherein the expression cassette comprising an engineered open reading frame (ORF) for transgene coding sequence which encodes for protein of interest, wherein the ORF is operably linked to regulatory control sequences which direct expression of the protein of interest in a cardiac cell, and wherein the regulatory control sequences comprises a hybrid cardiac promoter comprising a nucleic acid sequence of SEQ ID NO: 3 or a sequence at least 99% identical to SEQ ID NO: 3 comprising an enhancer which is a CMV IE enhancer, a spacer sequence, and a promoter which is a chicken TnT promoter.
  • ORF engineered open reading frame
  • the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3’, AAV5’ ITR - hybrid cardiac promoter - transgene -polyA - AAV3’ ITR. In certain embodiments, the rAAV comprises vector genome comprising a nucleic acid molecule comprising, 5’ to 3’, AAV5’ ITR - CMV IE promoter - spacer sequence - chicken TnT promoter - transgene -polyA - AAV3’ ITR.
  • the rAAV comprises a vector genome comprising a nucleic acid molecule comprising, 5’ to 3’, AAV5’ ITR - CMV IE promoter - spacer sequence - chTnT promoter - transgene - rabbit beta globin polyA - AAV3’ ITR.
  • the AAV capsid for the compositions and methods described herein is chosen based on the target cell.
  • the AAV capsid transduces a heart cell.
  • other AAV capsid may be chosen.
  • the Clade F AAV capsid is selected from an AAVhu68 capsid [See, e.g., US2020/0056159; PCT/US21/55436; SEQ ID NO: 4 and 5 for nucleic acid sequence; SEQ ID NO: 6 for amino acid sequence], an AAVhu95 capsid [See, e.g., US Provisional Application No. 63/251,599, filed October 2, 2201; SEQ ID NOs: 7 and 8 (hu95 nucleic acid sequence) and SEQ ID NO: 9 (hu95 amino acid sequence), or an AAVhu96 capsid [See, e.g., US Provisional Application No.
  • the AAV capsid is a non-clade F capsid, for example a Clade A, B, C, D, or E capsid.
  • the non-Clade F capsid is an AAV1 or a variation thereof.
  • the AAV capsid transduces a target cell other than the heart cells.
  • the AAV capsid is a Clade A capsid (e.g., AAV1, AAV6, AAVrh91), a Clade B capsid (e.g., AAV 2), a Clade C capsid (e.g., hu53), a Clade D capsid (e.g., AAV7), or a Clade E capsid (e.g., rhlO).
  • AAV1 AAV1, AAV6, AAVrh91
  • AAV 2 e.g., AAV 2
  • a Clade C capsid e.g., hu53
  • a Clade D capsid e.g., AAV7
  • a Clade E capsid e.g., rhlO
  • the term “clade” as it relates to groups of AAV refers to a group of AAV which are phylogenetically related to one another as determined using a Neighbor-Joining algorithm by a bootstrap value of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the AAV vpl amino acid sequence.
  • the Neighbor-Joining algorithm has been described in the literature. See, e.g., M. Nei and S. Kumar, Molecular Evolution and Phylogenetics (Oxford University Press, New York (2000). Computer programs are available that can be used to implement this algorithm.
  • the MEGA v2.1 program implements the modified Nei-Gojobori method.
  • the sequence of an AAV vpl capsid protein one of skill in the art can readily determine whether a selected AAV is contained in one of the clades identified herein, in another clade, or is outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jun; 78(10): 6381-6388, which identifies Clades A, B, C, D, E and F, and provides nucleic acid sequences of novel AAV, GenBank Accession Numbers AY530553 to AY530629. See, also, WO 2005/033321.
  • a rAAV is composed of an AAV capsid and a vector genome.
  • An AAV capsid is an assembly of a heterogeneous population of vpl, a heterogeneous population of vp2, and a heterogeneous population of vp3 proteins.
  • the term “heterogeneous” or any grammatical variation thereof refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences.
  • heterogeneous refers to a population consisting of elements that are not the same, for example, having vpl, vp2 or vp3 monomers (proteins) with different modified amino acid sequences.
  • heterogeneous population refers to differences in the amino acid sequence of the vpl, vp2 and vp3 proteins within a capsid.
  • the AAV capsid contains subpopulations within the vpl proteins, within the vp2 proteins and within the vp3 proteins which have modifications from the predicted amino acid residues. These subpopulations include, at a minimum, certain deamidated asparagine (N or Asn) residues.
  • certain subpopulations comprise at least one, two, three or four highly deamidated asparagines (N) positions in asparagine - glycine pairs and optionally further comprising other deamidated amino acids, wherein the deamidation results in an amino acid change and other optional modifications.
  • AAV capsids are provided which have a heterogeneous population of AAV capsid isoforms (i.e., VP1, VP2, VP3) which contain multiple highly deamidated “NG” positions.
  • the highly deamidated positions are in the locations identified below, with reference to the predicted full-length VP1 amino acid sequence.
  • the capsid gene is modified such that the referenced “NG” is ablated and a mutant “NG” is engineered into another position.
  • target cell and “target tissue” can refer to any cell or tissue which is intended to be transduced by the subject AAV vector or in which expression of a transgene is desired.
  • the term may refer to any one or more of heart (i.e., cardiac), muscle, liver, lung, airway epithelium, central nervous system, neurons, or eye (ocular cells).
  • target cell is intended to reference the cells of the subject being treated for cardiac disease (i.e., cardiomyopathy).
  • the vector is delivered to a target cell ex vivo. In certain embodiments, the vector is delivered to the target cell in vivo.
  • an rAAV production system useful for producing a rAAV as described herein.
  • the production system comprises a cell culture comprising (a) a nucleic acid sequence encoding an AAV capsid protein; (b) the vector genome; and (c) sufficient AAV rep functions and helper functions to permit packaging of the vector genome into the AAV capsid.
  • the cell culture is a human embryonic kidney 293 cell culture.
  • the AAV rep is from a different AAV.
  • the AAV rep is from AAV2.
  • the AAV rep coding sequence and cap genes are on the same nucleic acid molecule, wherein there is optionally a spacer between the rep sequence and cap gene.
