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WO2025133247A1 - Procédés de ré-administration de vecteur aav - Google Patents

Procédés de ré-administration de vecteur aav Download PDF

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Publication number
WO2025133247A1
WO2025133247A1 PCT/EP2024/088101 EP2024088101W WO2025133247A1 WO 2025133247 A1 WO2025133247 A1 WO 2025133247A1 EP 2024088101 W EP2024088101 W EP 2024088101W WO 2025133247 A1 WO2025133247 A1 WO 2025133247A1
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Prior art keywords
seq
peptide
aavpo1
vector
gene
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Ana BUJ-BELLO
Edith RENAUD-GABARDOS
Philippe Veron
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Institut National de la Sante et de la Recherche Medicale INSERM
Genethon
UNIVERSITE EVRY VAL D'ESSONNE
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Institut National de la Sante et de la Recherche Medicale INSERM
Genethon
UNIVERSITE EVRY VAL D'ESSONNE
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the invention pertains to the field of gene therapy.
  • the invention relates to methods for adeno-associated virus (AAV) vector re-administration using a recombinant porcine AAV serotype 1 (AAVpo1) vector in combination with a recombinant AAV serotype 9, rh10, or rh74 (AAV9, AAVrh10, AAVrh74,) vector and their application in gene therapy of diseases, in particular muscle and/or nervous system disorders, more particularly neuromuscular diseases such as genetic neuromuscular diseases.
  • AAV Recombinant Adeno-Associated Virus
  • rAAV or AAV vectors are widely used for in vivo gene transfer and clinical trials using AAV vectors are currently taking place for the treatment of a number of diseases.
  • AAV is a non-pathogenic virus belonging to the genus Dependoparvovirus within the family Parvoviridae.
  • AAV is a non-enveloped virus composed of a capsid of ⁇ 25 nm of diameter and a single strand DNA of 4.7 kb.
  • the genome carries two genes, rep and cap, flanked by two palindromic regions named Inverted terminal Repeats (ITR) that serve as the viral origins of replication and the packaging signal.
  • ITR Inverted terminal Repeats
  • the cap gene codes for three structural proteins VP1, VP2 and VP3 that compose the AAV capsid. VP1, VP2 and VP3 share the same C-terminal end which is all of VP3.
  • VP1 has a 735 amino acid sequence (GenBank YP_680426); VP2 (598 amino acids) starts at the Threonine 138 (T138) and VP3 (533 amino acids) starts at the methionine 203 (M203).
  • the rep gene encodes four proteins required for viral replication Rep78, Rep68, Rep52 and Rep40.
  • Tissue specificity is determined by the capsid serotype and commonly used AAV serotypes isolated from human (AAV2, 3, 5, 6) and non-human primates (AAV1, 4, 7-11) can transduce specific organs more efficiently than others, such as AAV6, AAV8, AAV9 and AAV-rh74 in muscle tissue and AAV2, AAV9, AAVrh10, AAVcy.10, AAV-PHP.B, AAV- PHP.EB and clade F AAVHSC such as AAVHSC7, AAVHSC15 and AAVHSC17 in nervous tissue.
  • AAV vector immunogenicity represents a major limitation to gene transfer with AAV vectors.
  • AAV2 is seroprevalent in up to 80 % of the human population (Fu et al., Hum Gene Ther Clin Dev., 2017 Dec;28(4):187-196; Stanford et al., Res Pract Thromb Haemost., 2019, 3: 261-267).
  • the high-level neutralising antibodies generated after the administration of commonly used AAV vector serotypes prevents the possibility of vector re-administration.
  • a peptide-modified AAVpo1 (AAVpo1.A1) is further capable of transducing the central nervous system (brain and spinal cord) and achieving transgene expression levels in different muscle groups and in the central nervous system that were at least equivalent if not superior to that of AAV9 vector while at the same time being detargeted from the liver (WO 2021/219762).
  • AAV adeno-associated virus
  • AAV vector re- administration should be possible after an initial administration of AAV vector using appropriate combinations of AAVpo1 and current AAV vector serotypes.
  • the examples of the present application show that AAVpo1 is not neutralized by antibodies produced after AAV8, AAV9, AAVrh74 or AAVrh10 injection ( Figure 1 and Figure 3), allowing a re-administration with AAVpo1 vector after an initial injection of AAV8, AAV9, AAVrh74 or AAVrh10 vector ( Figure 6 and Figure 7).
  • the invention relates to a combination of a recombinant porcine adeno-associated virus serotype 1 (AAVpo1) vector and a recombinant adeno-associated virus serotype 9, rh10 or rh74 (AAV9, AAVrh10, AAVrh74) vector, for use in the gene therapy of diseases, preferably wherein the AAV9, AAVrh10 or AAVrh74 vector is administered at an initial time point and the AAVpo1 vector is administered at a later time point.
  • AAVpo1 porcine adeno-associated virus serotype 1
  • AAV9, AAVrh10, AAVrh74 recombinant adeno-associated virus serotype 9, rh10 or rh74 vector
  • the AAVpo1, AAV9, AAVrh10 or AAVrh74 serotype is selected from the group consisting of: AAV capsids comprising a sequence having at least 95 % identity with anyone of SEQ ID NO: 1 and 3 to 5 and hybrid or peptide-modified derivatives thereof having at least 90 % identity with any one of SEQ ID NO: 1 and 3 to 5.
  • the peptide-modified AAV serotype comprises a peptide comprising at least the sequence RGD, preferably. a 7mer peptide comprising at least the sequence RGD.
  • the peptide-modified AAV serotype comprises a peptide selected from the group consisting of SEQ ID NO: 6 to 60 and 77 to 82; preferably SEQ ID NO: 6, 15, 33 to 60 and 77 to 82; more preferably SEQ ID NO: 6, 15, 36, 44, 50, 56, 58, 60, 77 and 79.
  • the AAVpo1 serotype has at least 95 % identity with SEQ ID NO: 61 and comprises the peptide A1 (SEQ ID NO: 15).
  • the AAV9 serotype has at least 95 % identity with SEQ ID NO:3 and comprises a peptide selected from the group consisting of SEQ ID NO: 6 to 60 and 77 to 82, preferably selected from the group consisting of SEQ ID NO: 6, 15, 33 to 60 and 77 to 82 and more preferably SEQ ID NO: 6, 15, 36, 44, 50, 56, 58, 60, 77 and 79.
  • the AAV9 serotype is hybrid AAV9rh74 having at least 95 % identity with SEQ ID NO: 62; preferably comprising the peptide P1 (SEQ ID NO: 6) or a peptide of any one of SEQ ID NO: 77 to 82; more preferably having at least 95 % identity with SEQ ID NO: 64 or 65 and comprising the peptide P1 (SEQ ID NO: 6) or having at least 95 % identity with SEQ ID NO: 83 and comprising the peptide SEQ ID NO: 77.
