WO2023108129A1 - Troponin c (tnnc1) gene therapy using aav vector - Google Patents

Troponin c (tnnc1) gene therapy using aav vector Download PDF

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Publication number
WO2023108129A1
WO2023108129A1 PCT/US2022/081282 US2022081282W WO2023108129A1 WO 2023108129 A1 WO2023108129 A1 WO 2023108129A1 US 2022081282 W US2022081282 W US 2022081282W WO 2023108129 A1 WO2023108129 A1 WO 2023108129A1
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Prior art keywords
polynucleotide
tnnc1
vector
promoter
disorder
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English (en)
French (fr)
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Christopher Dean HERZOG
Chester Bittencort SACRAMENTO
David RICKS
Raj PRABHAKAR
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Spacecraft Seven LLC
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Spacecraft Seven LLC
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Priority to EP22905416.8A priority Critical patent/EP4444893A4/en
Priority to CA3240616A priority patent/CA3240616A1/en
Priority to CN202280090177.8A priority patent/CN118613591A/zh
Priority to JP2024534532A priority patent/JP2024545507A/ja
Priority to US18/718,023 priority patent/US20240335566A1/en
Publication of WO2023108129A1 publication Critical patent/WO2023108129A1/en
Anticipated expiration legal-status Critical
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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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • 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
    • 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
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • TNNC1 located at 3p21.1, encodes cardiac muscle troponin C, the calcium-binding subunit responsible for sensing myofilament Ca 2+ and regulating contraction.
  • Troponin C neutralizes the suppression of the contractile interaction between myosin and actin induced by troponin I-tropomyosin.
  • TNNC1 loss of function (LOF) mutations decrease Ca 2+ sensitivity and binding, leading to dilated cardiomyopathy (DCM).
  • DCM dilated cardiomyopathy
  • TNNC1 gain of function (GOF) mutations increase Ca 2+ sensitivity and binding, leading to hypercontractility and hypertrophic cardiomyopathy (HCM).
  • HCM hypertrophic cardiomyopathy
  • Clinical manifestations of TNNC1 DCM include heart failure (e.g., a mean ejection fraction (EF) of less than 30%), left ventricular dilation, the need for a heart transplant, and risk of sudden cardiac death.
  • the average age of onset of TNNC1 DCM is about 30 years, but it can present earlier and even in pediatric patients.
  • Clinical manifestations of TNNC1 HCM are dyspnea, syncope, angina, arrhythmia, left ventricular hypertrophy (LVH), and left ventricular outflow tract obstruction (LVOTO).
  • FIG.1 shows a diagram illustrating a non-limiting example of a vector genome.
  • FIG.2 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 58.
  • the underlined portion is the expression cassette (SEQ ID NO: 64).
  • FIG.3 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 59.
  • the underlined portion is the expression cassette (SEQ ID NO: 65).
  • FIG.4 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 60.
  • the underlined portion is the expression cassette (SEQ ID NO: 66).
  • FIG.5 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 61.
  • the underlined portion is the expression cassette (SEQ ID NO: 67).
  • FIG.6 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 62.
  • FIG.7 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 57.
  • the MHCK7 promoter as described herein is labelled “Enhancer/MHCK7” in the diagram.
  • FIG.8 shows a diagram illustrating a non-limiting example of a vector genome.
  • the full polynucleotide sequence of the vector genome is SEQ ID NO: 58.
  • FIG.9 shows a western blot (WB) of human TnC protein expression in transduced CHO-Lec2 cells.
  • the WB shows human TnC (top panel) or loading control, GAPDH (bottom panel).
  • Lane 1 is AAV9-MHCK7-TNNC1 (transduced with MOI of 3E5)
  • lane 2 is AAVrh.74- MHCK7-TNNC1 (transduced with MOI of 3E5)
  • lane 3 is AAV9-hTnT-TNNC1 (transduced with MOI of 3E6)
  • lane 4 is AAVrh.74-hTnT-TNNC1 (transduced with MOI of 3E6)
  • lane 5 is a control of non-transduced cells.
  • FIG.10A shows the survival of male treated mice and
  • FIG.10B shows female survival. All AAV-injected animals lived considerably longer than formulation buffer (FB) injected D73N +/- controls.
  • FB comprises Phosphate Buffered Saline (PBS) with 0.01% Pluronic F-68 and does not include an AAV.
  • FIG.11A and FIG.11B show bar graphs illustrating that significant mitigation of the disease-related increase of End Diastolic Diameter was observed in all male AAV-injected groups, with the greatest effect noted in the AAV9-MHCK7 group compared to formulation buffer (FB)-injected D73N +/- control animals (FIG.11A). An apparent but nonsignificant effect was observed in female mice, most notably in the AAV9-hTnT-TNNC1 group (FIG.11B).
  • Statistical analyses One-way ANOVA followed by Tukey’s multiple comparisons test were performed (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001).
  • FIG.12A and FIG.12B show bar graphs illustrating significant mitigation of the disease-related increase in End Systolic Diameter (FIG.12A) in male mice treated with AAV9- MHCK7-TNNC1 and AAVrh.74-TNNC1 compared to FB injected D73N +/- controls was observed.
  • Statistical analyses One-way ANOVA followed by Tukey’s multiple comparisons test were performed (*p ⁇ 0.05, ****p ⁇ 0.0001).
  • FIG.13A and FIG.13B show bar graphs illustrating significant mitigation of the disease-related progression of dilated cardiomyopathy was revealed by greater Ejection Fraction (%) in AAV9-MHCK7-TNNC1 injected animals compared to FB injected D73N +/- control male mice (FIG.13A).
  • Statistical analyses One-way ANOVA followed by Tukey’s multiple comparisons test were performed (*p ⁇ 0.05).
  • FIG.14A and FIG.14B show bar graphs illustrating significant mitigation of the normal progression of dilated cardiomyopathy was revealed by greater Fractional Shortening (%) in AAV9-MHCK7-TNNC1 injected animals compared to FB injected D73N +/- control male mice (FIG.14A).
  • Statistical analyses One-way ANOVA followed by Tukey’s multiple comparisons test were performed (*p ⁇ 0.05).
  • DETAILED DESCRIPTION OF THE INVENTION [0022] The present disclosure provided gene therapy vectors for TNNC1 that deliver a polynucleotide encoding TNNC1 or a functional variant thereof, along with methods of use, and other compositions and methods.
  • the promoter is an MHCK7 promoter. In some embodiments, the AAV vector is an AAV9 vector. In some embodiments, the promoter is an MHCK7 promoter and the AAV vector is an AAV9 vector. In some embodiment, the promoter is an MHCK7 promoter and the AAV vector is a AAVrh.74 vector. In some embodiments, the promoter is a hTNNT2 promoter. In some embodiments, the AAV vector is an AAV9 vector. In some embodiments, the promoter is an hTNNT2 promoter and the AAV vector is an AAV9 vector. In some embodiment, the promoter is an hTNNT2 promoter and the AAV vector is a AAVrh.74 vector.
  • This disclosure further provides methods of treating a disease or disorder in a subject by administering a gene therapy vector of the disclosure.
  • the disease or disorder is TNNC1 DCM or TNNC1 HCM.