  • the vector genomes can be carried on any suitable vector, e.g., a plasmid, which is delivered to a packaging host cell.
  • a suitable vector e.g., a plasmid
  • the plasmids useful in this invention may be engineered such that they are suitable for replication and packaging in vitro in prokaryotic cells, insect cells, mammalian cells, among others. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art.
  • a plasmid useful in producing an rAAV particle comprises a vector genome comprising a AAV 5’ ITR, an expression cassette, and a AAV3’ ITR, wherein expression cassette comprises the engineered nucleic acid sequence comprising open reading frame (ORF) for a transgene sequence which encode a protein of interest, wherein the ORF is operably linked to regulatory control sequences which direct expression of the protein in a cardiac cell, and wherein the regulatory control sequences comprise a hybrid cardiac promoter comprising nucleic acid sequence of SEQ ID NO: 3 or a sequence at least 99% identical to SEQ ID NO: 3 comprising an enhancer which is a CMV IE enhancer, a spacer sequence, and a promoter which is a chTnT promoter.
  • ORF open reading frame
  • a gene therapy vector refers to a rAAV as described herein, which is suitable for use in treating a patient.
  • the ITRs are the only AAV components required in cis in the same construct as the nucleic acid molecule containing the gene.
  • the cap and rep genes can be supplied in trans.
  • the selected genetic element may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • Stable AAV packaging cells can also be made.
  • the methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012).
  • AAV intermediate or “AAV vector intermediate” refers to an assembled rAAV capsid which lacks the desired genomic sequences packaged therein. These may also be termed an “empty” capsid. Such a capsid may contain no detectable genomic sequences of an expression cassette, or only partially packaged genomic sequences which are insufficient to achieve expression of the gene product. These empty capsids are non-functional to transfer the gene of interest to a host cell.
  • the recombinant adeno-associated virus (AAV) described herein may be generated using techniques which are known. See, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; US 7588772 B2.
  • Such a method involves culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; an expression cassette composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the expression cassette into the AAV capsid protein.
  • ITRs AAV inverted terminal repeats
  • a production cell culture useful for producing a recombinant AAV having a capsid selected from AAVhu68, AAVhu95 or AAVhu96 is provided.
  • a cell culture contains a nucleic acid which expresses the AAVhu68 capsid protein in the host cell (e.g., SEQ ID NO: 4 or SEQ ID NO: 5; a nucleic acid molecule suitable for packaging into the AAVhu68 capsid, e.g., a vector genome which contains AAV ITRs and a non- AAV nucleic acid sequence encoding a gene operably linked to regulatory sequences which direct expression of the gene in a host cell; and sufficient AAV rep functions and adenovirus helper functions to permit packaging of the vector genome into the recombinant AAVhu68, or AAVhu95 capsid (e.g., SEQ ID NO: 7 or SEQ ID NO: 8), AAVhu96 capsid (e.g., SEQ
  • the cell culture is composed of mammalian cells (e.g., human embryonic kidney 293 cells, among others) or insect cells (e.g., Spodoptera frugiperda (SI9) cells).
  • mammalian cells e.g., human embryonic kidney 293 cells, among others
  • insect cells e.g., Spodoptera frugiperda (SI9) cells.
  • baculovirus provides the helper functions necessary for packaging the vector genome into the recombinant AAVhu68, AAVhu95 or AAVhu96 capsid.
  • rep functions are provided by an AAV other than AAV2, selected to complement the source of the ITRs.
  • cells are manufactured in a suitable cell culture (e.g., HEK 293 or Sf9) or suspension.
  • Methods for manufacturing the gene therapy vectors described herein include methods well known in the art such as generation of plasmid DNA used for production of the gene therapy vectors, generation of the vectors, and purification of the vectors.
  • the gene therapy vector is an AAV vector and the plasmids generated are an AAV cis-plasmid encoding the AAV vector genome and the gene of interest, an AAV trans-plasmid containing AAV rep and cap genes, and an adenovirus helper plasmid.
  • the vector generation process can include method steps such as initiation of cell culture, passage of cells, seeding of cells, transfection of cells with the plasmid DNA, post-transfection medium exchange to serum free medium, and the harvest of vector-containing cells and culture media.
  • the harvested vector-containing cells and culture media are referred to herein as crude cell harvest.
  • the gene therapy vectors are introduced into insect cells by infection with baculovirus-based vectors.
  • Zhang et al., 2009 Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production, Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety.
  • the crude cell harvest may thereafter be subject method steps such as concentration of the vector harvest, diafiltration of the vector harvest, microfluidization of the vector harvest, nuclease digestion of the vector harvest, filtration of microfluidized intermediate, crude purification by chromatography, crude purification by ultracentrifugation, buffer exchange by tangential flow filtration, and/or formulation and filtration to prepare bulk vector.
  • An affinity chromatography purification followed anion exchange resin chromatography are used to purify the vector drug product and to remove empty capsids.
  • GC genome copies
  • the number of particles (pt) per 20 pL loaded is then multiplied by 50 to give particles (pt) /mL.
  • Pt/mL divided by GC/mL gives the ratio of particles to genome copies (pt/GC).
  • Pt/mL-GC/mL gives empty pt/mL.
  • Empty pt/mL divided by pt/mL and x 100 gives the percentage of empty particles.
  • the methods include subjecting the treated AAV stock to SDS- polyacrylamide gel electrophoresis, consisting of any gel capable of separating the three capsid proteins, for example, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is separated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon.
  • Anti-AAV capsid antibodies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV capsid monoclonal antibody, most preferably the Bl anti-AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293).
  • a secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detection molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase.
  • a method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • a detection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detection kit.
  • samples from column fractions can be taken and heated in SDS-PAGE loading buffer containing reducing agent (e.g., DTT), and capsid proteins were resolved on pre-cast gradient polyacrylamide gels (e.g., Novex).
  • Silver staining may be performed using SilverXpress (Invitrogen, CA) according to the manufacturer's instructions or other suitable staining method, i.e., SYPRO ruby or coomassie stains.