  • the recombinant AAVpo1 vector and the recombinant AAV9, AAVrh74 or AAVrh10 vector encode a transgene of interest for therapy.
  • the transgene encoded by the recombinant AAVpo1 vector and the transgene encoded by the recombinant AAV9, AAVrh10 or AAVrh74 vector are the same.
  • the transgene encoded by the recombinant AAVpo1 vector and the transgene encoded by the recombinant AAV9, AAVrh10 or AAVrh74 vector are different.
  • the transgene of interest for therapy is selected from the group consisting of: (i) therapeutic genes; (ii) genes encoding therapeutic proteins or peptides such as therapeutic antibodies or antibody fragments and genome editing enzymes; and (iii) genes encoding therapeutic RNAs such as interfering RNAs, guide RNAs for genome editing and antisense RNAs capable of exon skipping.
  • the recombinant AAV serotype for the initial administration and the recombinant AAV serotype for the later administration are chosen respectively from : (i) natural AAV9 capsid of SEQ ID NO: 3 and AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61 ; (ii) AAVrh10 capsid, in particular natural AAVrh10 capsid of SEQ ID NO: 4 and AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61 (iii) peptide-modified AAV9 capsid comprising a peptide chosen from SEQ ID NO: 33 to 60 and AAVpo1 capsid modified with peptide A1 of SEQ ID NO: 61; (iv) AAVrh74 capsid, in particular natural AAVrh74 capsid of SEQ ID NO: 5 and AAVpo1 capsid modified with peptide A1 of SEQ ID
  • the combination is administered by systemic route, preferably intravascular route; more preferably intravenous route.
  • the combination according to the present disclosure is used in the gene therapy of muscle and/or nervous system diseases or disorders.
  • the target cells for gene therapy are in the nervous system (central and/or peripheral), muscles (cardiac, smooth and/or skeletal), or a combination thereof.
  • the disease is selected from the group consisting of: neurological diseases, muscular diseases, and combination thereof.
  • the disease is a neuromuscular disease affecting the nervous system, preferably a genetic neuromuscular disease affecting the nervous system.
  • the genetic neuromuscular disease is selected from the group comprising : (i) myopathies, such as congenital myopathies, myasthenic syndromes, metabolic myopathies, distal myopathies, muscular dystrophies with or without cardiomyopathy; and (ii) spinal muscular atrophies (SMAs) and motor neuron diseases; preferably SMN1-related spinal muscular atrophy, spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), spinobulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS).
  • myopathies such as congenital myopathies, myasthenic syndromes, metabolic myopathies, distal myopathies, muscular dystrophies with or without cardiomyopathy
  • SMAs spinal muscular atrophies
  • motor neuron diseases preferably SMN1-related spinal muscular atrophy, spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), spinobulbar muscular atrophy (SBMA) and amyotrophic
  • the target gene for gene therapy is selected from the group comprising: DMD, DYSF, FKRP, ⁇ -sarcoglycan (SGCA), ⁇ -sarcoglycan (SGCB), ⁇ -sarcoglycan (SGCG), Calpain 3 (CAPN3), Anoctamin 5 (ANO5), MTM1, DNM2, BIN1, GAA, AGL, ColQ, DOK7, SMN1, ASAH1, MCEP2, and AR genes.
  • the invention also relates to a product containing a recombinant AAVpo1 vector and a recombinant AAV9, AAVrh10 or AAVrh74 vector as a combined preparation for sequential use in the treatment of a disorder according to the present disclosure.
  • the invention relates to a combination of a recombinant porcine adeno-associated virus serotype 1 (AAVpo1) vector and a recombinant adeno-associated virus serotype 9, rh10 or rh74 (AAV9, AAVrh10, AAVrh74) vector for use in the gene therapy of diseases in an individual in need thereof, preferably wherein the AAV9, AAVrh10, or AAVrh74 vector is administered at an initial time point and the AAVpo1 vector is administered at a later time point.
  • AAV vector refers to an AAV vector particle.
  • porcine AAV serotype 1 (AAVpo1) vector” or AAVpo1 vector refers to an AAV vector comprising a porcine AAV serotype 1 capsid protein.
  • the term “recombinant AAV serotype 9, rh10, rh74 (AAV9, AVrh10, AAVrh74) vector” or AAV9, AAVrh10 AAVrh74 vector” or AAV9, AAVrh10 AAVrh74 vector refers to an AAV vector comprising an AAV serotype 9, rh10, rh74 capsid protein.
  • AAV serotype refers to an AAV capsid serotype.
  • AAV serotype 9, rh10, rh74 refers to AAV9, AAVrh10 or AAVrh74 capsid.
  • AAVpo1 serotype refers to AAVpo1 capsid.
  • AAV serotype includes natural AAV (i.e. wild-type AAV) and artificial AAV serotypes such as variants and hybrid capsids derived from natural AAV serotypes, as well as peptide-modified AAV serotypes derived from natural, variant or hybrid AAV serotypes.
  • AAV serotype refers to a functional AAV capsid which is able to transduce cells of the target tissue or organ and express a transgene in said target tissue or organ.
  • AAV capsid refers to AAV, VP1, VP2 and/or VP3 protein.
  • target tissue or organ refers to an individual tissue or organ or plurality of tissues or organs that can be targeted by the combination of recombinant AAVpo1 serotype vector and recombinant AAV9, AAVrh10 or AAVrh74 serotype vector according to the present disclosure.
  • the target tissue or organ is targeted for gene therapy, i.e., for treating diseases by sequential administration of the combination of AAV vectors according to the present disclosure.
  • disease or disorder refers to a disease that can be treated by gene therapy using the combination of AAV vectors according to the present disclosure.
  • a disease includes in particular, a disease associated with a gene mutation and therefore eligible to AAV gene therapy.
  • “a”, “an”, and “the” include plural referents, unless the context clearly indicates otherwise.
  • AAVpo1 (GenBank accession number FJ688147 as accessed on 24 July 2016) comprises the Cap gene from position 780 to 2930 of the partial viral genome sequence (2977 bp): VP1 CDS is from positions 780 to 2930; VP2 CDS is from positions 1188 to 2930 and VP3 CDS is from positions 1329 to 2930.
  • the AAVpo1 capsid protein (VP1) has the sequence GenBank accession number ACN42940.1 as accessed on 24 July 2016 or SEQ ID NO: 1.
  • AAV9 capsid corresponds to the amino acid sequence GenBank accession number AY530579.1 or protein ID number AAS99264.1 accessed on 24 June 2004 (SEQ ID NO: 3).
  • AAVrh10 capsid (GenBank accession number AY243015.1 or protein ID number AAO88201.1 accessed on 14 May 2003) corresponds to the amino acid sequence SEQ ID NO: 4.
  • AAVrh74 capsid corresponds to the amino acid sequence SEQ ID NO: 5.
  • AAVpo1, AAV9, AAVrh74 or AAVrh10 serotype includes the natural (wild- type) serotype as listed above (SEQ ID NO: 1 and 3 to 5) as well as any artificial serotype including any variant, hybrid and/or peptide-modified AAV capsid derived from said serotype.