  • a polynucleotide encoding a TNNC1 or functional variant thereof may be employed in generating a gene therapy vector.
  • the resulting vector may be employed in treating diseases or disorders, e.g. TNNC1 DCM, TNNC1 HCM or others.
  • DEFINITIONS [0025] The section headings are for organizational purposes only and are not to be construed as limiting the subject matter described to particular aspects or embodiments.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • sequence “identity” may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety).
  • NCBI National Center for Biotechnology Information
  • the percentage can be calculated by optimally aligning the sequence of interest to the reference sequence; comparing the two sequences over the entire length of the reference sequence; determining the number of positions at which the identical amino acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions; dividing the number of matched positions by the total number of positions in the reference sequence adjusted by adding the number of gap positions introduced into the reference sequence in generating the alignment; and multiplying the result by 100 to yield the percentage of sequence identity.
  • thymine (T) and uracil (U) can be considered equivalent.
  • Identity calculation can be performed manually or by the BLAST algorithm.
  • an “AAV vector” or “rAAV vector” refers to a recombinant vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs).
  • AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a plasmid encoding and expressing rep and cap gene products.
  • AAV vectors can be packaged into infectious particles using a host cell that has been stably engineered to express rep and cap genes.
  • an “AAV virion” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector.
  • the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector.”
  • production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle.
  • promoter refers to a polynucleotide sequence capable of promoting initiation of RNA transcription from a polynucleotide in a eukaryotic cell.
  • vector genome refers to the polynucleotide sequence packaged by the vector (e.g., an rAAV virion), including flanking sequences (e.g., in AAV, inverted terminal repeats).
  • flanking sequences e.g., in AAV, inverted terminal repeats.
  • expression cassette and “polynucleotide cassette” refer to the portion of the vector genome between the flanking ITR sequences.
  • “Expression cassette” implies that the vector genome comprises at least one gene encoding a gene product operable linked to an element that drives expression (e.g., a promoter), including any regulatory elements and/or enhancer elements. “Polynucleotide cassette” refers to the portion of the vector genome that comprises at least one gene encoding a gene product operatively linked to an element that drives expression (e.g., a promoter), including any regulatory elements and/or enhancer elements.
  • the term “patient in need” or “subject in need” refers to a patient or subject at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a recombinant gene therapy vector or gene editing system disclosed herein.
  • a patient or subject in need may, for instance, be a patient or subject diagnosed with a disorder associated with heart.
  • a subject may have a mutation in a TNNC1 gene or deletion of all or a part of the TNNC1 gene, or of gene regulatory sequences, that causes aberrant expression of the TNNC1 protein.
  • Subject and “patient” are used interchangeably herein.
  • the subject treated by the methods described herein may be an adult or a child. Subjects may range in age.
  • the term “variant” or “functional variant” refer, interchangeably, to a protein that has one or more amino-acid substitutions, insertions, or deletions compared to a parental protein that retains one or more desired activities of the parental protein.
  • “genetic disruption” refers to a partial or complete loss of function or aberrant activity in a gene.
  • a subject may suffer from a genetic disruption in expression or function in the TNNC1 gene that decreases expression or results in loss or aberrant function of the TNNC1 protein in at least some cells (e.g., cardiac cells) of the subject.
  • “Genetic Disruption” also refers to changes in a gene that lead to gain of function mutations, for example, gain of function mutations in the TNNC1 protein.
  • “treating” refers to ameliorating one or more symptoms of a disease or disorder.
  • the term “preventing” refers to delaying or interrupting the onset of one or more symptoms of a disease or disorder or slowing the progression of TNNC1-related disease or disorder, e.g., TNNC1 DCM and/or TNNC1 HCM.
  • TNNC1 PROTEIN OR POLYNUCLEOTIDE [0038] The present disclosure contemplates compositions and methods of use related to TNNC1 protein.
  • TNNC1 DCM or TNNC1 HCM Various mutations in TNNC1 are known to be associated with TNNC1 DCM or TNNC1 HCM.
  • mutations associated with TNNC1 DCM or TNNC1 HCM include, without limitation: Y5H, A8V, L29Q, A31S, C84Y, E134D, D132N, D145E, I148V, G159D, G159R, M103I, and/or any combination thereof.
  • the native sequence of human TNNC1 is shown below.
  • a TNNC1 isoform has a sequence of 161 amino acid residues (GenBank NP_003271.1): (SEQ ID NO: 1).
  • the TNNC1 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.
  • the TNNC1 protein is encoded by a polynucleotide that comprises a sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2.
  • the TNNC1 protein has no mutations associated with disease.
  • the TNNC1 protein is a wild-type or native TNNC1 protein, e.g. human TNNC1.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) virion, comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an TNNC1 or a functional variant thereof, operatively linked to a promoter.
  • rAAV adeno-associated virus
  • the disclosure provides a recombinant adeno- associated virus (rAAV) virion, comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an TNNC1, operatively linked to a promoter.
  • the TNNC1 protein comprises a polypeptide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.
  • the polynucleotide encoding the TNNC1 may comprise a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: [0042]
  • the polynucleotide sequence encoding the vector genome may comprise a Kozak sequence, including but not limited to (SEQ ID NO: 3). Kozak sequence may overlap the polynucleotide sequence encoding an TNNC1 protein or a functional variant thereof.
  • the vector genome may comprise a polynucleotide sequence (with Kozak underlined) at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: [0043]
  • the Kozak sequence is an alternative Kozak sequence comprising or consisting of any one of: [0044]
  • the vector genome comprises no Kozak sequence.
  • the polynucleotide sequence may be codon-optimized.
  • VECTOR GENOME [0045]
  • the AAV virions of the disclosure comprise a vector genome.
  • the vector genome may comprise an expression cassette (or a polynucleotide cassette for gene-editing applications not requiring expression of the polynucleotide sequence).
  • Any suitable inverted terminal repeats may be used.
  • the ITRs may be from the same serotype as the capsid or a different serotype (e.g., AAV2 ITRs may be used).
  • the 5 ⁇ ITR comprises an AAV ITR.
  • the 5 ⁇ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: (SEQ ID NO: 11).
  • the 5 ⁇ ITR comprises an AAV2 ITR. In some embodiments, the 5 ⁇ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: (SEQ ID NO: 12). [0048] In some embodiments, the 5 ⁇ ITR comprises an AAV ITR. In some embodiments, the 5 ⁇ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: (SEQ ID NO: 13).
  • the 5 ⁇ ITR comprises an AAV ITR. In some embodiments, the 5 ⁇ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: (SEQ ID NO: 14).
  • the 3 ⁇ ITR comprises an AAV ITR. In some embodiments, the 5 ⁇ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: (SEQ ID NO: 15).
  • the 3 ⁇ ITR comprises an AAV2 ITR.
  • the 5 ⁇ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: (SEQ ID NO: 16).
  • the 3 ⁇ ITR comprises an AAV2 ITR.
  • the 5 ⁇ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: [0053]
  • the 3 ⁇ ITR comprises an AAV2 ITR.
