WO2022029543A1 - Administration par vecteur de virus adéno-associé de micro-dystrophine pour traiter la dystrophie musculaire - Google Patents

Administration par vecteur de virus adéno-associé de micro-dystrophine pour traiter la dystrophie musculaire Download PDF

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WO2022029543A1
WO2022029543A1 PCT/IB2021/056698 IB2021056698W WO2022029543A1 WO 2022029543 A1 WO2022029543 A1 WO 2022029543A1 IB 2021056698 W IB2021056698 W IB 2021056698W WO 2022029543 A1 WO2022029543 A1 WO 2022029543A1
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seq
promoter
aav
spc5
muscle
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PCT/IB2021/056698
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English (en)
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Ajit Gupta
Pankti JASANI
Niraj Mishra
Lakshmikanth Gandikota
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Intas Pharmaceuticals Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C07K14/4707—Muscular dystrophy
    • C07K14/4708—Duchenne dystrophy
    • 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
    • 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
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the present invention relates to gene therapy vectors which are useful in the treatment of dystrophic diseases, especially Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD).
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy
  • Muscular dystrophy is a collection of heterogeneous genetic disorders characterized by weak skeletal muscles and their progressive degeneration.
  • Duchenne Muscular Dystrophy is one of the most common forms of muscular dystrophy, affecting 1 in every 3500-5000 live male births arising due to mutations in the DMD gene which encodes for Dystrophin protein.
  • BMD Becker muscular dystrophy
  • MD1, A3990 and mDys5R extremely truncated microdystrophin genes
  • AAV Adeno- associated virus
  • the current invention relates to development of an AAV based viral vector system containing an expression cassette of micro-dystrophin (abbreviated as p-dys like MD1 and A3990) that is packaged in the optimized viral capsids for muscle tissue tropism (i.e. different serotypes of AAV; AAV9 and AAVpol).
  • p-dystrophin abbreviated as p-dys like MD1 and A3990
  • Such vector can be used for the treatment of DMD and Becker’s Muscular Dystrophy (BMD).
  • the principal object of the present invention is to provide recombinant AAV vector comprising polynucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.
  • Another object of the present invention is to provide recombinant AAV vector comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein under regulation of muscle-specific control element.
  • Another object of the present invention relates to viral genome of recombinant AAV particle which comprises: i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin (MD1 and A3990); and iv) SV 40 Poly A transcription terminator.
  • ITR inverted terminal repeat
  • object functional micro-dystrophin is MD1. In another object functional microdystrophin is A3990.
  • Another object of the present invention relates to viral genome of recombinant AAV particle comprising nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding: i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5-
  • Another object of the present invention is to provide pharmaceutical formulation comprising:
  • AAV vector genome comprising nucleotide sequence encoding microdystrophin gene (MD1 and A3990);
  • a pharmaceutically acceptable carrier diluents, excipients, or buffer.
  • Another object of the present invention aims at alleviating or curing devastating Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide called microdystrophin gene (MD1 and A3990).
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy
  • Another object of the present invention is to provide a method of treatment for muscular dystrophy or dystrophinopathy comprises administering a therapeutically effective amount of the recombinant AAV vector containing nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.
  • the principal aspect of the present invention is to provide recombinant AAV vector comprising polynucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.
  • Another aspect of the present invention is to provide recombinant AAV vector comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein under regulation of muscle-specific control element.
  • Another aspect of the present invention relates to viral genome of recombinant AAV particle which comprises; i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990) gene; iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin (MD1 and A3990); and iv) SV 40 Poly A transcription terminator.
  • ITR inverted terminal repeat
  • functional micro-dystrophin is MD1. In another aspect functional microdystrophin is A3990.
  • Another aspect of the present invention relates to viral genome of recombinant AAV particle comprising nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding: i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5-
  • AAV vector genome comprising nucleotide sequence encoding microdystrophin gene (MD1 and A3990);
  • a pharmaceutically acceptable carrier diluents, excipients, or buffer.
  • Another aspect of the present invention aims at alleviating or curing devastating Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide called micro dystrophin (MD1 and A3990).
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy
  • Another aspect of the present invention is to provide a method of treatment for muscular dystrophy or dystrophinopathy comprises administering a therapeutically effective amount of the recombinant AAV vector containing nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.