  • the invention encompasses the use of AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid or serotype having at least at least 95%, 96%, 97%, 98% or 99% identity with AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid sequence as listed above (SEQ ID NO: 1 and 3 to 5).
  • the invention also encompasses the use of hybrid and/or peptide-modified AAV capsids derived from said AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid or serotype, wherein the hybrid and/or peptide-modified AAV capsid has at least at least 90% identity with AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid sequence as listed above (SEQ ID NO: 1 and 3 to 5).
  • the hybrid and/or peptide-modified AAV capsid may have 91%, 92%, 93%, 94%, 95% or more (96%, 97%, 98% or 99%) identity with AAVpo1, AAV9, AAVrh74, AAVrh10 capsid sequence as listed above (SEQ ID NO: 1 and 3 to 5).
  • a peptide-modified AAV capsid according to the invention (i) is derived from an initial AAV capsid (without the peptide(s)) having at least at least 95%, 96%, 97%, 98% or 99% identity with AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid sequence as listed above (SEQ ID NO: 1 and 3 to 5) and (ii) has at least 90% identity with AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid sequence as listed above (SEQ ID NO: 1 and 3 to 5).
  • a hybrid AAV capsid according to the invention is derived from at least two different AAV serotypes including at least one AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid or serotype having at least 95%, 96%, 97%, 98% or 99% identity with AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid sequence as listed above (SEQ ID NO: 1 and 3 to 5) and (ii) has at least 90% identity with said at least one AAVpo1, AAV9, AAVrh74 or AAVrh10 capsid sequence as listed above (SEQ ID NO: 1 and 3 to 5).
  • An artificial serotype according to the invention has a seroneutralization profile that is similar to that of the natural serotype it is derived from. This means that the artificial serotype is neutralized by AAV antibodies with titers similar to those of the natural serotype. Preferably, similar titers differ from each other by a dilution factor of less than 2; preferably less than 3. A 3.16-fold (10 0.5 - fold) dilution is called a half-logarithmic dilution or half-log dilution. [00035]
  • identity refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules.
  • the percentage of identity between two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Generally, a comparison is made when two sequences are aligned to give maximum identity.
  • the identity may be calculated by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA or CLUSTALW.
  • the AAVpo1, AAV9, AAVrh10 or AAVrh74 serotype is peptide-modified, in particular with a peptide which enhances transduction of cells of the target tissue or organ.
  • Libraries of AAV capsid variants displaying short random peptides on the surface of various AAV serotypes have been generated to screen for gene therapy vectors with altered cell specificities and/or transduction efficiencies (Review in Büning et al., Molecular Therapy: Methods & Clinical Development, 2019, 12, 248).
  • peptides able to target AAV vectors to a desired organ obtained by library screening are disclosed in the art (Michelfelder et al., PLoS ONE, 2009, 4, e5122; Kienle EC (Dissertation for the degree of Doctor of natural Sciences, Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany, 2014); WO 2019/207132; Börner et al., Molecular Therapy, April 2020, 28, 1017-1032; Weinmann et al., Nature communications, 2020, 11, 5432; Tabebordbar et al., Cell, 2021, 184, 4919-4938).
  • the targeting peptide consists generally of a sequence of up to 30 amino acids.
  • the peptide may consist of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids.
  • the peptide consists of a sequence of up to 15 amino acids, preferably 3 to 10 amino acids, in particular 7 or 10 amino acids.
  • Non-limiting examples of targeting peptides include the peptides disclosed in Börner et al (Table S2 and Table S3) ; Weinmann et al., Tabebordbar et al.; Ai Vu Hong et al.
  • AAV capsids comprising the insertion of a peptide comprising an RGD motif, which is known to bind several different cell-surface integrins, have been reported to improve gene delivery in muscle following systemic administration.
  • Peptides containing RGD motif are disclosed in Tabebordbar et al., Cell, 2021, 184, 4919-4938; Ai Vu Hong et al.
  • AAV capsids for muscle transduction in mice non-human primates and/or human primary myotubes display a peptide comprising a RGDL motif : RGDLGLS or P1 (AAVMYO or AAV9P1), RGDLTTP (MyoAAV 1A), GPGRGDQTTL (MyoAAV 2A), SNSRGDYNSL (MyoAAV 4A), ENRRGDFNNT (MyoAAV 4E), SAQRGDYVGL (MyoAAV 3A), QERRGDYTSM (MyoAAV 4C) inserted into the variable region VIII (WO 2019/207132; Weinmann et al., Nature communications, 2020, 11, 5432; Tabebordbar et al., Cell, 2021, 184, 4919-4938); or RGDLGRL (4um9), RGDLGEL (4um9_modified), RGDLATI (5ffo), RGDLAEL (5ffo_modified), RGDLQVL (5nem) and
  • the AAVpo1, AAV9, AAVrh10 or AAVrh74 serotype is modified with a peptide selected from the group consisting of : P1 (RGDLGLS), A1 (MPLGAAG), MyoAAV peptide (SEQ ID NO: 33 to 60) and SEQ ID NO: 77 to 82, in particular SEQ ID NO: 36 (MyoAAV 1A peptide), SEQ ID NO: 44 (MyoAAV 2A peptide), SEQ ID NO: 50 (MyoAAV 3A peptide), SEQ ID NO: 56 (MyoAAV 4A peptide), SEQ ID NO: 58 (MyoAAV 4C peptide), SEQ ID NO: 60 (MyoAAV 4E peptide), SEQ ID NO: 77 (4um9 peptide) and SEQ ID NO: 79 (5ffo peptide).
  • P1 RDLGLS
  • A1 MPLGAAG
  • MyoAAV peptide SEQ ID NO: 33 to 60
  • the AAVpo1 serotype is natural AAVpo1; variant AAVpo1, hybrid AAVpo1 or peptide-modified derivative thereof, preferably natural AAVpo1 or peptide-modified AAVpo1.
  • the AAVpo1 serotype (natural, hybrid or variant capsid) has at least 95 % identity with SEQ ID NO: 1.
  • the peptide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 6 to 60 and 77 to 82; preferably SEQ ID NO: 15, 33 to 60 and 77 to 82.
  • the peptide comprises or consists of the sequence MPLGAAG (peptide A1 or SEQ ID NO: 15).
  • the AAVpo1 serotype has at least 90 % identity with SEQ ID NO: 1, preferably at least 95 % identity with SEQ ID NO: 1, and comprises a peptide chosen from SEQ ID NO: 15, 33 to 60 and 77 to 82, preferably SEQ ID NO: 15.
  • said peptide- modified AAVpo1 capsid protein comprises a sequence selected from the group consisting of the sequence SEQ ID NO: 61 and the sequences having at least 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 61 which comprise said peptide of SEQ ID NO: 15, and the fragment thereof corresponding to VP2 or VP3 capsid protein.