  • the 5 ⁇ ITR comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: [0054]
  • the vector genome comprises one or more filler sequences, e.g., at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to: Promoters [0055]
  • the polynucleotide sequence encoding an TNNC1 protein or functional variant thereof is operably linked to a promoter.
  • the promoter is an MHCK7 promoter, which includes enhancer/promoter regions of murine muscle creatine kinase (MCK) and enhancer region of ⁇ -myosin heavy-chain genes.
  • MCK murine muscle creatine kinase
  • PGK phosphoglycerate kinase
  • CAG rabbit beta-globin gene
  • the promoter may be a synthetic promoter.
  • Exemplary synthetic promoters are provided by Schlabach et al. PNAS USA.107(6):2538–43 (2010).
  • a polynucleotide sequence encoding an TNNC1 protein or functional variant thereof is operatively linked to an inducible promoter.
  • An inducible promoter may be configured to cause the polynucleotide sequence to be transcriptionally expressed or not transcriptionally expressed in response to addition or accumulation of an agent or in response to removal, degradation, or dilution of an agent.
  • the agent may be a drug.
  • the agent may be tetracycline or one of its derivatives, including, without limitation, doxycycline.
  • the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically-regulated promoter, a physically-regulated promoter (i.e., a promoter that responds to presence or absence of light or to low or high temperature).
  • Inducible promoters include heavy metal ion inducible promoters (such as the mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and the promoters from T7 phage which are active in the presence of T7 RNA polymerase. This list of inducible promoters is non-limiting.
  • the promoter is a tissue-specific promoter, such as a promoter capable of driving expression in a cardiac cell to a greater extent than in a non-cardiac cell.
  • tissue-specific promoter is a selected from any various cardiac cell-specific promoters including but not limited to, desmin (Des), alpha-myosin heavy chain ( ⁇ -MHC), myosin light chain 2 (MLC-2), cardiac troponin C (cTnC), cardiac troponin T (hTNNT2), muscle creatine kinase (CK) and combinations of promoter/enhancer regions thereof, such as MHCK7.
  • the promoter is a ubiquitous promoter.
  • a “ubiquitous promoter” refers to a promoter that is not tissue-specific under experimental or clinical conditions.
  • the ubiquitous promoter is any one of CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, chicken beta-actin, and human beta-actin promoters.
  • the promoter sequence is selected from Table 3.
  • the promoter comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 21- 35. Table 3
  • promoters are the SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements.
  • HSV tk Herpes Simplex Virus thymidine kinase
  • CMV cytomegalovirus
  • LTR elements various retroviral promoters including LTR elements.
  • a large variety of other promoters are known and generally available in the art, and the sequences of many such promoters are available in sequence databases such as the GenBank database.
  • vectors of the present disclosure further comprise one or more regulatory elements selected from the group consisting of an enhancer, an intron, a poly-A signal, a 2A peptide encoding sequence, a WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element), and a HPRE (Hepatitis B posttranscriptional regulatory element).
  • the vector comprises a CMV enhancer.
  • the vectors comprise one or more enhancers.
  • the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a ⁇ - actin enhancer sequence, or an EF1- ⁇ enhancer sequence.
  • the sequence of the CMV immediate early (IE) enhancer is: (SEQ ID NO: 36).
  • the vector comprises an enhancer that is linked to a promoter.
  • the vector may comprise an MHCK7 promoter and enhancer.
  • the MHCK7 promoter and enhancer is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the following sequence: (SEQ ID NO: 21).
  • the vectors comprise one or more introns.
  • the intron is a rabbit globin intron sequence, a chicken ⁇ -actin intron sequence, a synthetic intron sequence, an SV40 intron, or an EF1- ⁇ intron sequence.
  • the vectors comprise a polyA sequence.
  • the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence.
  • the poly-A signal may be a bovine growth hormone polyadenylation signal (bGHpA).
  • the vectors comprise one or more transcript stabilizing element.
  • the transcript stabilizing element is a WPRE sequence, a HPRE sequence, a scaffold-attachment region, a 3 ⁇ UTR, or a 5 ⁇ UTR.
  • the vectors comprise both a 5 ⁇ UTR and a 3 ⁇ UTR.
  • the vector comprises a 5 ⁇ untranslated region (UTR) selected from Table 4.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 38-48. Table 4
  • the vector comprises a 3 ⁇ untranslated region selected from Table 5.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 49-57. Table 5
  • the vector comprises a polyadenylation (polyA) signal selected from Table 6.
  • the polyA signal comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS 52-56.
  • Table 6 Illustrative vector genomes are depicted in FIGs.1-6 and provided as SEQ ID NOs: 57-62. The expression cassette of each sequence, shown underlined in FIGs.1-6, are SEQ ID NOs: 63-68.
  • the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 57-62, optionally with or without the ITR sequences.
  • the vector genome comprises, consists essentially of, or consists of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 57-62.
  • the disclosure also contemplates expression cassettes of the illustrative vector genomes depicted in FIGs 1-6 and sequences comprising these, e.g., the sequences set forth in SEQ ID NOs: 57-62, but lacking the 5’ and 3’ ITRs, and variants thereof sharing 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the foregoing.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 59.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61.
  • the vector genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 62.
  • the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 63.
  • the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64.
  • the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65.
  • the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 66.
  • the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 67.
  • the expression cassette comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68.
  • ADENO-ASSOCIATED VIRUS VECTOR AND USES THEREOF [0074] Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including two ⁇ 145-nucleotide inverted terminal repeat (ITRs). There are multiple known variants of AAV, also sometimes called serotypes when classified by antigenic epitopes.
  • the nucleotide sequences of the genomes of the AAV serotypes are known.
  • the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077;
  • the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983);
  • the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829;
  • the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829;
  • the AAV-5 genome is provided in GenBank Accession No. AF085716;
  • the complete genome of AAV-6 is provided in GenBank Accession No.
  • AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004).
  • the sequence of the AAVrh.74 genome is provided in U.S. Patent 9,434,928, incorporated herein by reference.
  • Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs.
  • Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes.
  • the two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene.
  • Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.
  • the cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins.
  • a single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). [0075] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic.
  • AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).
  • the AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible.
  • the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA.
  • the rep and cap proteins may be provided in trans.
  • Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.
  • Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral proteins, which mediate cell transduction.
  • Such recombinant viruses may be produced by techniques known in the art, e.g., by transfecting packaging cells or by transient transfection with helper plasmids or viruses.
  • virus packaging cells include but are not limited to HeLa cells, SF9 cells (optionally with a baculovirus helper vector), 293 cells, etc.
  • a Herpesvirus-based system can be used to produce AAV vectors, as described in US20170218395A1. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in W095/14785, W096/22378, U.S. Pat. No.5,882,877, U.S. Pat. No.6,013,516, U.S. Pat.
  • AAV vectors useful in the practice of the present invention can be packaged into AAV virions (viral particles) using various systems including adenovirus-based and helper-free systems.
  • Standard methods in AAV biology include those described in Kwon and Schaffer. Pharm Res. (2008) 25(3):489-99; Wu et al. Mol. Ther. (2006) 14(3):316-27. Burger et al. Mol. Ther. (2004) 10(2):302-17; Grimm et al. Curr Gene Ther. (2003) 3(4):281-304; Deyle DR, Russell DW.