  • Figure 1 Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-MD1 with its transgene and other regulatory elements.
  • Figure 2 Schematic representation of (A) vector map & (B) cassette of plntas- hMHCK7-MDl with its transgene and other regulatory elements.
  • Figure 3 Schematic representation of (A) vector map & (B) cassette of plntas-Sk- CRM4-mDes-MDl with its transgene and other regulatory elements.
  • Figure 4 Schematic representation of (A) vector map & (B) cassette of plntas-Sk- CRM4-hDes-MDl with its transgene and other regulatory elements.
  • Figure 5 Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-hDes-MDl with its transgene and other regulatory elements.
  • Figure 6 Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-hCK-MDl with its transgene and other regulatory elements.
  • Figure 7 Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-A3990 with its transgene and other regulatory elements.
  • Figure 8 Schematic representation of (A) vector map & (B) cassette of plntas- hMHCK7-A3990 with its transgene and other regulatory elements.
  • Figure 9 Schematic representation of (A) vector map & (B) cassette of plntas-Sk- CRM4-mDes-A3990 with its transgene and other regulatory elements.
  • Figure 10 Schematic representation of (A) vector map & (B) cassette of plntas-Sk- CRM4-hDes-A3990 with its transgene and other regulatory elements.
  • Figure 11 Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-hDes-A3990 with its transgene and other regulatory elements.
  • Figure 12 Schematic representation of (A) vector map & (B) cassette of p!ntas-SPc5- 12-hCK-A3990 with its transgene and other regulatory elements.
  • Figure 13 Schematic representation of (A) vector map & (B) cassette of plntas-ACG- R2C9wt.
  • Figure 14 Schematic representation of (A) vector map & (B) cassette of plntas-ACG- R2Cpolwt.
  • Figure 15 Determination of Promoter Activity: Comparison of a Specific Promoter Activity amongst Different Cell Lines, viz. C2C12, RCDMD, HEK293FT and Huh- 7. Black dotted line represents 100% Flue activity cutoff derived under the regulation of CMV promoter.
  • Figure 16 Determination of Promoter Activity: Comparison of Activity of All Promoters within a Specific Cell Line to Understand the Cell Line Specific Activity. Black dotted line represents 100% Flue activity cutoff derived under the regulation of CMV promoter.
  • the rAAV vectors are designed to express microdystrophin (MD1 and A3990) protein in mammalian, and more particularly, human cells. These micro-dystrophin (MD1 and A3990) are particularly well suited for treatment of DMD and BMD. As described herein, rAAV micro-dystrophin (MD1 and A3990) constructs have been developed which have demonstrated high yield, expression levels, and/or activity.
  • wild-type serotypes There are several naturally occurring (“wild-type”) serotypes and over 100 known variants of AAV, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid proteins, and thus in their gene delivery properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.
  • AAV Adeno-associated virus
  • viruses including, without limitation, the virus itself and derivatives thereof. Except where otherwise indicated, the terminology refers to all subtypes or serotypes and both replication-competent and recombinant forms.
  • AAV includes, without limitation, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3 A (AAV-3 A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), AAV type 10 (AAV- 10 or AAV10 or AAVrhlO or AAV rhlO or AAV-rhlO), avian AAV, bovine AAV, porcine AAV like AAVpol, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV.
  • Primary AAV refers to AAV that infect prima
  • AAV vector refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs).
  • ITRs AAV terminal repeat sequences
  • AAV refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.
  • AAV "rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation/capsid proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes.”
  • helper virus refers to a virus that allows efficient AAV (e.g. wild-type AAV) replication and packaging in a mammalian cell.
  • a variety of such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses and poxviruses such as vaccinia.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC.
  • Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • RNA refers to a polynucleotide that performs a function of some kind in the cell.
  • a gene can contain an open reading frame that is capable of encoding a gene product.
  • a gene product is a protein, which is transcribed and translated from the gene.
  • RNA e.g. a functional RNA product, e.g., an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA for nucleases, etc., which is transcribed but not translated.
  • operatively linked refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner.