  • the concentration of AAV vector genomes (vg) in column fractions can be measured by quantitative real time PCR (Q-PCR).
  • Samples are diluted and digested with DNase I (or another suitable nuclease) to remove exogenous DNA. After inactivation of the nuclease, the samples are further diluted and amplified using primers and a TaqManTM fluorogenic probe specific for the DNA sequence between the primers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct) is measured for each sample on an Applied Biosystems Prism 7700 Sequence Detection System. Plasmid DNA containing identical sequences to that contained in the AAV vector is employed to generate a standard curve in the Q-PCR reaction. The cycle threshold (Ct) values obtained from the samples are used to determine vector genome titer by normalizing it to the Ct value of the plasmid standard curve. End-point assays based on the digital PCR can also be used.
  • DNase I or another
  • an optimized q-PCR method which utilizes a broad spectrum serine protease, e.g., proteinase K (such as is commercially available from Qiagen). More particularly, the optimized qPCR genome titer assay is similar to a standard assay, except that after the DNase I digestion, samples are diluted with proteinase K buffer and treated with proteinase K followed by heat inactivation. Suitably samples are diluted with proteinase K buffer in an amount equal to the sample size.
  • the proteinase K buffer may be concentrated to 2-fold or higher. Typically, proteinase K treatment is about 0.2 mg/mL, but may be varied from 0.1 mg/mL to about 1 mg/mL.
  • the treatment step is generally conducted at about 55 °C for about 15 minutes, but may be performed at a lower temperature (e.g., about 37 °C to about 50 °C) over a longer time period (e.g., about 20 minutes to about 30 minutes), or a higher temperature (e.g., up to about 60 °C) for a shorter time period (e.g., about 5 to 10 minutes).
  • heat inactivation is generally at about 95 °C for about 15 minutes, but the temperature may be lowered (e.g., about 70 to about 90 °C) and the time extended (e.g., about 20 minutes to about 30 minutes). Samples are then diluted (e.g., 1000-fold) and subjected to TaqMan analysis as described in the standard assay.
  • droplet digital PCR may be used.
  • ddPCR droplet digital PCR
  • methods for determining single-stranded and self-complementary AAV vector genome titers by ddPCR have been described. See, e.g., M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2): 115-25. doi: 10.1089/hgtb.2013.131. Epub 2014 Feb 14.
  • the manufacturing process for rAAV as described herein involves method as described in US Provisional Patent Application No. 63/371,597, filed August 16, 2022, and US Provisional Patent Application No. 63/371,592, filed August 16, 2022, which are incorporated herein by reference in its entirety.
  • the method for separating rAAVhu68 (or AAVhu95 or AAVhu96) particles having packaged genomic sequences from genome-deficient AAVhu68 (or AAVhu95 or AAVhu96) intermediates involves subjecting a suspension comprising recombinant AAVhu68 (or AVhu95 or AAVhu96) viral particles and AAVhu68 (or AVhu95 or AAVhu96) capsid intermediates to fast performance liquid chromatography, wherein the AAVhu68 (or AVhu95 or AAVhu96) viral particles and AAVhu68 (or AVhu95 or AAVhu96) intermediates are bound to a strong anion exchange resin equilibrated at a pH of about 10.2, and subjected to a salt gradient while monitoring eluate for ultraviolet absorbance at about 260 nanometers (nm) and about 280 nm.
  • the pH may be in the range of about 10 to 10.4.
  • the AAV full capsids are collected from a fraction which is eluted when the ratio of A260/A280 reaches an inflection point.
  • the diafiltered product may be applied to an affinity resin (Life Technologies) that efficiently captures the AAV serotype. Under these ionic conditions, a significant percentage of residual cellular DNA and proteins flow through the column, while AAV particles are efficiently captured.
  • the produced rAAV is suspended in a suitable physiologically compatible composition (e.g., a buffered saline).
  • a suitable physiologically compatible composition e.g., a buffered saline
  • This composition may be frozen for storage, later thawed and optionally diluted with a suitable diluent.
  • the vector may be prepared as a composition which is suitable for delivery to a patient without proceeding through the freezing and thawing steps.
  • NAb titer a measurement of how much neutralizing antibody (e.g., anti- AAV Nab) is produced which neutralizes the physiologic effect of its targeted epitope (e.g., an AAV).
  • Anti -AAV NAb titers may be measured as described in, e.g., Calcedo, R., et al., Worldwide Epidemiology of Neutralizing Antibodies to Adeno-Associated Viruses. Journal of Infectious Diseases, 2009. 199(3): p. 381-390, which is incorporated by reference herein.
  • sc refers to self-complementary.
  • Self-complementary AAV refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template.
  • dsDNA double stranded DNA
  • a “replication-defective virus” or “viral vector” refers to a synthetic or artificial viral particle in which an expression cassette containing a gene of interest is packaged in a viral capsid or envelope, where any viral genomic sequences also packaged within the viral capsid or envelope are replication-deficient; i.e., they cannot generate progeny virions but retain the ability to infect target cells.
  • the genome of the viral vector does not include genes encoding the enzymes required to replicate (the genome can be engineered to be "gutless" - containing only the gene of interest flanked by the signals required for amplification and packaging of the artificial genome), but these genes may be supplied during production. Therefore, it is deemed safe for use in gene therapy since replication and infection by progeny virions cannot occur except in the presence of the viral enzyme required for replication.
  • the capsid protein is a non-naturally occurring capsid.
  • Such an artificial capsid may be generated by any suitable technique, using a selected AAV sequence (e.g., a fragment of a vpl capsid protein) in combination with heterologous sequences which may be obtained from a different selected AAV, noncontiguous portions of the same AAV, from a non-AAV viral source, or from a non- viral source.
  • An artificial AAV may be, without limitation, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a “humanized” AAV capsid.
  • Pseudotyped vectors wherein the capsid of one AAV is replaced with a heterologous capsid protein, are useful in the invention.
  • AAV2/5 and AAV2/8 are exemplary pseudotyped vectors.
  • the selected genetic element may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion.
  • rAAV particles are referred to as DNase resistant.