  • SEQ ID NO: 61 corresponds to the AAVpo1 capsid modified by the peptide A1, named AAVpo1.A1, disclosed in WO 2021/219762.
  • VP2 corresponds to the amino acid sequence from K136 to the end of SEQ ID NO: 61.
  • VP3 corresponds to the amino acid sequence from M184 to the end of SEQ ID NO: 61.
  • said peptide-modified AAVpo1 capsid protein comprises the sequence SEQ ID NO: 61, or a fragment thereof corresponding to VP2 or VP3 capsid protein.
  • the AAV9 serotype is natural (wild-type) AAV9 or peptide-modified AAV9 comprising a peptide chosen from SEQ ID NO: 3 to 60 and 77 to 82, preferably the P1 peptide, the A1 peptide, a MyoAAV peptide chosen from SEQ ID NO: 33 to 60, or a RGDLXXL/I peptide chosen from SEQ ID NO: 77 to 82, in particular SEQ ID NO: 36, 44, 50, 56, 58, 60, 77 and 79.
  • AAV9 serotype is hybrid AAV9.rh74, P1-modified AAV9.rh74 or AAV9.rh74 modified with RGDLXXL/I peptide 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem or 5nem_modified.
  • AAV9.rh74 modified with RGDLXXL/I peptide is preferably modified with peptide 4um9.
  • AAV9.rh74 capsid is disclosed in WO 2019/193119 and corresponds to the amino acid sequence SEQ ID NO: 62.
  • AAV9rh74 capsids are disclosed in WO2022/053630 and WO 2020/200499 and correspond to SEQ ID NO: 64 and SEQ ID NO: 65, respectively.
  • AAV9.rh74 modified with RGDLXXL/I peptide 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem or 5nem_modified are disclosed in WO 2023/237748 and correspond to SEQ ID NO: 83 to 88, respectively.
  • SEQ ID NO: 83 is LICA1.
  • the AAV9 serotype is AAV9 variant or peptide-modified derivative thereof.
  • the peptide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 6 to 60 and 77 to 82, preferably SEQ ID NO: 6, 15, 33 to 60, and 77 to 82.
  • AAV9 variant includes in particular: AAV-PHP such as AAV-PHP.A, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S; AAV9BI; AAV9LD; AAVS1and AAVS10 (El Andari et al., Science Advances, 2022, 8, 1-21; WO2019207132A1).
  • Peptide- modified AAV9 variant includes in particular AAVMYO2 (SEQ ID NO: 66) and AAVMYO3 (SEQ ID NO: 67), corresponding to P1-modified AAVS1 and AAVS10 capsids respectively (El Andari et al.; WO2019207132A1).
  • the AAV9 serotype has at least 90 % identity with SEQ ID NO: 3; preferably at least 95 % identity with SEQ ID NO: 3.
  • the AAV9 serotype has at least 90 % identity with SEQ ID NO: 3, preferably at least 95 % identity with SEQ ID NO: 3, and comprises a peptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 6 to 60 and 77 to 82, preferably SEQ ID NO: 6, 15, 33 to 60 and 77 to 82; more preferably the peptide P1 (SEQ ID NO: 6); more preferably comprising a sequence having at least 95 % identity with any one of SEQ ID NO: 63, 66 or 67 which comprises said P1 peptide (SEQ ID NO: 6); still more preferably comprising any one of SEQ ID NO: 63, 66 and 67.
  • AAV9 serotype is hybrid AAV9rh74, preferably having at least 95 % identity with SEQ ID NO: 62 and/or comprising a peptide comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 6 to 60 and 77 to 82, preferably SEQ ID NO: 6, 15, 33 to 60 and 77 to 82; more preferably the peptide P1 (SEQ ID NO: 6) or a RGDLXXL/I peptide chosen from 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem and 5nem_modified, preferably peptide 4um9 (SEQ ID NO: 77).
  • hybrid AAV9rh74 serotype comprises a sequence having at least 95 % identity with SEQ ID NO: 62, or a sequence having at least 95 % identity with SEQ ID NO: 64 or 65 which comprises said peptide P1; more preferably comprising any one of SEQ ID NO: 62, 64 or 65.
  • hybrid AAV9rh74 serotype comprises a sequence having at least 95 % identity with SEQ ID NO: 62, or a sequence having at least 95 % identity with SEQ ID NO: 83 to 88 which comprises said RGDLXXL/I peptide chosen from 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem and 5nem_modified, respectively; more preferably comprising any one of SEQ ID NO: 83 to 88; still more preferably SEQ ID NO: 83.
  • the AAVrh10 serotype is natural AAVrh10; variant AAVrh10, hybrid AAVrh10 or peptide-modified derivative thereof.
  • AAVrh10 serotype (natural, hybrid or variant capsid) comprises a peptide chosen from SEQ ID NO: 6 to 60 and 77 to 82, preferably the P1 peptide, the A1 peptide or a MyoAAV peptide chosen from SEQ ID NO: 33 to 60 and the RGDLXXL/I peptide chosen from SEQ ID NO: 77 to 82, in particular SEQ ID NO: 36, 44, 50, 56, 58, 60, 77 and 79.
  • the AAVrh10 serotype has at least 95 % identity with SEQ ID NO: 4.
  • the AAVrh10 serotype has at least 90 % identity with SEQ ID NO: 4, preferably at least 95 % identity with SEQ ID NO: 4, and comprises the peptide P1 (SEQ ID NO: 6), a MyoAAV peptide chosen from SEQ ID NO: 33 to 60 or a RGDLXXL/I peptide chosen from SEQ ID NO: 77 to 82, in particular SEQ ID NO: 36, 44, 50, 56, 58, 60, 77 and 79.
  • the AAVrh74 serotype is natural AAVrh74; variant AAVrh74, hybrid AAVrh74 or peptide-modified derivative thereof.
  • AAVrh74 serotype (natural, hybrid or variant capsid) comprises a peptide chosen from SEQ ID NO: 6 to 60 and 77 to 82, preferably the P1 peptide, the A1 peptide, a MyoAAV peptide chosen from SEQ ID NO: 33 to 60 or a RGDLXXL/I peptide chosen from SEQ ID NO: 77 to 82, in particular SEQ ID NO: 36, 44, 50, 56, 58, 60, 77 and 79.
  • AAVrh74 serotype is hybrid AAV9.rh74 or peptide-modified derivative thereof as disclosed above.
  • the AAVrh74 serotype has at least 95 % identity with SEQ ID NO: 5. In some particular embodiments, the AAVrh74 serotype has at least 90 % identity with SEQ ID NO: 5, preferably at least 95 % identity with SEQ ID NO: 5, and comprises the peptide P1 (SEQ ID NO: 6), a MyoAAV peptide chosen from SEQ ID NO: 33 to 60 or a RGDLXXL/I peptide chosen from SEQ ID NO: 77 to 82, in particular SEQ ID NO: 36, 44, 50, 56, 58, 60, 77 and 79; more preferably comprising SEQ ID NO: 6 or SEQ ID NO: 77.