  • a promoter is operatively linked to a coding region if the promoter initiates transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • pharmaceutically acceptable refers to compounds and compositions which are suitable for administration to humans and/or animals without undue adverse side effects such as toxicity and/or irritation and/or allergic response and commensurate with a reasonable benefit/risk ratio.
  • the principal embodiment of the present invention is to provide recombinant AAV vector comprising polynucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.
  • Another embodiment of the present invention is to provide recombinant AAV vector comprising a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein under the regulation of muscle-specific control element.
  • Another embodiment of the present invention relates to viral genome of recombinant AAV particle which comprises; i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5- 12-hCK promoter which drives transcription of micro-dystrophin gene (MD1 and A3990); and iv) SV 40 Poly A transcription terminator.
  • ITR inverted terminal repeat
  • functional micro-dystrophin is MD1. In another embodiment functional micro-dystrophin is A3990.
  • Another embodiment of the present invention relates to viral genome of recombinant AAV particle comprising nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding: i) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence; ii) Functional micro-dystrophin gene (MD1 and A3990); iii) Muscle-specific expression control element selected from SPc5-12 promoter or hMHCK7 promoter or Sk-CRM4-mDes or Sk-CRM4-hDes or SPc5-12-hDes or SPc5-
  • AAV vector genome comprising nucleotide sequence encoding microdystrophin gene (MD1 and A3990);
  • a pharmaceutically acceptable carrier diluents, excipients, or buffer.
  • Another embodiment of the present invention aims at alleviating or curing the devastating Duchenne muscular dystrophy (DMD) as well as Becker muscular dystrophy (BMD) by expressing a shorter but functional dystrophin polypeptide called micro dystrophin (MD1 and A3990).
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy
  • Another embodiment of the present invention is to provide a method of treatment for muscular dystrophy or dystrophinopathy comprises administering a therapeutically effective amount of the recombinant AAV vector containing nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 encoding a functional microdystrophin protein.
  • said sequence can be an isolated nucleic acid encoding a microdystrophin having substantial homology or identity (60%, 70%, 80%, 90% 95% or even 99%) to the peptides disclosed herein, especially of sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12.
  • the nucleotide sequence of an isolated nucleic acid encoding a peptide of the invention is “substantially homologous/identical”, that is, about 60% homologous/identical , more preferably about 70% homologous/identical, even more preferably about 80% homologous/identical, more preferably about 90% homologous/identical, even more preferably, about 95% homologous/identical, and even more preferably about 97%, 98% or even 99% homologous/identical to a nucleotide sequence of an isolated nucleic acid encoding the functional microdystrophin, especially of sequence SEQ ID NO: 01 or SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or SEQ ID NO: 05 or SEQ ID NO: 06 or SEQ ID NO: 07 or SEQ ID NO: 08 or SEQ ID NO: 09 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12.
  • ITRs Vectors having ITRs from a different source than its capsid are termed "pseudotyped".
  • inverted terminal repeats (ITR) from AAV2 may be selected for generation of pseudotyped AAV and in certain embodiments, ITRs from a source other than AAV2 may be selected for this construct to generate another pseudotyped AAV.
  • ITRs from the same source as the capsid may be selected.
  • any homologous or heterologous combination of AAV ITRs - AAV capsid could be used to generate recombinant AAV or pseudotyped recombinant AAV.
  • ITRs may be selected to generate a self-complementary AAV, such as defined infra.
  • DNA sequence packaged inside the virus contains ITR sequences at 5’ and 3’ end along with different muscle-specific promoters described as follows:
  • SPc5-12 promoter is a synthetic muscle-specific promoter containing enhancer element with binding sites for the various skeletal muscle-specific transcription factors like MEF-1, MEF-2, TEF-1 and SRE followed by chicken alpha-actin promoter (reported in Patent US6410228B1).
  • hMHCK7 promoter is a hybrid promoter consisting of enhance and promoter elements of human muscle creatine kinase (hCK) and mouse alpha-myosin heavychain gene (MyHC), respectively (reported in Trask et al., JBC, 1988 and Patent application W02018170408A1).
  • Sk-CRM4-mDes promoter contains muscle-specific transcription factor binding sites for EF2, CEBP, LRF, MyoD and SREBP followed by mouse desmin enhancer and promoter (reported in Patent application WO2015110449).