  • DNase endonuclease
  • other endo- and exo- nucleases may also be used in the purification steps described herein, to remove contaminating nucleic acids.
  • Such nucleases may be selected to degrade single stranded DNA and/or double-stranded DNA, and RNA.
  • Such steps may contain a single nuclease, or mixtures of nucleases directed to different targets, and may be endonucleases or exonucleases.
  • nuclease-resistant indicates that the AAV capsid has fully assembled around the expression cassette which is designed to deliver a gene to a host cell and protects these packaged genomic sequences from degradation (digestion) during nuclease incubation steps designed to remove contaminating nucleic acids which may be present from the production process.
  • a “subpopulation” of vp proteins refers to a group of vp proteins which has at least one defined characteristic in common and which consists of at least one group member to less than all members of the reference group, unless otherwise specified.
  • a “subpopulation” of vpl proteins is at least one (1) vpl protein and less than all vpl proteins in an assembled AAV capsid, unless otherwise specified.
  • a “subpopulation” of vp3 proteins may be one (1) vp3 protein to less than all vp3 proteins in an assembled AAV capsid, unless otherwise specified.
  • vpl proteins may be a subpopulation of vp proteins; vp2 proteins may be a separate subpopulation of vp proteins, and vp3 are yet a further subpopulation of vp proteins in an assembled AAV capsid.
  • vpl, vp2 and vp3 proteins may contain subpopulations having different modifications, e.g., at least one, two, three or four highly deamidated asparagines, e.g., at asparagine - glycine pairs.
  • a pharmaceutical composition comprising a vector as described herein in a formulation buffer.
  • a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer.
  • the rAAV is formulated at about 1 x 10 9 genome copies (GC)/mL to about 1 x 10 14 GC/mL.
  • the rAAV is formulated at about 3 x 10 9 GC/mL to about 3 x 10 13 GC/mL.
  • the rAAV is formulated at about 1 x 10 9 GC/mL to about 1 x 10 13 GC/mL.
  • the rAAV is formulated at least about 1 x 10 11 GC/mL.
  • compositions comprising an rAAV or a vector as described herein and an aqueous suspension media.
  • the suspension is formulated for intravenous delivery, intrathecal administration, or intracerebroventricular administration.
  • the compositions contains at least one rAAV stock and an optional carrier, excipient and/or preservative.
  • a “stock” of rAAV refers to a population of rAAV. Despite heterogeneity in their capsid proteins due to deamidation, rAAV in a stock are expected to share an identical vector genome.
  • a stock can include rAAV having capsids with, for example, heterogeneous deamidation patterns characteristic of the selected AAV capsid proteins and a selected production system.
  • the stock may be produced from a single production system or pooled from multiple runs of the production system. A variety of production systems, including but not limited to those described herein, may be selected.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present invention into suitable host cells.
  • the rAAV vector delivered vector genomes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • a composition in one embodiment, includes a final formulation suitable for delivery to a subject, e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
  • a final formulation suitable for delivery to a subject e.g., is an aqueous liquid suspension buffered to a physiologically compatible pH and salt concentration.
  • one or more surfactants are present in the formulation.
  • the composition may be transported as a concentrate which is diluted for administration to a subject.
  • the composition may be lyophilized and reconstituted at the time of administration.
  • a suitable surfactant, or combination of surfactants may be selected from among non-ionic surfactants that are nontoxic.
  • a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Plutonic® F68 [BASF], also known as Poloxamer 188, which has a neutral pH, has an average molecular weight of 8400.
  • Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of poly oxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Poly oxy capryllic glyceride), poly oxy 10 oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.
  • the formulation contains a poloxamer.
  • Poloxamer 188 is selected.
  • the surfactant may be present in an amount up to about 0.0005 % to about 0.001% (based on weight ratio, w/w %) of the suspension. In another embodiment, the surfactant may be present in an amount up to about 0.0005 % to about 0.001% (based on volume ratio, v/v %) of the suspension. In yet another embodiment, the surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension, wherein n % indicates n gram per 100 mL of the suspension.
  • the composition includes a carrier, diluent, excipient and/or adjuvant.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water.
  • the buffer/carrier should include a component that prevents the rAAV, from sticking to the infusion tubing but does not interfere with the rAAV binding activity in vivo.
  • a suitable surfactant, or combination of surfactants may be selected from among non-ionic surfactants that are nontoxic.
  • a difunctional block copolymer surfactant terminating in primary hydroxyl groups is selected, e.g., such as Poloxamer 188 (also known under the commercial names Pluronic® F68 [BASF], Lutrol® F68, Synperonic® F68, Kolliphor® P188) which has a neutral pH, has an average molecular weight of 8400.
  • Poloxamers may be selected, i.e., nonionic triblock copolymers composed of a central hydrophobic chain of poly oxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)), SOLUTOL HS 15 (Macrogol-15 Hydroxystearate), LABRASOL (Poly oxy capryllic glyceride), poly oxy -oleyl ether, TWEEN (polyoxyethylene sorbitan fatty acid esters), ethanol and polyethylene glycol.
  • the formulation contains a poloxamer.
  • copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content.
  • Poloxamer 188 is selected.
  • the surfactant may be present in an amount up to about 0.0005 % to about 0.001% of the suspension.
  • the composition containing the rAAV comprising a transgene and regulatory control sequences comprising a hybrid cardiac promoter comprising a CMV IE enhancer, a spacer sequence, and a chicken TnT promoter is delivered at a pH in the range of 6.8 to 8, or 7.2 to 7.8, or 7.5 to 8.
  • the composition containing the rAAV comprising a transgene and regulatory control sequences comprising a hybrid cardiac promoter comprising nucleic acid sequence of SEQ ID NO: 3 or a sequence at least 99% identical to SEQ ID NO: 3 is delivered at a pH in the range of 6.8 to 8, or 7.2 to 7.8, or 7.5 to 8.
  • the composition containing the rAAV is delivered intravenously at a pH of about 6.5 to about 7.5 may be desired. In certain embodiments, the composition containing the rAAV is delivered intravenously at a pH of about 6.8 to about 7.2 may be desired. However, other pHs within the broadest ranges and these subranges may be selected for other route of delivery.