  • AAV DNA in the rAAV genomes may be from any AAV variant or serotype for which a recombinant virus can be derived including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV- 10, AAV-11, AAV- 12, AAV-13 and AAVrh10.
  • Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692.
  • rAAV comprises a self-complementary genome.
  • an rAAV comprising a “self-complementary” or “double stranded” genome refers to an rAAV which has been engineered such that the coding region of the rAAV is configured to form an intra-molecular double-stranded DNA template, as described in McCarty et al.
  • scAAV Self- complementary recombinant adeno-associated virus
  • rAAV comprising a self-complementary genome can only hold about half of that amount ( ⁇ 2.4kb).
  • the rAAV vector comprises a single stranded genome.
  • a “single standard” genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAVs have singled stranded DNA genomes. There have been some indications that rAAVs should be scAAVs to achieve efficient transduction of cells.
  • the present disclosure contemplates, however, rAAV vectors that maybe have singled stranded genomes, rather than self-complementary genomes, with the understanding that other genetic modifications of the rAAV vector may be beneficial to obtain optimal gene transcription in target cells.
  • the present disclosure relates to single-stranded rAAV vectors capable of achieving efficient gene transfer to anterior segment in the mouse eye. See Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE 12(8): e0182473 (2017).
  • the rAAV vector is of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh.74.
  • Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692.
  • Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).
  • the rAAV vector is of the serotype AAV9.
  • said rAAV vector is of serotype AAV9 and comprises a single stranded genome. In some embodiments, said rAAV vector is of serotype AAV9 and comprises a self-complementary genome. In some embodiments, a rAAV vector comprises the inverted terminal repeat (ITR) sequences of AAV2. In some embodiments, the rAAV vector comprises an AAV2 genome, such that the rAAV vector is an AAV-2/9 vector, an AAV-2/6 vector, or an AAV-2/8 vector. [0082] Full-length sequences and sequences for capsid genes for most known AAVs are provided in US Patent No.8,524,446, which is incorporated herein in its entirety.
  • AAV vectors may comprise wild-type AAV sequence or they may comprise one or more modifications to a wild-type AAV sequence.
  • an AAV vector comprises one or more amino acid modifications, e.g., substitutions, deletions, or insertions, within a capsid protein, e.g., VP1, VP2 and/or VP3.
  • the modification provides for reduced immunogenicity when the AAV vector is provided to a subject.
  • Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as endothelial cells or more particularly endothelial tip cells.
  • the rAAV is directly injected into the intracerebroventricular space of the subject.
  • the rAAV virion is an AAV2 rAAV virion.
  • the capsid many be an AAV2 capsid or functional variant thereof.
  • the AAV2 capsid shares at least 98%, 99%, or 100% identity to a reference AAV2 capsid, e.g., (SEQ ID NO: 69).
  • the rAAV virion is an AAV9 rAAV virion.
  • the capsid many be an AAV9 capsid or functional variant thereof.
  • the AAV9 capsid shares at least 98%, 99%, or 100% identity to a reference AAV9 capsid, e.g., (SEQ ID NO: 70).
  • the rAAV virion is an AAV6 rAAV virion.
  • the capsid many be an AAV9 capsid or functional variant thereof.
  • the AAV6 capsid shares at least 98%, 99%, or 100% identity to a reference AAV6 capsid, e.g., (SEQ ID NO: 71).
  • the rAAV virion is an AAVrh.10 rAAV virion.
  • the capsid many be an AAV9 capsid or functional variant thereof.
  • the AAVrh.10 capsid shares at least 98%, 99%, or 100% identity to a reference AAVrh.10 capsid, e.g., (SEQ ID NO: 72).
  • the capsid protein is encoded by a polynucleotide supplied on a plasmid in trans to the transfer plasmid.
  • AAVrh.74 capsid coding sequence [0091]
  • the disclosure further provides protein sequences for AAVrh.74 VP1, VP2, and VP3, including SEQ ID NOs: 74-76, respectively, and homologs or functional variants thereof.
  • the AAVrh.74 capsid comprises the amino acid sequence set forth in SEQ ID NO: 74.
  • the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh.74 VP1 which is set forth in SEQ ID NO: 74.
  • the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh.74 VP2 which is set forth in SEQ ID NO: 75.
  • the rAAV vector comprises a polypeptide that comprises, or consists essentially of, or yet further consists of a sequence, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to amino acid sequence of AAVrh.74 VP3 which is set forth in SEQ ID NO: 76.
  • the rAAV virion is an AAV-PHP.B rAAV virion or a neutrotrophic variant thereof, such as, without limitation, those disclosed in Int’l Pat. Pub. Nos. WO 2015/038958 A1 and WO 2017/100671 A1.
  • the AAV capsid may comprise at least 4 contiguous amino acids from the sequence TLAVPFK (SEQ ID NO: 78) or KFPVALT (SEQ ID NO: 79), e.g., inserted between a sequence encoding for amino acids 588 and 589 of AAV9.
  • the capsid many be an AAV-PHP.B capsid or functional variant thereof.
  • the AAV-PHP.B capsid shares at least 98%, 99%, or 100% identity to a reference AAV-PHP.B capsid, e.g., [0098]
  • AAV capsids used in the rAAV virions of the disclosure include those disclosed in Pat. Pub. Nos. WO 2009/012176 A2 and WO 2015/168666 A2.
  • the present inventors have determined that an AAV9 vector, AAVrh.74, or an AAVrh.10 vector will confer desirable cardiac tropism on the vector.
  • an AAV9 vector, AAVrh.74, or an AAVrh.10 vector may provide desired specificity to cardiac cells.
  • PHARMACEUTICAL COMPOSITIONS AND KITS [0100]
  • the disclosure provides pharmaceutical compositions comprising the rAAV virion of the disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • various solutions can be employed, such as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose.
  • Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as Poloxamer 188, e.g., at 0.001% or 0.01%.
  • a dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
  • the pharmaceutical forms suitable for injectable use include but are not limited to sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form is sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • the disclosure comprises a kit comprising an rAAV virion of the disclosure and instructions for use.
  • the disclosure provides a method of increasing wildtype TNNC1 expression and/or activity in a cell, comprising contacting the cell with an rAAV of the disclosure. In another aspect, the disclosure provides a method of increasing wildtype TNNC1 expression and/or activity in a subject, comprising administering to an rAAV of the disclosure.
  • the cell and/or subject is deficient in TNNC1 messenger RNA or TNNC1 protein expression levels and/or activity and/or comprises a loss-of-function mutation in TNNC1.
  • the cell and/or the subject has a gain of function mutation in TNNC1.
  • the cell and/or the subject has a mutation selected from the group consisting of Y5H, A8V, L29Q, A31S, C84Y, E134D, D132N, D145E, I148V, G159D, G159R, or any combination thereof, relative to a human wildtype TNNC1 gene.
  • the cell may be a cardiac cell, e.g. a cardiomyocyte cell.
  • the method promotes survival of cardiac cell, e.g. a cardiomyocyte cell, in cell culture and/or in vivo. In some embodiments, the method promotes and/or restores function of the heart.