  • Sk-CRM4-hDes promoter contains muscle-specific transcription factor binding sites for EF2, CEBP, LRF, MyoD and SREBP followed by human desmin enhancer and promoter (reported in Li and Paulin, JBC, 1991 and Patent application WO2015110449).
  • SPc5-12-hDes promoter contains enhancer element with binding sites for the various skeletal muscle-specific transcription factors like MEF-1, MEF-2, TEF-1 and SRE followed by human desmin (hDes) promoter (reported in Li and Paulin, JBC, 1991 and Patent US6410228B1).
  • SPc5-12-hCK promoter contains enhancer element with binding sites for the various skeletal muscle-specific transcription factors like MEF-1, MEF-2, TEF-1 and SRE followed by hCK promoter (reported in Trask et al., JBC, 1988 and Patent US6410228B1).
  • microdystrophin All promoters are downstream linked with intron and Kozak sequence followed by the nucleotide sequence encoding microdystrophin (MD1 or A3990) along with SV40 Poly (A) transcription termination sequence.
  • the construct is intended to produce a fully functional microdystrophin polypeptide sequence; MD1 encoding N-terminal Actin-Binding Domain, Hinge regions - Hl , H2 and H4 and Rod domains - R1 -3 and R24, Cys-rich region and A3990 encoding N-terminal Actin-Binding Domain, Hinge regions - Hl, H3 and H4 and Rod domains - Rl-2 and R22-24, and Cys-rich region; that will provide continuous source of functional microdystrophin in the muscle tissues of DMD patients, augmenting with non- functional dystrophin protein.
  • the expression cassettes can be carried on any suitable vector, e.g. a plasmid, which is delivered either to a packaging host cell (encoding rep and cap of AAV) along with helper plasmid or helper virus carrying adenovirus helper element or a mammalian cell line along with helper and packaging plasmids (transient triple transfection).
  • a plasmid which is delivered either to a packaging host cell (encoding rep and cap of AAV) along with helper plasmid or helper virus carrying adenovirus helper element or a mammalian cell line along with helper and packaging plasmids (transient triple transfection).
  • the plasmids useful may be engineered such that they are suitable for replication and packaging in prokaryotic cells, mammalian cells, or both. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art.
  • transducing plasmid vector constructs (1) pIntas-SPc5-12-MDl, (2) plntas- SPc5-12-A3990, (3) pIntas-hMHCK7-MDl, (4) pIntas-hMHCK7-A3990, (5) plntas- Sk-CRM4-mDes-MDl, (6) pIntas-Sk-CRM4-mDes-A3990, (7) pintas- Sk-CRM4- hDes-MDl, (8) pintas- Sk-CRM4-hDes-A3990, (9) pIntas-SPc5-12-hDes-MDl, (10) pIntas-SPc5-12-hDes-A3990, (11) P Intas-SPc5-12-hCK-MDl and (12) pIntas-SPc5- 12-hCK-A3990 were synthesized by combining six different
  • a gene cassette was synthesized and cloned in the vector backbone (reporter plasmid and micro-dystrophin (pDys) variants plasmid MD1 or A3990) carrying a high-copy- number functional origin of replication for E. coli and kanamycin resistance gene as selection marker by GenScript in trans-conformation that comprises:
  • Cassette has one of the genetic promoters selected from A to F as mentioned below:
  • A. SPc5-12 Promoter A chimeric synthetic muscle-specific promoter (as disclosed in US6410228B1) that contains enhancer element with binding sites for the various skeletal muscle-specific transcription factors like MEF-1, MEF-2, TEF-1 and SRE followed by chicken alpha-actin promoter.
  • B. SPc5-12-hDes Promoter A chimeric synthetic muscle-specific promoter (as described in Li and Paulin, JBC, 1991 and US6410228B1) that contains enhancer element of SPc5-12 followed by human desmin (hDes) promoter.
  • C. SPc5-12-hCK Promoter A chimeric synthetic promoter (as described in Trask et al., JBC, 1988 and US6410228B1) that contains that contains enhancer element of SPc5-12 followed by human muscle creatine kinase (hCK) promoter.