  • the formulation may contain a buffered saline aqueous solution not comprising sodium bicarbonate.
  • a formulation may contain a buffered saline aqueous solution comprising one or more of sodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride and mixtures thereof, in water, such as a Harvard’s buffer.
  • the buffer is PBS.
  • compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • compositions according to the present invention may comprise a pharmaceutically acceptable carrier, such as defined above.
  • a pharmaceutically acceptable carrier such as defined above.
  • the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and/or admixed with suitable excipients designed for delivery to the subject via injection, or for delivery by another route and/or device.
  • the dosage of the vector (e.g., rAAV) is about 1 x 10 9 GC/kg mass to about 1 x 10 14 GC/kg, including all integers or fractional amounts within the range and the endpoints.
  • the dosage is adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed.
  • the levels of expression of the transgene product can be monitored to determine the frequency of dosage resulting in viral vectors, preferably AAV vectors containing the minigene.
  • dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention.
  • phrases “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • the term “dosage” or “amount” can refer to the total dosage or amount delivered to the subject in the course of treatment, or the dosage or amount delivered in a single unit (or multiple unit or split dosage) administration.
  • the replication-defective virus compositions can be formulated in dosage units to contain an amount of replication-defective virus that is in the range of about 1.0 x 10 9 GC to about 1.0 x 10 16 GC (to treat an average subject of 70 kg in body weight) including all integers or fractional amounts within the range, and preferably 1.0 x 10 12 GC to 1.0 x 10 14 GC for a human patient.
  • the compositions are formulated to contain at least IxlO 9 , 2xl0 9 , 3xl0 9 , 4xl0 9 , 5xl0 9 , 6xl0 9 , 7xl0 9 , 8xl0 9 , or 9xl0 9 GC per dose including all integers or fractional amounts within the range.
  • the compositions are formulated to contain at least IxlO 10 , 2xlO 10 , 3xl0 10 , 4xlO 10 , 5xl0 10 , 6xlO 10 , 7xlO 10 , 8xl0 10 , or 9x10 10 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least IxlO 11 , 2X10 11 , 3X10 11 , 4X10 11 , SxlO 11 , OxlO 11 , 7X10 11 , SxlO 11 , or 9xlO xl GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least 1x10 , 2x10 , 3x10 , 5xl0 12 , 6xl0 12 , 7xl0 12 , 8xl0 12 , or 9xl0 12 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least IxlO 13 , 2xl0 13 , 3xl0 13 , 4xl0 13 , 5xl0 13 , 6xl0 13 , 7xl0 13 , 8xl0 13 , or 9xl0 13 GC per dose including all integers or fractional amounts within the range.
  • the compositions are formulated to contain at least IxlO 14 , 2xl0 14 , 3xl0 14 , 4x1014, 5xl0 14 , 6xl0 14 , 7xl0 14 , 8xl0 14 , or 9xl0 14 GC per dose including all integers or fractional amounts within the range.
  • compositions are formulated to contain at least IxlO 15 , 2x10 15 , 3xl0 15 , 4xl0 15 , 5xl0 15 , 6xl0 15 , 7xl0 15 , 8xl0 15 , or 9xl0 15 GC per dose including all integers or fractional amounts within the range.
  • the dose can range from IxlO 10 to about IxlO 12 GC per dose including all integers or fractional amounts within the range.
  • compositions in the pharmaceutical composition described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.
  • a method is provided herein is a method of treating a human subject diagnosed with cardiac disease (i.e. , cardiomyopathy). Further provided herein are uses of an rAAV, as described herein, in the manufacture (preparing) of a medicament for the treatment a human subject diagnosed with cardiac disease (i.e., cardiomyopathy).
  • the method comprises administering to a subject a suspension of a vector or an rAAV as described herein.
  • the method comprises administering to a subject a suspension of a rAAV as described herein in a formulation buffer at a dose of about I x lO 9 genome copies (GC)/kg to about I x lO 14 GC/kg.
  • the rAAV is formulated at 3 x 10 13 GC/kg.
  • the methods and compositions described herein may be used for treatment of any of the stages of cardiomyopathy.
  • the patient is an infant, a toddler, or the patient is from 3 years to 6 years of age, from 3 years to 12 years of age, from 3 years to 18 years of age, from 3 years to 20 years of age.
  • patients are older than 18 years of age.
  • the patient is about 20 to 60.
  • the patient is about 40 to 50.
  • patients are older than 60 years of age.
  • the methods and compositions may be used for treatment of mitochondrial cardiomyopathy associated with Barth Syndrome.
  • Barth Syndrome is a rare, X-linked recessive disorder characterized by a loss of function mutation in TAZ gene (i.e., amenable gene therapy).
  • Bart Syndrome is associated with pediatric onset cardiomyopathy (i.e., by age 5) with neutropenia, mild mitochondrial myopathy (skeletal muscle weakness), and mild intellectual impairment. See also, Sabbah, H.N., Barth syndrome cardiomyopathy: targeting the mitochondria with elamipretide, Heart Failure Reviews (2021) 26:237-253, which is incorporated herein by reference in its entirety.
  • the methods and compositions may be used in treatment of autosomal dominant form of long-QT syndrome caused by a loss-of- function and partial dominant negative mutations in KCNQ1 gene (i.e., amenable to gene replacement or knockdown/replace approach).
  • the autosomal dominant form of long-QT syndrome is associated with syncope and sudden cardiac death usually occurring during exercise or emotional stress, and many patients remain at-risk despite standard of care (beta blockers, cardiac sympathetic denervation) and require Implantable Cardioverter Defibrillator (ICD). See also, Huang H., et al., Mechanisms of KCNQ1 channel dysfunction in long QT syndrome involving voltage sensor domain mutations, Sci. Adv. 2018, 4:1-12, epub March 7, 2018, which is incorporated herein by reference in its entirety.
  • the methods and compositions may be used in treatment of hypertrophic cardiomyopathy.