  • treatment with the rAAV virion results in at least two-fold, at least five-fold, at least ten-fold, or more TNNC1 protein levels detectable in cardiac fibroblasts (CFs) in the subject’s heart.
  • treatment with the rAAV virion results in at least two-fold, at least five-fold, at least ten-fold, or more TNNC1 protein levels detectable in cardiomyocytes in the subject’s heart.
  • treatment with the rAAV virion results in at least two-fold, at least five-fold, at least ten-fold, or more TNNC1 protein levels detectable in smooth muscle cells (SMCs) in the subject’s heart.
  • SMCs smooth muscle cells
  • treatment with the rAAV virion results in at least two-fold, at least five-fold, at least ten-fold, or more TNNC1 protein levels detectable in endothelial cells (ECs) in the subject’s heart. In certain embodiments, treatment with the rAAV virion results in at least two-fold, at least five-fold, at least ten-fold, or more TNNC1 protein levels detectable in the epicardium in the subject’s heart. In certain embodiments, treatment with the rAAV virion results in at least two-fold, at least five- fold, at least ten-fold, or more TNNC1 protein levels detectable in the myocardium in the subject’s heart.
  • treatment with the rAAV virion results in at least two- fold, at least five-fold, at least ten-fold, or more TNNC1 protein levels detectable in the endocardium in the subject’s heart METHODS OF TREATMENT [0108]
  • the disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of an rAAV virion of the disclosure.
  • the disease or disorder is a cardiac disease or disorder.
  • Illustrative cardiac disorders include heart failure, TNNC1 DCM, TNNC1 HCM, arrhythmogenic right ventricular cardiomyopathy (ARVC), Brugada syndrome (BrS) and idiopathic ventricular fibrillation, left ventricular non-compaction cardiomyopathy, or restrictive cardiomyopathy, hypertrophic cardiomyopathy.
  • the subject suffers from or is at risk for a TNNC1-related cardiomyopathy (e.g., TNNC1 DCM or TNNC1 HCM).
  • the AAV-mediated delivery of TNNC1 protein to the heart may increase life span, prevent or attenuate cardiac cell degeneration, heart failure, scarring or fibrosis, reduced ejection fraction, arrythmia, exercise intolerance, angina (chest pain), dyspnea (shortness of breath), edema, left ventricular hypertrophy, left ventricular noncompaction, ventricular dilation, syncope, sudden cardiac death, exertional myalgias and cramps.
  • the AAV-mediated delivery of TNNC1 protein to the heart may show improvement from, or prevent normal disease course detected by use of pathological electrocardiogram, echocardiogram, cardiac CT, cardiac MRI, heart biopsy, decrease in paroxysmal ventricular arrhythmias, decrease in sudden cardiac death, and/or decrease in or lack of further development of fibrosis and/or myofibrillar disarray in myocardium.
  • the methods of the disclosure may prevent a decrease in, restore, and/or increase left ventricular ejection fraction (LVEF) and or ejection fraction, percent factional shortening, left ventricular end-systolic dimension (LVESD), and left ventricular end-diastolic dimension (LVEDD), left ventricular outflow tract velocity time integral (LVOT VTI).
  • LVEF left ventricular ejection fraction
  • LVESD left ventricular end-systolic dimension
  • LVEDD left ventricular end-diastolic dimension
  • LVOT VTI left ventricular outflow tract velocity time integral
  • the methods of disclosure result in an increase (e.g., an increase of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 70%, or about 70% to about 100%) in wildtype TNNC1 protein expression in the subject.
  • the methods of the disclosure result in an increase (e.g., an increase of about 5% to about 25%, about 25% to about 50%, about 50% to about 100%, or about 100% to about 200%) in the ratio of wildtype to mutant TNNC1 protein in the subject.
  • the methods disclosed herein may provide efficient biodistribution in the heart. They may result in sustained in expression in all, or a substantial fraction of, cardiac cells, e.g., cardiomyocytes. Notably, the methods disclosed herein may provide long-lasting expression of TNNC1 protein throughout the life of the subject following AAV vector administration.
  • Combination therapies are also contemplated by the invention.
  • the AAV vector is administered at a dose of between about 1 ⁇ 10 12 and 5 ⁇ 10 14 vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered at a dose of between about 1 ⁇ 10 13 and 5 ⁇ 10 14 vg/kg.
  • the AAV vector is administered at a dose of between about 5 ⁇ 10 13 and 3 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered at a dose of between about 5 ⁇ 10 13 and 1 ⁇ 10 14 vg/kg.
  • the AAV vector is administered at a dose of less than about 1 ⁇ 10 12 vg/kg, less than about 3 ⁇ 10 12 vg/kg, less than about 5 ⁇ 10 12 vg/kg, less than about 7 ⁇ 10 12 vg/kg, less than about 1 ⁇ 10 13 vg/kg, less than about 3 ⁇ 10 13 vg/kg, less than about 5 ⁇ 10 13 vg/kg, less than about 7 ⁇ 10 13 vg/kg, less than about 1 ⁇ 10 14 vg/kg, less than about 3 ⁇ 10 14 vg/kg, less than about 5 ⁇ 10 14 vg/kg, less than about 7 ⁇ 10 14 vg/kg, less than about 1 ⁇ 10 15 vg/kg, less than about 3 ⁇ 10 15 vg/kg, less than about 5 ⁇ 10 15 vg/kg, or less than about 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered at a dose of about 1 ⁇ 10 12 vg/kg, about 3 ⁇ 10 12 vg/kg, about 5 ⁇ 10 12 vg/kg, about 7 ⁇ 10 12 vg/kg, about 1 ⁇ 10 13 vg/kg, about 3 ⁇ 10 13 vg/kg, about 5 ⁇ 10 13 vg/kg, about 7 ⁇ 10 13 vg/kg, about 1 ⁇ 10 14 vg/kg, about 3 ⁇ 10 14 vg/kg, about 5 ⁇ 10 14 vg/kg, about 7 ⁇ 10 14 vg/kg, about 1 ⁇ 10 15 vg/kg, about 3 ⁇ 10 15 vg/kg, about 5 ⁇ 10 15 vg/kg, or about 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered at a dose of 1 ⁇ 10 12 vg/kg, 3 ⁇ 10 12 vg/kg, 5 ⁇ 10 12 vg/kg, 7 ⁇ 10 12 vg/kg, 1 ⁇ 10 13 vg/kg, 3 ⁇ 10 13 vg/kg, 5 ⁇ 10 13 vg/kg, 7 ⁇ 10 13 vg/kg, 1 ⁇ 10 14 vg/kg, 3 ⁇ 10 14 vg/kg, 5 ⁇ 10 14 vg/kg, 7 ⁇ 10 14 vg/kg, 1 ⁇ 10 15 vg/kg, 3 ⁇ 10 15 vg/kg, 5 ⁇ 10 15 vg/kg, or 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered systemically at a dose of between about 1 ⁇ 10 12 and 5 ⁇ 10 14 vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered systemically at a dose of between about 1 ⁇ 10 13 and 5 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of between about 5 ⁇ 10 13 and 3 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered systemically at a dose of between about 5 ⁇ 10 13 and 1 ⁇ 10 14 vg/kg.