  • D. hMHCK7 Promoter A chimeric promoter (as described Trask et al., JBC, 1988 and W02018170408A1) that contains enhancer element of hCK followed by alphamyosin heavy-chain gene (MyHC) promoter.
  • MyHC alphamyosin heavy-chain gene
  • E. Sk-CRM4-mDes Promoter A chimeric synthetic muscle-specific promoter (as described in WO2015110449) that contains muscle-specific transcription factor binding sites for EF2, CEBP, LRF, MyoD and SREBP followed by mouse desmin enhancer and promoter.
  • Sk-CRM4-hDes Promoter A chimeric synthetic muscle-specific promoter (as described in Li and Paulin, JBC, 1991 and WO2015110449) that contains musclespecific transcription factor binding sites for EF2, CEBP, LRF, MyoD and SREBP followed by human desmin enhancer and promoter.
  • Chimeric Intron A chimera between introns from human P-globin and immunoglobulin heavy chain genes (As described in US6461606).
  • Coding sequence Coding sequences for micro-dystrophin (pDys) variants MD1 or A3990 or reporter genes i.e. synthetic luc2 version of the firefly luciferase gene FLuc (Genbank Accession number MK484108.1) and enhanced Green fluorescent protein (eGFP), connected with 2A peptide from porcine teschovirus-1 polyprotein i.e. P2A. All the above sequences were codon optimized for human expression before synthesis.
  • pDys micro-dystrophin
  • FLuc firefly luciferase gene
  • eGFP enhanced Green fluorescent protein
  • MD1 encodes N-terminal actin-binding domain, hinge regions - Hl, H2 and H4 and rod domains - Rl-3, and R24, and cys-rich region and A3990 encodes N-terminal actin-binding domain, hinge regions - Hl, H3 and H4 and rod domains - Rl-2, and R22-24, and cys-rich region.
  • Reporter constructs encoding FLuc-P2A-eGFP were generated for the promoter screening in vitro and in vivo.
  • SV40 Polyadenylation Signal It is mammalian terminator that promotes both polyadenylation and transcription termination (As described in US5635177).
  • Plasmid vector pintas- SPc5-12-MDl
  • Plasmid vector pintas- SPc5- 12- A3990
  • Reporter Plasmid vector pIntas-SPc5-12-FLuc-eGFP Combining the gene sequences of promoter SPc5-12 and reporter sequence FLuc- P2A-eGFP in the elementary transducing cassette, we generated reporter transfer plasmid p!ntas-SPc5-12-FLuc-eGFP.
  • the construct was proposed to be used for screening of skeletal muscle-specific promoter/enhancer combinations while exploiting either FLuc activity or eGFP or both as a measure of promoter/enhancer activity in muscle, kidney and liver cell lines of human and mouse origin viz.
  • HEK- 293 FT Human Embryonic Kidney cells
  • HepG2 Human Liver cells
  • Human-7 Human hepatoma-derived cell line
  • RCDMD Human DMD muscle cells
  • C2C12 mouse myoblast cells
  • Plasmid vector pintas- SPc5-12-hDes-A3990
  • Reporter Plasmid vector pIntas-SPc5-12-hDes-FLuc-eGFP
  • HEK- 293 FT Human Embryonic Kidney cells
  • HepG2 Human Liver cells
  • Human-7 Human hepatoma-derived cell line
  • RCDMD Human DMD muscle cells
  • C2C12 mouse myoblast cells
  • Plasmid vector pintas- SPc5-12-hCK-MDl
  • Plasmid vector pintas- SPc5-12-hCK-A3990
  • Reporter Plasmid vector pIntas-SPc5-12-hCK-FLuc-eGFP
  • Plasmid vector pIntas-hMHCK7-MDl
  • transfer plasmid pintas- hMHCK7- MD1 (SEQ ID NO: 02, Figure 2) that will encode —132 kDa fully functional microdystrophin protein, MD1.
  • Plasmid vector pIntas-hMHCK7-A3990
  • Reporter Plasmid vector pIntas-hMHCK7-FLuc-eGFP
  • reporter transfer plasmid pIntas-hMHCK7-FLuc-eGFP was proposed to be used for screening of skeletal muscle-specific promoter/enhancer combinations while exploiting either FLuc activity or eGFP or both as a measure of promoter/enhancer activity in muscle, kidney and liver cell lines of human and mouse origin viz.