  • the AAVrh74 serotype is hybrid AAV9rh74, preferably having at least 95 % identity with SEQ ID NO: 62 or having at least 95 % identity with SEQ ID NO: 64 or 65 and comprising peptide P1 (SEQ ID NO: 6); more preferably comprising a sequence selected from SEQ ID NO: 62, 64 and 65.
  • hybrid AAV9rh74 serotype comprises a sequence having at least 95 % identity with SEQ ID NO: 62, or a sequence having at least 95 % identity with SEQ ID NO: 83 to 88 which comprises said RGDLXXL/I peptide chosen from 4um9, 4um9_modified, 5ffo, 5ffo_modified, 5nem and 5nem_modified, respectively; more preferably comprising any one of SEQ ID NO: 83 to 88; still more preferably SEQ ID NO: 83.
  • the combination of rAAV for the initial and the later administrations comprises respectively: (i) natural AAV9 capsid and peptide-modified AAVpo1 capsid comprising the peptide A1 (AAVpo1A1); (ii) AAVrh10 capsid, in particular natural AAVrh10 capsid and peptide-modified AAVpo1 capsid comprising the peptide A1 (AAVpo1A1); (iii) peptide-modified AAV9 capsid comprising a MyoAAV peptide chosen from SEQ ID NO: 33 to 60 and peptide-modified AAVpo1 capsid comprising the peptide A1 (AAVpo1A1); (iv) AAVrh74 capsid, in particular natural AAVrh74 capsid and peptide-modified AAVpo1 capsid comprising the peptide A1 (AAVpo1A1).
  • the invention also relates to a recombinant porcine adeno-associated virus serotype 1 (AAVpo1) vector for use in the gene therapy of diseases according to the present disclosure, wherein the AAVpo1 vector is administered to a patient having a natural immunity to an AAV9, AAVrh10 and/or AAVrh74 vector according to the present disclosure.
  • AAVpo1 vector is administered to a patient having a natural immunity to an AAV9, AAVrh10 and/or AAVrh74 vector according to the present disclosure.
  • a patient having a natural immunity to an AAV9, AAVrh10 and/or AAVrh74 vector relates to a patient having pre-existing neutralizing antibodies against AAV9, AAVrh10 or AAVrh74 resulting from natural infection with adeno- associated virus (AAV).
  • the presence and/or level of neutralizing antibodies against AAV9, AAVrh10 or AAVrh74 in patient plasma or serum may be determined using appropriate neutralization assays that are well-known in the art and disclosed in the present application.
  • neutralization assay is disclosed in Meliani et al., Hum. Gene Ther. Methods, 2015, 26, 45-53).
  • the genome of the AAV vectors for use in combination according to the present disclosure either be a single-stranded or self-complementary double-stranded genome (McCarty et al, Gene Therapy, 2003, Dec.,10(26), 2112-2118).
  • Self-complementary vectors are generated by deleting the terminal resolution site (trs) from one of the AAV terminal repeats.
  • the modified vectors whose replicating genome is half the length of the wild-type AAV genome have the tendency to package DNA dimers.
  • the AAV genome is flanked by ITRs.
  • the AAV vector is a pseudotyped vector, i.e. its genome and capsid are derived from AAVs of different serotypes.
  • the vector comprises AAV2 rep proteins and/or AAV2 ITRs.
  • the AAV vectors for use in combination according to the present disclosure are produced by standard methods for producing AAV vectors that are well-known in the art (Review in Aponte-Ubillus et al., Applied Microbiology and Biotechnology, 2018, 102: 1045- 1054).
  • transgene of interest for therapy By “gene of interest for therapy”, “gene of therapeutic interest”, “gene of interest” or “heterologous gene of interest”, it is meant a therapeutic gene or a gene encoding a therapeutic protein, peptide or RNA or a combination thereof.
  • the transgene encoded by the recombinant AAVpo1 vector and the transgene encoded by the recombinant AAV9, AAVrh10 or AAVrh74 vector are the same.
  • the transgene encoded by the recombinant AAVpo1 vector and the transgene encoded by the recombinant AAV9, AAVrh10, or AAVrh74 vector are different.
  • the invention encompasses multiple vector systems such as dual vector systems, wherein the combination of vectors encodes overlapping fragments of the transgene that form a complete transgene after co-delivery of the combination of vectors into cells.
  • the gene of interest is any nucleic acid sequence capable of modifying a target gene or target cellular pathway, in cells of target tissue or organ.
  • the gene may modify the expression, sequence or regulation of the target gene or cellular pathway.
  • the target tissue or organ comprises muscles and/or nervous system.
  • the term “muscle” refers to cardiac muscle (i.e. heart), smooth muscle and skeletal muscle.
  • the term “muscle cells” refers to myocytes, myotubes, myoblasts, and/or satellite cells.
  • the term “nervous system” refers to both the central (CNS) and peripheral (PNS) nervous system.
  • the term “central nervous system or CNS” refers to the brain, spinal cord, retina, cochlea, optic nerve, and/or olfactory nerves.
  • the term CNS cells refer to any cells of the CNS including neurons and glial cells (oligodendrocytes, astrocytes, ependymal cells, microglia).
  • the PNS refers to the nerves and ganglia outside the brain and spinal cord.
  • the target tissue or organ may comprise essentially the nervous system (CNS and/or PNS), essentially muscles or both the nervous system and muscles.
  • the gene of interest is a functional version of a gene or a fragment thereof.
  • the functional version of said gene includes the wild-type gene, a variant gene such as variants belonging to the same family and others, or a truncated version, which preserves the functionality of the encoded protein at least partially.
  • a functional version of a gene is useful for replacement or additive gene therapy to replace a gene, which is deficient or non-functional in a patient.
  • a functional version of the dystrophin gene includes the various mini-dystrophin and micro-dystrophin gene constructs that are well- known in the art and other functional versions of the dystrophin gene.
  • the gene of interest is a gene which inactivates a dominant allele causing an autosomal dominant genetic disease.
  • a fragment of a gene is useful as recombination template for use in combination with a genome editing enzyme.
  • the gene of interest may encode a protein of interest for a particular application, (for example an antibody or antibody fragment, a genome-editing enzyme) or a RNA.
  • the protein is a therapeutic protein including a therapeutic antibody or antibody fragment, or a genome-editing enzyme.
  • the RNA is a therapeutic RNA.
  • the sequence of the gene of interest is optimized for expression in the treated individual, preferably a human individual.
  • Sequence optimization may include a number of changes in a nucleic acid sequence, including codon optimization, increase of GC content, decrease of the number of CpG islands, decrease of the number of alternative open reading frames (ARFs) and/or decrease of the number of splice donor and splice acceptor sites.
  • the gene of interest is a functional gene able to produce the encoded protein, peptide or RNA in the target cells of the disease.