  • the AAV vector is administered systemically at a dose of less than about 1 ⁇ 10 12 vg/kg, less than about 3 ⁇ 10 12 vg/kg, less than about 5 ⁇ 10 12 vg/kg, less than about 7 ⁇ 10 12 vg/kg, less than about 1 ⁇ 10 13 vg/kg, less than about 3 ⁇ 10 13 vg/kg, less than about 5 ⁇ 10 13 vg/kg, less than about 7 ⁇ 10 13 vg/kg, less than about 1 ⁇ 10 14 vg/kg, less than about 3 ⁇ 10 14 vg/kg, less than about 5 ⁇ 10 14 vg/kg, less than about 7 ⁇ 10 14 vg/kg, less than about 1 ⁇ 10 15 vg/kg, less than about 3 ⁇ 10 15 vg/kg, less than about 5 ⁇ 10 15 vg/kg, or less than about 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered systemically at a dose of about 1 ⁇ 10 12 vg/kg, about 3 ⁇ 10 12 vg/kg, about 5 ⁇ 10 12 vg/kg, about 7 ⁇ 10 12 vg/kg, about 1 ⁇ 10 13 vg/kg, about 3 ⁇ 10 13 vg/kg, about 5 ⁇ 10 13 vg/kg, about 7 ⁇ 10 13 vg/kg, about 1 ⁇ 10 14 vg/kg, about 3 ⁇ 10 14 vg/kg, about 5 ⁇ 10 14 vg/kg, about 7 ⁇ 10 14 vg/kg, about 1 ⁇ 10 15 vg/kg, about 3 ⁇ 10 15 vg/kg, about 5 ⁇ 10 15 vg/kg, or about 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered systemically at a dose of 1 ⁇ 10 12 vg/kg, 3 ⁇ 10 12 vg/kg, 5 ⁇ 10 12 vg/kg, 7 ⁇ 10 12 vg/kg, 1 ⁇ 10 13 vg/kg, 3 ⁇ 10 13 vg/kg, 5 ⁇ 10 13 vg/kg, 7 ⁇ 10 13 vg/kg, 1 ⁇ 10 14 vg/kg, 3 ⁇ 10 14 vg/kg, 5 ⁇ 10 14 vg/kg, 7 ⁇ 10 14 vg/kg, 1 ⁇ 10 15 vg/kg, 3 ⁇ 10 15 vg/kg, 5 ⁇ 10 15 vg/kg, or 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered intravenously at a dose of between about 1 ⁇ 10 12 and 5 ⁇ 10 14 vector genomes (vg) of the AAV vector per kilogram (vg) of total body mass of the subject (vg/kg). In some embodiments, the AAV vector is administered intravenously at a dose of between about 1 ⁇ 10 13 and 5 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of between about 5 ⁇ 10 13 and 3 ⁇ 10 14 vg/kg. In some embodiments, the AAV vector is administered intravenously at a dose of between about 5 ⁇ 10 13 and 1 ⁇ 10 14 vg/kg.
  • the AAV vector is administered intravenously at a dose of less than about 1 ⁇ 10 12 vg/kg, less than about 3 ⁇ 10 12 vg/kg, less than about 5 ⁇ 10 12 vg/kg, less than about 7 ⁇ 10 12 vg/kg, less than about 1 ⁇ 10 13 vg/kg, less than about 3 ⁇ 10 13 vg/kg, less than about 5 ⁇ 10 13 vg/kg, less than about 7 ⁇ 10 13 vg/kg, less than about 1 ⁇ 10 14 vg/kg, less than about 3 ⁇ 10 14 vg/kg, less than about 5 ⁇ 10 14 vg/kg, less than about 7 ⁇ 10 14 vg/kg, less than about 1 ⁇ 10 15 vg/kg, less than about 3 ⁇ 10 15 vg/kg, less than about 5 ⁇ 10 15 vg/kg, or less than about 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered intravenously at a dose of about 1 ⁇ 10 12 vg/kg, about 3 ⁇ 10 12 vg/kg, about 5 ⁇ 10 12 vg/kg, about 7 ⁇ 10 12 vg/kg, about 1 ⁇ 10 13 vg/kg, about 3 ⁇ 10 13 vg/kg, about 5 ⁇ 10 13 vg/kg, about 7 ⁇ 10 13 vg/kg, about 1 ⁇ 10 14 vg/kg, about 3 ⁇ 10 14 vg/kg, about 5 ⁇ 10 14 vg/kg, about 7 ⁇ 10 14 vg/kg, about 1 ⁇ 10 15 vg/kg, about 3 ⁇ 10 15 vg/kg, about 5 ⁇ 10 15 vg/kg, or about 7 ⁇ 10 15 vg/kg.
  • the AAV vector is administered intravenously at a dose of 1 ⁇ 10 12 vg/kg, 3 ⁇ 10 12 vg/kg, 5 ⁇ 10 12 vg/kg, 7 ⁇ 10 12 vg/kg, 1 ⁇ 10 13 vg/kg, 3 ⁇ 10 13 vg/kg, 5 ⁇ 10 13 vg/kg, 7 ⁇ 10 13 vg/kg, 1 ⁇ 10 14 vg/kg, 3 ⁇ 10 14 vg/kg, 5 ⁇ 10 14 vg/kg, 7 ⁇ 10 14 vg/kg, 1 ⁇ 10 15 vg/kg, 3 ⁇ 10 15 vg/kg, 5 ⁇ 10 15 vg/kg, or 7 ⁇ 10 15 vg/kg.
  • administration comprises systemic, local, direct injection, intravenous, intracardiac injection. Administration may be performed by cardiac catheterization.
  • the disclosure provides for local administration and systemic administration of an effective dose of rAAV and compositions of the invention.
  • systemic administration may be administration into the circulatory system so that the entire body is affected.
  • Systemic administration includes parental administration through injection, infusion or implantation.
  • Routes of administration for the compositions disclosed herein include intravenous (“IV”) administration, intraperitoneal (“IP”) administration, intramuscular (“IM”) administration, intralesional administration, or subcutaneous (“SC”) administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, a depot formulation, etc.
  • the methods of the disclosure comprise administering an AAV vector of the disclosure, or pharmaceutical composition thereof by intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic or intracranial administration.
  • administration of an rAAV of the present invention may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal. Administration includes, but is not limited to, injection into the heart.
  • the methods of the disclosure comprise intracardiac delivery.
  • Infusion may be performed using specialized cannula, catheter, syringe/needle using an infusion pump.
  • Administration may comprise delivery of an effective amount of the rAAV virion, or a pharmaceutical composition comprising the rAAV virion, to the heart. These may be achieved, e.g., via intravenous, intramuscular, intraarterial, intrarenal, intraurethral, intracardiac, intracoronary, intramyocardial, intradermal, epidural, subcutaneous, intraperitoneal, intraventricular, ionophoretic or intracranial administration.
  • the compositions of the disclosure may further be administered intravenously.
  • the method of treatment disclosed herein may reduce and/or prevent one or more symptoms including but not limited to ventricular hypertrophy, syncope, chest pain, left ventricular outflow tract obstruction, left ventricular dilation, reduced ejection fraction, systolic dysfunction, NYHA Class III-IV heart failure, ventricular tachycardia, exercise intolerance, angina.