  • HEK- 293 FT Human Embryonic Kidney cells
  • HepG2 Human Liver cells
  • Human-7 Human hepatoma-derived cell line
  • RCDMD Human DMD muscle cells
  • C2C12 mouse myoblast cells
  • Plasmid vector pIntas-Sk-CRM4-mDes-MDl
  • Reporter Plasmid vector pIntas-Sk-CRM4-mDes-FLuc-eGFP
  • HEK-293 FT Human Embryonic Kidney cells
  • HepG2 Human Liver cells
  • Human-7 Human hepatoma-derived cell line
  • RCDMD Human DMD muscle cells
  • C2C12 mouse myoblast cells
  • Plasmid vector pintas- Sk-CRM4-hDes-MDl
  • Plasmid vector pintas- Sk-CRM4-hDes-A3990
  • HEK-293 FT Human Embryonic Kidney cells
  • HepG2 Human Liver cells
  • Human-7 Human hepatoma-derived cell line
  • RCDMD Human DMD muscle cells
  • C2C12 mouse myoblast cells
  • Packaging Plasmid vector pIntas-ACG-R2C9
  • the packaging plasmid vector pIntas-ACG-R2C9 (SEQ ID NO: 13, Figure 13) consists of an AAV2 genomic expression cassette lacking 5’- and 3’-ITRs as well as AAV2 capsid i.e. consists of 5’UTR (untranslated Region), replicase (Rep2) coding sequence, spacer sequence and 3’UTR of AAV2 (AAV2 genome - Genbank Accession: NC_001401.2) along with AAV9 capsid coding sequence, AAV9 genome - Genbank Accession: AY530579.1) placed between Rep2 and 3’ UTR of AAV2 in a vector backbone with a functional origin of replication for E. coli and ampicillin as selection marker. Additionally, ACG was used as start codon instead of ATG.
  • Packaging Plasmid vector pIntas-ACG-R2Cpol
  • the packaging plasmid vector pintas- AC G-R2Cpol (SEQ ID NO: 14, Figure 14) consists of an AAV2 genomic expression cassette lacking 5’- and 3’-ITRs as well as AAV2 capsid i.e. consists of 5’UTR (untranslated Region), Replicase (Rep2) coding sequence, spacer sequence and 3’UTR of AAV2 (AAV2 genome - Genbank Accession: NC_001401.2) along with AAVpol capsid coding sequence (AAVpol genome - Genbank Accession: FJ688147.1) placed between Rep2 and 3’ UTR of AAV2 in a vector backbone with a functional origin of replication for E. coli and ampicillin as selection marker. Additionally, ACG was used as start codon instead of ATG.
  • Helper Plasmid pintas Helper
  • the helper plasmid vector plntas-Helper contains the sequences of E2A, E4 and VA helper genes from Adenovirus Type 5 (Genbank Accession: AF369965.1). The expression of the said genes is driven by inherent viral promoters.
  • the plasmid DNAs were transformed in an A. coli strain DH5 -alpha, ToplO and/or in Stbl3 or Stbl4. Transformants were scored over LB-kanamycin or LB-ampicillin plates. Plasmid DNAs were isolated from the transformants using miniprep (QIAprep Spin Miniprep Kit; Cat No. 27104) and restriction digestion analysis was performed to characterize the plasmid for the presence of gene of interest and vector integrity. Among several correct transformants, a high performing (high purity, yield as well as high content of covalently closed circular plasmid) clone was selected and final glycerol banks were prepared.
  • a vial from the glycerol stock was grown over night at 37 °C for 16 to 18 hours in LB medium containing desired antibiotic (kanamycin or ampicillin) and temperature shift was done at 42°C for 4 to 6 hours (optional).
  • Bacterial pellet was subsequently harvested by pelleting the biomass via centrifugation at 4 °C and large scale plasmid preparation was performed using maxiprep (Qiagen plasmid Maxi Kits; Cat No.12162, 12163 and 12165) or gigaprep (Qiagen Hi speed plasmid Giga EF Kit; Cat No.1054575) as per manufacturer’s recommendation. Briefly, bacterial pellets were re-suspended in re-suspension buffer using a pipette followed by alkaline lysis, neutralization and column purification.