  • the gene of interest is a human gene.
  • the AAV vectors comprise the gene of interest in a form expressible in cells of target organs.
  • the gene of interest is operably linked to appropriate regulatory sequences for expression of a transgene in the individual’s target cells, tissue(s) or organ(s).
  • the gene of interest is operably linked to at least two promoters wherein one of them is target cells specific- or inducible promoter which is functional in cells of a target tissue or organ and the other is target-specific or inducible promoter which is functional in cells of another target tissue or organ which is different from the first one.
  • the gene of interest may be inserted in an expression cassette further comprising additional regulatory sequences as disclosed above.
  • Muscle-specific promoters include without limitation, the desmin (Des) promoter, muscle creatine kinase (MCK) promoter, alpha-myosin heavy chain (alpha-MHC) promoter, myosin light chain 2 (MLC-2) promoter, cardiac troponin C (cTnC) promoter, human skeletal actin (HSA) promoter or synthetic muscle-specific promoter such as SpC5-12 promoter, CK6 promoter, or MHCK7 promoter.
  • Promoters for nervous system, such as CNS expression include promoters driving ubiquitous expression and promoters driving expression into neurons.
  • neuron-selective promoters include the promoters of Choline Acetyl Transferase (ChAT), Neuron Specific Enolase (NSE), Synapsin, Hb9 and ubiquitous promoters including Neuron-Restrictive Silencer Elements (NRSE).
  • Representative promoters driving selective expression in glial cells include the promoter of the Glial Fibrillary Acidic Protein gene (GFAP).
  • GFAP Glial Fibrillary Acidic Protein gene
  • RNA is advantageously complementary to a target DNA or RNA sequence or binds to a target protein.
  • the RNA is an interfering RNA such as a shRNA, a microRNA, a guide RNA (gRNA) for use in combination with a Cas enzyme or similar enzyme for genome editing, an antisense RNA capable of exon skipping such as a modified small nuclear RNA (snRNA) or a long non-coding RNA.
  • the interfering RNA or microRNA may be used to regulate the expression of a target gene involved in muscle and/or nervous system disease.
  • the guide RNA in complex with a Cas enzyme or similar enzyme for genome editing may be used to modify the sequence of a target gene, in particular to correct the sequence of a mutated/deficient gene or to modify the expression of a target gene involved in a disease, in particular a muscle and/or nervous system disorder.
  • the antisense RNA capable of exon skipping is used in particular to correct a reading frame and restore expression of a deficient gene having a disrupted reading frame.
  • the RNA is a therapeutic RNA.
  • the genome-editing enzyme according to the invention is any enzyme or enzyme complex capable of modifying a target gene or target cellular pathway.
  • the genome-editing enzyme may modify the expression, sequence or regulation of the target gene or cellular pathway.
  • the genome-editing enzyme is advantageously an engineered nuclease, such as with no limitations, a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector-based nuclease (TALENs), Cas enzyme from clustered regularly interspaced palindromic repeats (CRISPR)-Cas system and similar enzymes.
  • a meganuclease such as with no limitations, a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector-based nuclease (TALENs), Cas enzyme from clustered regularly interspaced palindromic repeats (CRISPR)-Cas system and similar enzymes.
  • ZFN zinc finger nuclease
  • TALENs transcription activator-like effector-based nuclease
  • CRISPR
  • the genome- editing enzyme in particular an engineered nuclease such as Cas enzyme and similar enzymes, may be a functional nuclease which generates a double-strand break (DSB) or single-stranded DNA break (nickase such as Cas9(D10A) in the target genomic locus and is used for site- specific genome editing applications, including with no limitations: gene correction, gene replacement, gene knock-in, gene knock-out, mutagenesis, chromosome translocation, chromosome deletion, and the like.
  • DSB double-strand break
  • nickase such as Cas9(D10A
  • the genome-editing enzyme such as Cas enzyme and similar enzyme may be a DNA base-editor such as cytosine base-editor and adenine base-editor or a prime-editor.
  • Base- editors can install all four transition mutations while Prime-editor expand the scope of donor- free precise DNA editing to not only all transition and transversion mutations, but small insertion and deletion mutations as well.
  • DNA base-editing and prime-editing tools enable precise nucleotide substitutions in a programmable manner, without requiring a donor template.
  • the genome-editing enzyme such as Cas enzyme and similar enzymes may be engineered to become nuclease-deficient and used as DNA-binding protein for various genome engineering applications such as with no limitation: transcriptional activation, transcriptional repression, epigenome modification, genome imaging, DNA or RNA pull-down and the like.
  • the combination of AAV vector serotypes according to the present disclosure is used in gene therapy of diseases, in particular targeted gene therapy directed to target tissue or organ.
  • the combination of AAV vector serotypes according to the present disclosure is preferably used in the form of separate pharmaceutical compositions comprising a therapeutically effective amount of AAV vector particles of each serotype, preferably AAV vector particles packaging a therapeutic gene of interest according to the present disclosure.
  • Gene therapy refers to a treatment of an individual which involves delivery of nucleic acid of interest into an individual's cells for the purpose of treating a disease. Delivery of the nucleic acid is generally achieved using a delivery vehicle, also known as a vector. The combination of rAAV vector particles according to the present disclosure may be employed to deliver a gene to a patient's cells.
  • Cell therapy refers to a process wherein cells stably transduced by a combination of rAAV vector particles according to the present disclosure are delivered to the individual in need thereof by any appropriate mean such as for example by intravenous injection (infusion), or injection in the tissue of interest (implantation or transplantation).
  • cell therapy comprises collecting cells from the individual, transducing the individual’s cells with the combination of rAAV vector particles according to the present disclosure, and administering the stably transduced cells back to the patient.
  • “cell” refers to isolated cell, natural or artificial cellular aggregate, bioartificial cellular scaffold and bioartificial organ or tissue.
  • Gene therapy can be performed by gene transfer, gene editing, exon skipping, RNA-interference, trans-splicing or any other genetic modification of any coding or regulatory sequences in the cell, including those included in the nucleus, mitochondria or as commensal nucleic acid such as with no limitation viral sequences contained in cells.
  • the two main types of gene therapy are the following: - a therapy aiming to provide a functional replacement gene for a deficient/abnormal gene: this is replacement or additive gene therapy; - a therapy aiming at gene or genome editing: in such a case, the purpose is to provide to a cell the necessary tools to correct the sequence or modify the expression or regulation of a deficient/abnormal gene so that a functional gene is expressed or an abnormal gene is suppressed (inactivated): this is gene editing therapy.
  • the gene of interest may be a functional version of a gene, which is deficient or mutated in a patient, as is the case for example in a genetic disease.