  • the methods and compositions may be used in treatment of hypertrophic cardiomyopathy caused by loss-of-function mutations in the MYBPC3 gene (i.e., amenable to gene therapy). See also, Mearini G., et al., Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice, Nature Communication, 2014, 5:5515, epub December 2, 2014, which is incorporated herein by reference in its entirety.
  • the methods and compositions may be used in treatment of long WT syndrome type 2 caused by a loss-of-function mutation in hERG (Kv 11.1; also, Kv 11.1 voltage-gated potassium channel) gene.
  • hERG Kv 11.1
  • Kv 11.1 also, Kv 11.1 voltage-gated potassium channel
  • Curran ME. et al., A Molecular Basis for Cardiac Arrhythmia: HERG Mutations Cause Long QT Syndrome, Cell, Voi.
  • the methods and compositions may be used for treatment of LMNA cardiomyopathy or a disease caused by loss-of-function mutation in the LMNA gene.
  • LMNA cardiomyopathy or a disease caused by loss-of-function mutation in the LMNA gene.
  • Kang, S., et al. Laminopathies; Mutations on single gene and various human genetic diseases, BMB Rep. 2018; 51(7): 327-337, which is incorporated herein by reference in its entirety.
  • US Provisional Patent Application No. 63/293,680 filed December 24, 2021, which is incorporated herein by reference in its entirety.
  • the methods and compositions may be used for treatment of heart failure, ischemia-reperfusion injury, myocardial infarction, ventricular remodeling, or a disease associated with extracellular superoxide dismutase 3 (SOD3 or EcSOD). See also, US Patent Application Publication No. US20130136729A1, which is incorporated herein by reference in its entirety.
  • the methods and compositions may be used for treatment of myocardial infarction, reduced ejection fraction of the heart or a disease associated with myc transcription factor, cyclin T 1 and cyclin-dependent kinase 9 (CDK9). See also, International Patent Application Publication No. W02020/165603A1, which is incorporated herein by reference in its entirety.
  • the methods and compositions may be used for treatment of heart failure, or heart tissue damage, or degeneration, or a disease associated with cyclin A2 protein. See also, International Patent Application Publication No. W02020/051296A1, which is incorporated herein by reference in its entirety.
  • the methods and compositions may be used for treatment of dilated cardiomyopathy (DCM), a heart failure, a cardiac fibrosis, a heart inflammation, an ischemic heart disease, a myocardial infarction, an ischemic/reperfusion (I/R)-related injuries, a transverse aortic constriction, or a disease associated with YY1 or BMP7 protein.
  • DCM dilated cardiomyopathy
  • a heart failure a cardiac fibrosis
  • a heart inflammation an ischemic heart disease
  • myocardial infarction an ischemic/reperfusion (I/R)-related injuries
  • I/R ischemic/reperfusion
  • the methods and compositions may be used for treatment of dilated cardiomyopathy, or a disease associated with cardiac Apoptosis Repressor with Caspase Recruitment Domain (cARC). See also, International Patent Application Publication No. W02021/016126A1, which is incorporated herein by reference in its entirety.
  • cARC Caspase Recruitment Domain
  • Symptoms of cardiomyopathy or a disease associated with a mutation in a LMNA gene, KCNQ1 gene, MYBPC3 gene, TAZ gene, or hERG gene include atrioventricular (AV) conduction block, atrial fibrillation, arrhythmia including atrial arrhythmia such as atrial flutter and atrial tachycardia, and ventricular arrhythmias including sustained ventricular tachycardias, and ventricular fibrillation (VF) and/or heart failure.
  • AV atrioventricular
  • arrhythmia including atrial arrhythmia such as atrial flutter and atrial tachycardia
  • ventricular arrhythmias including sustained ventricular tachycardias
  • VF ventricular fibrillation
  • the methods and compositions described herein may be used to ameliorate one or more symptoms of cardiomyopathy including increased average life span, and/or reduction in progression towards heart failure.
  • the rAAV or vectors comprise/s a trans gene which encodes for a protein is selected from transgenes associated with cardiomyopathies, such as, e.g., potassium voltage-gated channel subfamily Q member 1 protein (KCNQ1 gene; SEQ ID NO: 14), cardiac myosin binding protein C (MYBPC3 gene, SEQ ID NO: 15), tafazzin (TAZ, SEQ ID NO: 16), Kvll.l voltage-gated potassium channel protein (hERG gene, SEQ ID NO: 17), Lamin A (LMNA gene; SEQ ID NO: 13) and are useful for treating cardiomyopathies and the symptoms thereof.
  • KCNQ1 gene potassium voltage-gated channel subfamily Q member 1 protein
  • MYBPC3 gene cardiac myosin binding protein C
  • TEZ tafazzin
  • hERG gene SEQ ID NO: 17
  • Lamin A LMNA gene
  • co-therapies or co-treatments may be utilized, which comprise co-administration with another active agent.
  • the cotherapy may further comprise administration of beta blockers, angiotensin-converting enzyme (ACE) inhibitors, diuretics.
  • Diuretic agent used may be acetazolamine (Diamox) or other suitable diuretics.
  • the diuretic agent is administered at the time of gene therapy administration.
  • the diuretic agent is administered prior to gene therapy administration.
  • the diuretic agent is administered where the volume of injection is 3 mL.
  • the co-treatment may further comprise implantable cardioverter defibrillators (ICD), pacemakers (PM) and/or cardiac resynchronization therapy (CRT).
  • ICD implantable cardioverter defibrillators
  • PM pacemakers
  • CRT cardiac resynchronization therapy
  • an immunosuppressive co-therapy may be used in a subject in need.
  • Immunosuppressants for such co-therapy include, but are not limited to, a glucocorticoid, steroids, antimetabolites, T-cell inhibitors, a macrolide (e.g., a rapamycin or rapalog), and cytostatic agents including an alkylating agent, an antimetabolite, a cytotoxic antibiotic, an antibody, or an agent active on immunophilin.