  • promoter activity was assessed in cell lines from different origins including C2C12, RCDMD, HEK293FT, and Huh-7 or HepG2 by a promoter reporter assay (as described in Piekarowicz et. al., Mol Ther Methods Clin Dev. 2019) for screening promoter for muscle-specific expression.
  • C2C12 mouse myoblast cells
  • RCDMD Immmortalized Human Duchenne Muscular Dystrophy Skeletal Muscle Cells
  • HEK293FT Human Embryonic Kidney cells
  • Huh-7 human hepatocyte cell
  • HepG2 Human Hepatic cells
  • EXAMPLE 4 SINGLE DNA TRANSIENT TRANSFECTION IN DIFFERENT CELL LINES
  • pintas- SPc5-12-FLuc-eGFP, pIntas-SPc5-12-hCK-Fluc-eGFP and plntas- hMHCK7-FLuc-eGFP reporter constructs showed FLuc activity higher than positive control in both C2C12 and RCDMD.
  • the promoters were ranked as follows:
  • the best candidate promoter would be the one which gave good microdystrophin expression both in mouse and human muscle tissue.
  • the promoters selected for further studies with micro-dystrophin constructs were pIntas-SPc5-12- MD1, pIntas-SPc5-12-A3990, pIntas-SPc5-12-hCK-MDl, pIntas-SPc5-12-hCK- A3990, pIntas-hMHCK7-MDl, and pIntas-hMHCK7-A3990.
  • Recombinant reporter AAV was generated, purified, titrated for viral genome and capsid particles and promoter driven systemic expression was evaluated in vivo using Balb/c / C57BL/6 / SCID-mdx mouse. (As described in Sarcar et a/ Nat. Comm. 2019) with some modifications as mentioned below.
  • HEK 293 cells were propagated and maintained in 1000 mL shake flask in a suitable mammalian cell culture media.
  • An exemplary option of such kind of media can be DMEM medium (Gibco, Cat No. Al 5169-01) or BalanCD medium (Irvine Scientific, Cat No. 94137).
  • the cells were seeded at 0.8 million/mL in 1000 mL shake flask with 300 mL media and maintained at 37 °C, 125 rpm and 8% CO2 for 48 hours or until the cell density reaches between 2.0-3.5 million/mL.
  • a triple co-transfection of HEK 293 cells was done with the either of the AAV reporter transgene plasmid (as identified in EXAMPLE 5) along with packaging plasmid vector (p!ntas-ACG-R2C9 or pintas- AC G-R2Cpol : plasmids encoding capsid proteins for AAV serotype 9 or AAV serotype pol) and helper plasmid (plntas- Helper) in the ratio of 1: 1:2.
  • the transfection of DNA was done using PEI-MAX (Polysciences Inc., Cat no. 24765-1).
  • Transfected HEK 293 cells were maintained at 37 °C, 125 rpm and 8% CO2 for 72 to 96 hours in CO2 shaker incubator. At the termination time culture was harvested and stored in -80 °C freezer till the purification.
  • HEK293 cell culture harvests post transfection (from Example 6) were lysed and clarified by centrifugation followed by 0.2 pm filtration.
  • the AAV preparations were purified by methods using affinity chromatography, anion exchange chromatography, CsCl or lodixanol gradient purification (separation of filled capsid with empty capsid).
  • the purified product was characterized for vector genome titer.
  • In-vivo screening of the AAV vectors containing different promoters driving FLuc expression was performed in Balb/c / C57BL/6 / SCID-mdx mice. Six to eight weeks old animals were dehaired one day before intramuscular or intravenous (tail vein) administration of recombinant reporter AAV9 or AAVpol (50-100 pl of purified reporter AAV vectors [1 x 10 10 - 6 x 10 11 vg/mouse]). The experimental animals were live-imaged using non-invasive in vivo bioluminescence optical imaging system (IVIS, Perkin Elmer) at 2, 4 and 6 weeks post AAV-administration within one minute after intravenous administration of a D-luciferin substrate at a dose of 150 mg/kg of body weight.