  • EFFECTS OF RAAV ADMINISTRATION [0128]
  • administration of rAAV of the present disclosure may have beneficial effects for the subject.
  • administration of rAAV of the present disclosure may increase survivability of the subject compared to a subject that is not administered the rAAV of the present disclosure.
  • administration of rAAV of the present disclosure increases survivability by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject that is not administered the rAAV of the present disclosure.
  • administration of rAAV of the present disclosure increases survivability by between 1% and 90%, between 20% and 80%, between 30% and 80%, between 40% and 80%, between 50% and 80%, between 1% to 2% , between 2% to 3%, between 3% to 4%, between 4% to 5%, between 5% to 6%, between 6% to 7%, between 7% to 8%, between 8% to 9%, between 9% to 10%, between 10% to 15%, between 15% to 20%, between 20% to 35%, between 25% to 30%, between 30% to 35%, between 35% to 40%, between 40% to 45%, between 45% to 50%, between 50% to 55%, between 55% to 60%, between 60% to 65%, between 65% to 70%, between 70% to 75%, between 75% to 80%, between 80% to 85%, between 85% to 90%, between 90% to 95%, between 95% to 100%, between 100% to 200%, between 200% to 300%, between 300% to 400%, or between 400% to 500% compared to
  • administration of rAAV of the present disclosure prevents a decrease in the ejection fraction in a subject compared to a subject that is not administered the rAAV of the present disclosure.
  • administration of rAAV of the present disclosure prevents a decrease in the ejection fraction by at least about 1% , at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject that is not administered the rAAV of
  • administration of rAAV of the present disclosure prevents a decrease in the ejection fraction by between 1% and 90%, between 20% and 80%, between 30% and 80%, between 40% and 80%, between 50% and 80%, between 1% to 2% , between 2% to 3%, between 3% to 4%, between 4% to 5%, between 5% to 6%, between 6% to 7%, between 7% to 8%, between 8% to 9%, between 9% to 10%, between 10% to 15%, between 15% to 20%, between 20% to 35%, between 25% to 30%, between 30% to 35%, between 35% to 40%, between 40% to 45%, between 45% to 50%, between 50% to 55%, between 55% to 60%, between 60% to 65%, between 65% to 70%, between 70% to 75%, between 75% to 80%, between 80% to 85%, between 85% to 90%, between 90% to 95%, or between 95% to 100% compared to a subject that is not administered the rAAV of the present disclosure.
  • administration of rAAV of the present disclosure prevents a decrease in, restore, and/or increase left ventricular ejection fraction (LVEF) in a subject compared to a subject that is not administered the rAAV of the present disclosure.
  • LVEF left ventricular ejection fraction
  • administration of rAAV of the present disclosure prevents a decrease in the ejection fraction by at least about 1% , at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to a subject that is not administered the rAAV of the present disclosure.
  • administration of rAAV of the present disclosure prevents a decrease in, restore, and/or increase in left ventricular end-systolic dimension (LVESD) in a subject compared to a subject that is not administered the rAAV of the present disclosure.
  • LESD left ventricular end-systolic dimension
  • administration of rAAV of the present disclosure prevents an increase in end-diastolic diameter (EDD) in a subject by at least about 1% , at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject that is not administered the rAAV of the present disclosure.
  • EDD end-diastolic diameter
  • administration of rAAV of the present disclosure prevents a decrease in, restore, and/or increase in left ventricular end-systolic dimension (LVESD) in a subject by between 1% and 90%, between 20% and 80%, between 30% and 80%, between 40% and 80%, between 50% and 80%, between 1% to 2% , between 2% to 3%, between 3% to 4%, between 4% to 5%, between 5% to 6%, between 6% to 7%, between 7% to 8%, between 8% to 9%, between 9% to 10%, between 10% to 15%, between 15% to 20%, between 20% to 35%, between 25% to 30%, between 30% to 35%, between 35% to 40%, between 40% to 45%, between 45% to 50%, between 50% to 55%, between 55% to 60%, between 60% to 65%, between 65% to 70%, between 70% to 75%, between 75% to 80%, between 80% to 85%, between 85% to 90%, between 90% to 95%, between 95% to 100%, between 1% and 90%, between 20% and
  • administration of rAAV of the present disclosure prevents a decrease in, restore, and/or increase in left ventricular end-diastolic dimension (LVEDD) in a subject compared to a subject that is not administered the rAAV of the present disclosure.
  • LVED left ventricular end-diastolic dimension
  • administration of rAAV of the present disclosure prevents an increase in LVPW in a subject by at least about 1% , at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject that is not administered the rAAV of the present disclosure.
  • administration of rAAV of the present disclosure prevents a decrease in, restore, and/or increase in left ventricular end-diastolic dimension (LVEDD) in a subject by between 1% and 90%, between 20% and 80%, between 30% and 80%, between 40% and 80%, between 50% and 80%, between 1% to 2% , between 2% to 3%, between 3% to 4%, between 4% to 5%, between 5% to 6%, between 6% to 7%, between 7% to 8%, between 8% to 9%, between 9% to 10%, between 10% to 15%, between 15% to 20%, between 20% to 35%, between 25% to 30%, between 30% to 35%, between 35% to 40%, between 40% to 45%, between 45% to 50%, between 50% to 55%, between 55% to 60%, between 60% to 65%, between 65% to 70%, between 70% to 75%, between 75% to 80%, between 80% to 85%, between 85% to 90%, between 90% to 95%, between 95% to 100%,
  • administration of rAAV of the present disclosure prevents a decrease in, restore, and/or increase in left ventricular outflow tract velocity time integral (LVOT VTI) in a subject compared to a subject that is not administered the rAAV of the present disclosure.
  • LVOT VTI left ventricular outflow tract velocity time integral
  • administration of rAAV of the present disclosure prevents an increase in LVPW in a subject by at least about 1% , at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% compared to a subject that is not administered the rAAV of the present disclosure.
  • administration of rAAV of the present disclosure prevents a decrease in, restore, and/or increase in left ventricular outflow tract velocity time integral (LVOT VTI) in a subject by between 1% and 90%, between 20% and 80%, between 30% and 80%, between 40% and 80%, between 50% and 80%, between 1% to 2% , between 2% to 3%, between 3% to 4%, between 4% to 5%, between 5% to 6%, between 6% to 7%, between 7% to 8%, between 8% to 9%, between 9% to 10%, between 10% to 15%, between 15% to 20%, between 20% to 35%, between 25% to 30%, between 30% to 35%, between 35% to 40%, between 40% to 45%, between 45% to 50%, between 50% to 55%, between 55% to 60%, between 60% to 65%, between 65% to 70%, between 70% to 75%, between 75% to 80%, between 80% to 85%, between 85% to 90%, between 90% to 95%, between 95% to 100%, between 100%
  • EXAMPLE 1 PRE-CLINICAL BIOACTIVITY AND EFFICACY [0141]
  • AAV vectors or respective expression cassettes are tested in vitro using cultured cardiomyocytes (e.g., induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from patients or primary cardiomyocytes collected from animal models) or other cells amenable to transfection or transduction with these constructs.