  • the immune suppressant may include a nitrogen mustard, nitrosourea, platinum compound, methotrexate, azathioprine, mercaptopurine, fluorouracil, dactinomycin, an anthracy cline, mitomycin C, bleomycin, mithramycin, IL-2 receptor- (CD25-) or CD3-directed antibodies, anti-IL-2 antibodies, ciclosporin, tacrolimus, sirolimus, IFN- P, IFN-y, an opioid, or TNF-a (tumor necrosis factor-alpha) binding agent.
  • the immunosuppressive therapy may be started 0, 1, 2, 3, 4, 5, 6, 7, or more days prior to or after the gene therapy administration.
  • Such immunosuppressive therapy may involve administration of one, two or more drugs (e.g., glucocorticoids, prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e., rapamycin)).
  • drugs e.g., glucocorticoids, prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e., rapamycin
  • Such immunosuppressive drugs may be administrated to a subject in need once, twice or for more times at the same dose or an adjusted dose.
  • Such therapy may involve coadministration of two or more drugs, the (e.g., prednelisone, micophenolate mofetil (MMF) and/or sirolimus (i.e., rapamycin)) on the same day.
  • One or more of these drugs may be continued after gene therapy administration, at the same dose or an adjusted dose.
  • Such therapy may be for about 1 week (7
  • the rAAV as described herein is administrated once to the subject in need. In another embodiment, the rAAV is administrated more than once to the subject in need.
  • “Patient” or “subject”, as used herein interchangeably, means a male or female mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research.
  • the subject of these methods and compositions is a human patient.
  • the subject of these methods and compositions is a male or female human patient.
  • compositions in the method described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.
  • a kit which includes a concentrated vector suspended in a formulation (optionally frozen), optional dilution buffer, and devices and components required for intravenous administration.
  • the kit may additional or alternatively include components for intravenous delivery.
  • the kit provides sufficient buffer to allow for injection. Such buffer may allow for about a 1:1 to a 1:5 dilution of the concentrated vector, or more.
  • higher or lower amounts of buffer or sterile water are included to allow for dose titration and other adjustments by the treating clinician.
  • one or more components of the device are included in the kit.
  • Suitable dilution buffer is available, such as, a saline, a phosphate buffered saline (PBS) or a glycerol/PBS. It should be understood that the compositions in kit described herein are intended to be applied to other compositions, regiments, aspects, embodiments and methods described across the Specification.
  • heterologous as used to describe a nucleic acid sequence or protein means that the nucleic acid or protein was derived from a different organism or a different species of the same organism than the host cell or subject in which it is expressed.
  • heterologous when used with reference to a protein or a nucleic acid in a plasmid, expression cassette, or vector, indicates that the protein or the nucleic acid is present with another sequence or subsequence which with which the protein or nucleic acid in question is not found in the same relationship to each other in nature.
  • the terms “increase” “decrease” “reduce” “ameliorate” “improve” “delay” or any grammatical variation thereof, or any similar terms indication a change means a variation of about 5 fold, about 2 fold, about 1 fold, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5 % compared to the corresponding reference (e.g., untreated control or a subject in normal condition without cardiomyopathy, unless otherwise specified.
  • RNA Ribonucleic acid
  • expression is used herein in its broadest meaning and comprises the production of RNA or of RNA and protein.
  • expression or “translation” relates in particular to the production of peptides or proteins. Expression may be transient or may be stable.
  • administration refers to delivery of composition described herein to a subject.
  • the term “about” refers to a variant of ⁇ 10% from the reference integer and values therebetween, unless otherwise specified.
  • “about” 500 pM includes ⁇ 50 (i.e., 450 - 550, which includes the integers therebetween).
  • the term “about” is inclusive of all values within the range including both the integer and fractions.
  • the term “about” when used to modify a numerical value means a variation of ⁇ 10%, ( ⁇ 10%, e.g., ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ⁇ 7, ⁇ 8, ⁇ 9, ⁇ 10, or values therebetween) from the reference given, unless otherwise specified.
  • E ⁇ # or the term “e+#” is used to reference an exponent.
  • 5E10 or “5el0” is 5 x 10 10 . These terms may be used interchangeably.
  • rAAV comprising GFP gene or a Test Transgene 1 (TT1, encoding for Test Protein 1, TP1) under various hybrid promoters were which were generated and used to evaluate promoter-driven cardiac transgene expression in mice.
  • TT1 Test Transgene 1
  • the rAAV are generated using triple transfection techniques, utilizing (1) a cis plasmid encoding AAV2 rep proteins and the AAVhu68 VP1 cap gene, (2) a cis plasmid comprising adenovirus helper genes not provided by the packaging cell line which expresses adenovirus Ela, and (3) a trans plasmid containing the vector genome for packaging in the AAV capsid. See, e.g., US 2020/0056159.
  • the trans plasmid is designed to contain either the vector genome comprising TT1 encoding for TP1.
  • the vector genome contains an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR at the extreme 5’ and 3’ end, respectively.
  • the ITRs flank the sequences of the expression cassette packaged into the AAV capsid which have sequences encoding a GFP protein or a Test Protein 1 (TP1).
  • the expression cassette further comprises regulatory sequences operably linked to the protein coding sequences, which comprises a hybrid cardiac promoter of SEQ ID NO: 3 (comprising a CMV IE enhancer (SEQ ID NO: 1), a spacer, and a chicken cardiac troponin T (chTnT) (SEQ ID NO: 2)), and a rabbit beta-globin (RBG) poly A.
  • hybrid cardiac promoter comprising enhancer/promoter elements (as described in Table 1 below) for expression levels and cardiac specificity when administered intravenously in mice using AAVhu68 capsid comprising GFP transgene.
  • Our goals were to maintain robust cardiomyocyte expression as achieved with (similar or greater) with CB7 hybrid ubiquitous promoter, to demonstrate conduction system expression, to reduce off- target expression (especially hepatic), to improve production yields of rAAV vectors as compared with rAAV comprising CB7 promoter.
  • rAAV comprising a hybrid cardiac promoter comprising a chicken troponin T (chTnT) promoter showed increased yield in comparison to production yield (e.g., 1 cell stack (CS) prep) of rAAV comprising CB7 promoter.