  • IVIS non-invasive in vivo bioluminescence optical imaging system
  • mice Four- weeks-old SCID/mdx mice were injected intravenously with 1-5 X 10 11 VG therapeutic AAV vector (AAV9/AAVpol) encoding pDes-MDl and / or pDes- A3990. Control mice were injected with vehicle either carrying empty AAV vector or no AAV vector. Additionally, the age matched C57BL/6 mice were used as positive control. Four/eight/sixteen weeks post-injection, the treated, control mice and C57BL/6 mice were subjected to a treadmill test to determine correction of the dystrophic phenotype.
  • AAV9/AAVpol AAV9/AAVpol
  • AAV vector treated and control SCID-mdx mice and/or C57BL/6 mice injected with AAV vectors (AAV9/AAVpol) encoding MD1 or p Des- 3990 were euthanized 16 weeks post vector administration and the extensor digitorum longus muscle was isolated from each mouse.
  • Force test an in vitro muscle test using an Aurora 1200 A was performed on freshly isolated muscles after electrical stimulation at 30 °C chamber filled with Krebs-Ringer bicarbonate buffer solution containing 10 mM glucose and continuously gassed with 99% O2. The electrical stimulation was induced with a series of 150 Hz pulses for 0.5 s (evoking titanic force of 0.2 ms pulses).

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Abstract

L'invention concerne un système de vecteur viral à base de VAAr contenant une cassette d'expression de micro-dystrophie (abrégé en MD1 de type μ-dys ∆3990) qui est empaqueté dans les capsides virales optimisées pour un tropisme vers le tissu musculaire (c'est-à-dire, différents sérotypes de VAA; AAV9 et AAVpo1). Un tel vecteur peut être utilisé pour le traitement de la DMD et de la dystrophie musculaire de Becker (BMD).
PCT/IB2021/056698 2020-08-06 2021-07-26 Administration par vecteur de virus adéno-associé de micro-dystrophine pour traiter la dystrophie musculaire WO2022029543A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023015304A1 (fr) * 2021-08-05 2023-02-09 Insmed Incorporated Particules de virus adéno-associés et leurs procédés d'utilisation
WO2023248251A1 (fr) * 2022-06-24 2023-12-28 Indian Institute Of Technology Kanpur Vecteur aav optimisé pour la thérapie génique de la dystrophie musculaire

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015197232A1 (fr) * 2014-06-27 2015-12-30 Genethon Traitement systémique efficace de pathologies musculaires dystrophiques
WO2017221145A1 (fr) * 2016-06-21 2017-12-28 Bamboo Therapeutics, Inc. Gènes optimisés de mini-dystrophine et cassettes d'expression et leur utilisation
WO2018170408A1 (fr) * 2017-03-17 2018-09-20 Research Institute At Nationwide Children's Hospital, Inc. Administration par vecteur à virus adéno-associé de micro-dystrophine spécifique du muscle pour traiter la dystrophie musculaire
WO2019195362A1 (fr) * 2018-04-03 2019-10-10 Curators Of The University Of Missouri Dystrophines à régions charnières 1 et/ou 4 modifiées pour la thérapie de la dystrophinopathie

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015197232A1 (fr) * 2014-06-27 2015-12-30 Genethon Traitement systémique efficace de pathologies musculaires dystrophiques
WO2017221145A1 (fr) * 2016-06-21 2017-12-28 Bamboo Therapeutics, Inc. Gènes optimisés de mini-dystrophine et cassettes d'expression et leur utilisation
WO2018170408A1 (fr) * 2017-03-17 2018-09-20 Research Institute At Nationwide Children's Hospital, Inc. Administration par vecteur à virus adéno-associé de micro-dystrophine spécifique du muscle pour traiter la dystrophie musculaire
WO2019195362A1 (fr) * 2018-04-03 2019-10-10 Curators Of The University Of Missouri Dystrophines à régions charnières 1 et/ou 4 modifiées pour la thérapie de la dystrophinopathie

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023015304A1 (fr) * 2021-08-05 2023-02-09 Insmed Incorporated Particules de virus adéno-associés et leurs procédés d'utilisation
WO2023248251A1 (fr) * 2022-06-24 2023-12-28 Indian Institute Of Technology Kanpur Vecteur aav optimisé pour la thérapie génique de la dystrophie musculaire

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