  • iPSC-CMs induced pluripotent stem cell cardiomyocytes
  • TNNC1 is assessed by ELISA, immunofluorescence, immunohistochemistry, and Western blot.
  • Vector DNA is detected by PCR and TNNC1 transgene mRNA is detected by qRT-PCR.
  • TNNC1 transgene either following AAV vector transduction and/or transfection with vector plasmids
  • Selected vectors are tested in vivo using mutant mouse models of cardiomyopathy.
  • a D73N +/- knock-in mouse model exhibits severe DCM phenotype. This mouse model exhibits one or more DCM elements of human disease.
  • the D73N +/- knock-in mouse (described in, e.g., McConnell et al. Front.
  • Physiol.2015; 6:242 has a mutation in the regulatory N-domain of cardiac troponin C (cTnC) which increases the rate of Ca 2+ dissociation and reduces Ca 2+ sensitivity.
  • the mechanism of this phenotype is caused by the substitution of an acidic Asp (D) with neutral Asn (N) in the X position of the second Ca 2+ binding loop.
  • D acidic Asp
  • N neutral Asn
  • the D73N +/- knock-in mouse recapitulates the DCM phenotype starting at 4 weeks with increased left ventricular (LV) size with thinner walls and fibrosis observed at 12 weeks.
  • DCM phenotypes observed include left ventricular ejection fraction (LVEF) reduced to ⁇ 28% at 4 weeks, reduced fractional shortening (FS), impaired LV systolic function, and prolonged QRS and QT intervals.
  • LVEF left ventricular ejection fraction
  • FS reduced fractional shortening
  • impaired LV systolic function impaired LV systolic function
  • prolonged QRS and QT intervals The mouse model exhibits a reduction in survival by 6 weeks with a median survival of ⁇ 12 weeks and 100% mortality by 19 weeks.
  • Table 7 An inducible I61Q +/- knock-in mouse model exhibits moderately severe DCM phenotype. This mouse model exhibits one or more DCM elements of human disease.
  • the I61Q +/- knock-in mouse (described in, e.g., Davis et al., Cell, 2016; 165(5):1147-1159) has a doxycycline inducible cardiac single amino acid variant of cTnC.
  • the I61Q +/- knock-in mouse recapitulates the DCM phenotypes including reduced cardiac function, reduced Ca 2+ binding and tension causing eccentric hypertrophy and LV dilation, increased diastolic LV chamber dimension, decreased septal wall thickness, increased cardiac mass, increased myocyte length-to- width ratios, and heart failure observed at ⁇ 6 weeks.
  • the mouse model exhibits 50% survival at ⁇ 3 months, 25 -30% survival at 4 months, and 100% mortality by 8 months.
  • Benefit of AAV-mediated TNNC1 expression is evidenced by increase in survival, mitigate decrease in body weight, mitigation of the normal progression of cardiomyopathy (e.g., TNNC1 DCM or TNNC1 HCM) observed on echocardiograms from left and/or right ventricle (e.g., LVESD, LVEDD), mitigation of enlarged size of right and/or left ventricle and/or mitigation of typical decrease in left ventricular ejection fraction and/or fractional shortening in the mouse models.
  • cardiomyopathy e.g., TNNC1 DCM or TNNC1 HCM
  • LVESD left and/or right ventricle
  • Electrophysiological evidence of functional benefit of AAV-mediated delivery of TNNC1 protein is demonstrated by mitigation of disease-related disrupted calcium dynamics in affected cardiomyocytes, most notably on measures of L-type calcium current, sarcoplasmic reticulum calcium leak, diastolic calcium leak, as well as standard measures of calcium transients in affected (e.g., TNNC1-deficient) cardiomyocytes such as time to peak amplitude and relaxation time constants. For example, aberrant Ca 2+ sensitivity in force generation of the myocytes may become more similar to that observed in a healthy control.
  • Histological analyses reveals the benefit by diminished appearance of disease-related myofiber disarray and/or fibrosis, hypertrophy, apoptotic cells, reduction in the ⁇ H2AX marker of DNA damage and reduction in disease-related change in atrial size and absolute size of heart. Additionally, benefit may also be revealed by diminished or normalized ⁇ -myosin heavy chain levels, B-type natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and MYH7 levels in the myocardium relative to non AAV-TNNC1 treated, diseased controls. Benefit may also be revealed through cardiac histopathology analysis.
  • BNP B-type natriuretic peptide
  • ANP atrial natriuretic peptide
  • MYH7 MYH7
  • EXAMPLE 2 PRE-CLINICAL TRANSGENE EXPRESSION
  • FIG.7 and FIG.8 Expression cassettes illustrated in FIG.7 and FIG.8 were tested.
  • AAV vectors or respective plasmid expression cassettes were tested in vitro using cultured CHO-Lec2 (mutant cells that have a 70–90% deficiency of sialic acid in their glycoproteins and gangliosides that make this cell more susceptible to AAV9 transduction).
  • Subsequent expression of human Cardiac Troponin C transgene protein (TnC) in transduced CHO-Lec2 cells was assessed by Western blot (FIG.9).
  • TnC Cardiac Troponin C transgene protein
  • FIG.9 Western blot
  • the D73N +/- knock-in mouse model exhibits a severe dilated cardiomyopathy (DCM) phenotype
  • DCM severe dilated cardiomyopathy
  • This mouse model exhibits one or more DCM elements of human disease.
  • the D73N +/- knock-in mouse (described in, e.g., McConnell et al. Front. Physiol.2015; 6:242) has a mutation in the regulatory N-domain of TNNC1 which increases the rate of Ca 2+ dissociation and reduces Ca 2+ sensitivity.
  • the mechanism of this phenotype is caused by the substitution of an acidic Asp (D) with neutral Asn (N) in the X position of the second Ca 2+ binding loop.
  • the D73N +/- knock-in mouse recapitulates the DCM phenotype starting at 4 weeks of age with increased left ventricular (LV) size, thinner ventricular walls, and subsequent fibrosis observed at 12 weeks of age. Additional markers of a DCM phenotype observed in this mouse model include reduced left ventricular ejection fraction (LVEF) by ⁇ 28% at 4 weeks, reduced fractional shortening (FS), impaired LV systolic function, and prolonged QRS and QT intervals.
  • This mouse model also exhibits a reduction in survival that may emerge as early as 6 weeks of life with a median survival of ⁇ 12 weeks and 100% mortality by 21-22 weeks (a variable which may be influenced experimentally by level of stress caused by frequency of handling the animal).
  • Echocardiography revealed significant benefit of AAV-mediated overexpression of human TnC on cardiac function, evidenced by reduced end diastolic diameter (EDd) in all AAV injected male groups compared to FB injected D73N +/- controls (FIG.11A) and reduced end systolic diameter (ESd) in AAV9-MHCK7-TNNC1 and AAVrh74-MHCK7-TNNC1 treated males compared to FB injected D73N +/- controls (FIG. 12A).
  • EDd end diastolic diameter
  • ESd reduced end systolic diameter

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JP2024545507A (ja) 2024-12-09
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