  • CS cell stack
  • FIG. 2A shows results of the cardiac promoter evaluation in mice, plotted as pg of GFP over pg protein as examined from heart tissue collected at 14 days post treatment with vehicle control, or with rAAVhu68.eGFP vectors comprising CB7 hybrid promoter, MH-MH hybrid promoter (rat a-myosin heavy enhancer with rat a- myosin heavy promoter), TnT/MH-MH hybrid promoter (chicken TnT enhancer with rat a-myosin heavy promoter), CMV-MH hybrid promoter (CMV IE enhancer with rat a-myosin heavy promoter), TnT promoter (chicken TnT promoter), MH/TnT-TnT hybrid promoter (rat a-myosin heavy enhancer with chicken TnT promoter), or CMV- TnT promoter hybrid promoter (CMV IE enhancer with chicken TnT promoter).
  • CMV IE enhancer with chicken TnT promoter CMV- T
  • FIG. 2B shows results of the cardiac promoter evaluation in mice, plotted as pg of GFP over pg protein as examined from liver tissue collected at 14 days post treatment with vehicle control, or with rAAVhu68.eGFP vectors comprising CB7 hybrid promoter, MH-MH hybrid promoter (rat a-myosin heavy enhancer with rat a-myosin heavy promoter), TnT/MH-MH hybrid promoter (chicken TnT enhancer with rat a-myosin heavy promoter), CMV-MH hybrid promoter (CMV IE enhancer with rat a-myosin heavy promoter), TnT promoter(chicken TnT promoter), MH/TnT-TnT hybrid promoter (rat a-myosin heavy enhancer with chicken TnT promoter), or CMV-TnT hybrid promoter (CMV IE enhancer with chicken TnT promoter).
  • CMV IE enhancer with chicken TnT promoter CMV-TnT
  • FIG. 1 A shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP ofNHP heart tissue (septum) collected on day 21 post administration with AAVhu68-eGFP comprising CB7 promoter.
  • FIG. IB shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP ofNHP heart tissue (left ventricle) collected on day 21 post administration with AAVhu68-eGFP comprising CB7 hybrid promoter.
  • FIG. 1 A shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP ofNHP heart tissue (septum) collected on day 21 post administration with AAVhu68-eGFP comprising CB7 promoter.
  • FIG. 1C shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP ofNHP heart tissue (septum) collected on day 21 post administration with AAVhu68-eGFP comprising CMV IE enhancer and chicken TnT promoter.
  • FIG. ID shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-GFP ofNHP heart tissue (left ventricle) collected on day 21 post administration with AAVhu68-eGFP comprising CMV IE enhancer and chicken TnT promoter.
  • EXAMPLE 2 Pilot Study examining efficacy of rAAV comprising TT1 knockout mouse model.
  • test transgene 1 encoding for a test protein 1 - TP1
  • hybrid cardiac promoter comprising CMV IE enhancer and chicken cardiac TnT selective promoter (chTnT).
  • Lamin A knock out (KO) and wild type (WT) newborn littermates were injected intravenously with either vehicle (PBS) or AAVhu68.TTl at a dose of 5 x IO 10 GC (5el0 GC, about 3 x 10 13 GC/kg (3el3 GC/kg)).
  • Mice survival was examined, and body weights were measured throughout study, at the indicated days.
  • liver and heart tissues were collected and analyzed with immunohistochemical staining with anti -TP 1 antibody to examine expression TP1 expression (FIGs. 3A-3C).
  • FIG. 3A shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-TPl of FRG mouse liver tissue, following administration at newborn stage with AAVhu68.TTl intravenously at a dose of 5 x IO 10 GC (approximately 3 x 10 13 GC/kg).
  • FIG. 3B shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-TPl of mouse heart tissue, following administration at newborn stage with vehicle control.
  • FIG. 3A shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-TPl of FRG mouse liver tissue, following administration at newborn stage with AAVhu68.TTl intravenously at a dose of 5 x IO 10 GC (approximately 3 x 10 13 GC/kg).
  • FIG. 3B shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-TPl of mouse heart tissue
  • 3C shows a representative microscopy image from immunohistochemical (IHC) analysis of staining with anti-TPl of mouse heart tissue, following administration at newborn stage with AAVhu68.TTl intravenously at a dose of 5 x IO 10 GC (approximately 3 x 10 13 GC/kg).
  • IHC immunohistochemical

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Abstract

L'invention concerne un AAV recombinant (rAAV) comprenant une capside d'AAV et un génome de vecteur conditionné en son sein, le génome de vecteur comprenant une répétition terminale inversée (ITR) en 5' d'AAV, et une cassette d'expression comprenant un cadre de lecture ouvert (ORF) pour un transgène sous le contrôle d'une séquence régulatrice comprenant un promoteur cardiaque hybride, qui dirige l'expression du transgène dans une cellule cardiaque cible, et une ITR en 3' d'AAV. L'invention concerne également une composition pharmaceutique comprenant un rAAV tel que décrit ici dans une solution tampon, et une méthode de traitement de diverses pathologies cardiaques.
PCT/US2022/082384 2021-12-24 2022-12-24 Compositions et méthodes comprenant un promoteur spécifique du coeur WO2023122804A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2015142941A1 (fr) * 2014-03-17 2015-09-24 Avalanche Biotechnologies, Inc. Composés et procédés pour améliorer l'expression des gènes dans les cônes rétiniens
US20180055908A1 (en) * 2015-02-20 2018-03-01 Institut Pasteur Prevention and/or treatment of hearing loss or impairment

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015142941A1 (fr) * 2014-03-17 2015-09-24 Avalanche Biotechnologies, Inc. Composés et procédés pour améliorer l'expression des gènes dans les cônes rétiniens
US20180055908A1 (en) * 2015-02-20 2018-03-01 Institut Pasteur Prevention and/or treatment of hearing loss or impairment

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Title
CHEN ET AL.: "Enhancing the Utility of Adeno-Associated Virus Gene Transfer through Inducible Tissue-Specific Expression", HUMAN GENE THERAPY METHODS, vol. 24, August 2013 (2013-08-01), pages 270 - 278, XP055348961, DOI: 10.1089/hgtb.2012.129 *

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