  • Gene or genome editing uses one or more gene(s) of interest, such as: (i) a gene encoding a therapeutic RNA as defined above such as an interfering RNA like a shRNA or a microRNA, a guide RNA (gRNA) for use in combination with a Cas enzyme or similar enzyme, or an antisense RNA capable of exon skipping such as a modified small nuclear RNA (snRNA); and (ii) a gene encoding a genome-editing enzyme as defined above such as an engineered nuclease like a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector-based nuclease (TALENs), Cas enzyme or similar enzymes; or a combination of such genes, and maybe also a fragment of a functional version of a gene for use as recombination template, as defined above.
  • a therapeutic RNA as defined above such as an interfering RNA like a shRNA or a microRNA, a guide
  • Gene therapy is used for treating various inherited (genetic) or acquired diseases or disorders affecting the structure or function of target tissue or organ.
  • the diseases may be caused by trauma, infection, degeneration, structural or metabolic defects, tumors, inflammatory or autoimmune disorders, stroke or other causes.
  • the disease affects one or more of the following target tissue or organs: muscles including skeletal, smooth or cardiac muscles; nervous system including brain or spinal cord; the disease may affect muscles including skeletal or cardiac muscles; nervous system including brain or spinal cord; or both.
  • the disease is a myopathy such as skeletal and/or cardiomyopathy, preferably a genetic myopathy.
  • the disease is a neurological disorder (nervous system disease), preferably a genetic neurological disorder.
  • the neurological disorder may affect the CNS and/or PNS such as the brain, spinal cord or both.
  • CNS diseases include for example Alzheimer, Parkinson, Frontotemporal dementia, Huntington disease and others.
  • the disease is a neuromuscular disorder, preferably a genetic neuromuscular disorder.
  • Neuromuscular disease or disorder is a very broad term encompassing a range of conditions that impair the functioning of the muscles, either directly, being pathologies of the voluntary muscle, or indirectly, being pathologies of the peripheral nervous system or neuromuscular junctions.
  • Some neuromuscular diseases are also associated with central nervous system disease, such as ALS or spinal muscular atrophies.
  • Examples of mutated genes in genetic neurological disorders, that can be targeted by gene therapy using the combination of AAV vectors of the invention are listed in the following table: [00082] Genetic neurological disorders Disease Gene Friedreich ataxia FXN Ataxia-telangectasia ATM Niemann pick disease SMPD1, NPC1,2 Spinocerebellar ataxia ATXN1-3, PLEKHG4, SPTBN2, CACNA1A, ATXN 7 Marinesco-Sjoren SIL1 Juvenile Parkinsonism SNCA, PARK2, PINK1, GCH1, TH Dystonia TOR1A, GCH1 Huntington’s disease HTT Wilson disease ATP7B Pantothenate kinase- PANK2 associated neurodegeneration Lysosomal Storage Disorders GBA, SMPD1, NPC1, NPC2 (LSDs) Fragile X syndrome FMR1 Rett Syndrome MECP2 Ad
  • said neurological disease is selected from the group consisting of: Spinal muscular atrophy (SMN1, ASAH1 genes), Amyotrophic lateral sclerosis (SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, OPTN and others); Hereditary paraplegia (SPAST (SPG4), SPG7, and other SPG genes such as SPG11, SPG20 and SPG21; in particular SPAST (SPG4) and SPG7) and Charcot-Marie-Tooth, Type 4B1 (MTMR2).
  • said gene is selected from the group consisting of: SMN1, ASAH1, DNM2, MTMR2 and SPAST genes.
  • said gene is selected from the group consisting of: SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4 and OPTN.
  • Examples of mutated genes in genetic myopathies that can be targeted by gene therapy using the combination of AAV vectors of the invention are listed in the following tables: [00085] Muscular dystrophies Gene Protein DMD Dystrophin EMD Emerin FHL1 Four and a half LIM domain 1 LMNA Lamin A/C SYNE1 Spectrin repeat containing, nuclear envelope 1 (nesprin 1) SYNE2 Spectrin repeat containing, nuclear envelope 2 (nesprin 2) TMEM43 Transmembrane protein 43 TOR1AIP1 Torsin A interacting protein 1 DUX4 Double homeobox 4 SMCHD1 Structural maintenance of chromosomes flexible hinge domain containing 1 PTRF Polymerase I and transcript release factor MYOT Myotilin CAV3 Caveolin 3 DNAJB6 HSP-40 homologue, sub
  • the above listed genes may be used as target for gene editing.
  • Gene editing is used to correct the sequence of a mutated gene or modify the expression or regulation of a deficient/abnormal gene so that a functional gene is expressed in muscle cells.
  • the gene of interest is chosen from those encoding therapeutic RNAs such as interfering RNAs, guide RNAs for genome editing and antisense RNAs capable of exon skipping, wherein the therapeutic RNAs target the preceding list of genes.
  • Tools such as CRISPR/Cas9 may be used for that purpose.
  • the target gene for gene therapy is a gene responsible for a neurological disorder, a muscular disorder, or a neuromuscular disorder, as disclosed herein.
  • Disease that can treated by gene therapy using a combination of AAV vector serotypes according to the disclosure include in particular: Muscular dystrophies, Congenital muscular dystrophies, Congenital myopathies, Distal myopathies, Other myopathies, Myotonic syndromes, Ion Channel muscle diseases, Malignant hyperthermia, Metabolic myopathies, Hereditary Cardiomyopathies, Congenital myasthenic syndromes, Myastenia, Spinal muscular atrophies (SMAs) and Motor Neuron diseases, Hereditary paraplegia, Hereditary ataxia, Hereditary motor and sensory neuropathies and other neuromuscular disorders; the disease can be treating by targeting the gene associated with said diseases as listed in the Tables above.
  • Myopathies include hereditary cardiomyopathies, metabolic myopathies, other myopathies, distal myopathies, muscular dystrophies and congenital myopathies; muscular dystrophies include Duchenne muscular dystrophies; congenital myopathies include myotubular myopathy and centronuclear myopathies
  • SMAs Spinal muscular atrophies
  • motor neuron diseases include amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), pseudobulbar palsy, progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), spinal muscular atrophy (SMA) such as spinal muscular atrophy with progressive myoclonic epilepsy (SMA- PME), SMN1-related spinal muscular atrophy, spinobulbar muscular atrophy (SBMA) and monomelic atrophy (MMA), as well as some rarer variants resembling ALS; (iii) Myotonic syndrome includes myotonic dystrophy type 1
  • the genetic neuromuscular disease is selected from the group comprising : (i) myopathies, such as congenital myopathies, myasthenic syndromes, metabolic myopathies, distal myopathies, muscular dystrophies with or without cardiomyopathy; and (ii) spinal muscular atrophies (SMAs) and motor neuron diseases; preferably SMN1-related spinal muscular atrophy, spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), spinobulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS).
  • myopathies such as congenital myopathies, myasthenic syndromes, metabolic myopathies, distal myopathies, muscular dystrophies with or without cardiomyopathy
  • SMAs spinal muscular atrophies
  • motor neuron diseases preferably SMN1-related spinal muscular atrophy, spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME), spinobulbar muscular atrophy (SBMA) and amyotrophic
  • Dystrophinopathies are a spectrum of X-linked muscle diseases caused by pathogenic variants in DMD gene, which encodes the protein dystrophin.
  • Dystrophinopathies comprises Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD) and DMD-associated dilated cardiomyopathy.
  • DMD Duchenne muscular dystrophy
  • BMD Becker muscular dystrophy
  • DMD-associated dilated cardiomyopathy a dilated cardiomyopathy.
  • LGMDs The Limb-girdle muscular dystrophies (LGMDs) are a group of disorders that are clinically similar to DMD but occur in both sexes as a result of autosomal recessive and autosomal dominant inheritance.
  • Limb-girdle dystrophies are caused by mutation of genes that encode sarcoglycans and other proteins associated with the muscle cell membrane, which interact with dystrophin.
  • LGMD1 refers to genetic types showing dominant inheritance (autosomal dominant), whereas LGMD2 refers to types with autosomal recessive inheritance.
  • Pathogenic variants at more than 50 loci have been reported (LGMD1A to LGMD1G; LGMD2A to LGMD2W).
  • Calpainopathy (LGMD2A) is caused by mutation of the gene CAPN3 with more than 450 pathogenic variants described.
  • LGMD Emery-Dreifuss Muscular Dystrophy
  • HSPs Hereditary spastic paraplegias
  • MECP2 hereditary spastic paraplegia
  • MBD methyl-CpG-binding domain recognizes and binds 5-mC regions.
  • MECP2 gene is X-linked and subject to X inactivation.
  • MECP2 gene mutations are the cause of most cases of Rett syndrome, a progressive neurologic developmental disorder and one of the most common causes of cognitive disability in females.
  • A Androgen Receptor
  • SBMA is an X-linked, adult-onset neuromuscular condition caused by an abnormal polyglutamine (polyQ) tract expansion in androgen receptor (AR) protein.
  • AR isoform 2 is a naturally occurring variant encoding a truncated AR lacking the polyQ harboring domain.
  • the solution or suspension may comprise additives which are compatible with viral vectors and do not prevent viral vector particle entry into target cells.
  • the form must be sterile and must be fluid to the extent that easy syringe ability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • An example of an appropriate solution is a buffer, such as phosphate buffered saline (PBS) or Ringer lactate.
  • the administration is intracerebral, intracerebroventricular, intracisternal, and/or intrathecal administration, alone or combined with parenteral administration, preferably intravascular, intramuscular or subcutaneous administration; more preferably intravascular such as intravenous (IV) or intraarterial administration, and even more preferably intravenous (IV).
  • the administration is parenteral, preferably intravascular alone or combined with intracerebral, intracerebroventricular, intracisternal, and/or intrathecal administration.
  • the initial time point administration and the later time point administration may be by the same route or a different route.
  • the initial time point and the later time point of administration of the combination of vectors are separated by several weeks, months or years.
  • the vector used for the initial administration and the vector used for the later time point administration encode the same transgene and the later time point is characterized by a decreased transgene expression level or therapeutic efficacy of the AAV vector administered at the initial time point.
  • the sequential administration of the combination of AAV vector serotypes via the systemic route produces sustained, high level transgene expression in target organs as compared to the administration of the vector used for the initial administration.
  • Figure 2 Neutralization antibody titers against various rAAV serotypes in mice injected with rAAVpo1
  • Figure 3 Neutralization antibody titers against rAAVpo1 in rats injected with various rAAV serotypes
  • Figure 4 Neutralization antibody titers against various rAAV serotypes in rats injected with rAAVpo1
  • A Seroprevalence in percent.
  • B Seropositivity in percent.
  • Figure 6 Biodistribution in skeletal muscles from rats injected with AAV8, AAV9 or AAVrh10 carrying rat Mtm1 gene, followed by AAVpo1.A1-Luciferase gene administration, or rats injected with AAVpo1.A1 carrying rat Mtm1 gene, followed by AAV8-, AAV9- or AAVrh10-Luciferase administration, and controls.
  • B Vector copy number in diaphragm.
  • Figure 7 Luciferase activity in skeletal muscles from rats injected with AAV8, AAV9 or AAVrh10 carrying rat Mtm1 gene, followed by AAVpo1.A1-Luciferase administration, or rats injected with AAVpo1.A1 carrying rat Mtm1 gene, followed by AAV8-, AAV9- or AAVrh10-Luciferase administration, and controls.
  • A Luciferase activity quantified by Relative Luminescence Unit (RLU) normalized by ⁇ g of proteins in Tibialis Anterior.
  • B Luciferase activity quantified by Relative Luminescence Unit (RLU) normalized by ⁇ g of proteins in diaphragm.
  • RLU Relative Luminescence Unit
  • EXAMPLE 1 Materials and Methods [000154] The neutralizing antibody assay was performed as previously described (Meliani et al., Hum. Gene Ther. Methods, 2015, 26, 45-53).
  • the 2V6.11 cell line (Accession number CVCL_6355) which inducibly expresses the human adenovirus E4 ORF 634kDa oncoprotein was previously described in Mohammadi et al. (Nucleic Acids Res., 2004, 32, 2652-2659). This cell line was chosen for its highest efficacy of AAV transduction as shown previously in Meliani et al., precited. Recombinant AAV vector serotypes express luciferase reporter gene under control of CMV promoter.
  • AAV9P1 vector was injected intravenously at 5 x 10 13 vg/kg in 4 mice at 7 weeks of age.
  • AAVpo1.A1 is AAVpo1 wild- type modified with peptide A1, previously disclosed in WO 2021/219762.
  • AAVpo1.A1 is representative of AAVpo1.
  • AAV9P1 was previously disclosed in Weinmann et al., Nat Commun. 2020 Oct 28;11(1):5432. doi: 10.1038/s41467-020-19230-w and WO 2019/207132.
  • AAVpo1 vector such as AAVpo1.A1 vector could be used for re-administration after an initial administration of AAV9, AAVrh10 or AAVrh74 vector.
  • mice injected with AAVpo1.A1 develop high levels of neutralizing antibodies against the injected serotype (AAVpo1.A1) as well as against AAVpo1WT (titer between 1:316 and 1:3160).
  • Neutralizing titers against AAV9 and AAV5 were lower in a range, between 1:3.16 and 1:31.6 (except 1:100 and 1:316 in 2/5 mice for AAV9).
  • neutralizing titers against AAV8 were much lower (undetectable in 3/5 mice; titer of 1:1 in 2/5 mice; Figure 2). 3.
  • Rats injected with AAV9, AAVrh10 develop neutralizing antibodies against the injected serotype but do not develop neutralizing antibodies against AAVpo1.A1 or at low titer (undetectable in 11-12/15 rats; titer of 1:1 in 3-4/15 rats; Figure 3). These results indicate that AAVpo1 vector such as AAVpo1.A1 vector could be used for re-administration after an initial administration of AAV9 or AAVrh10 vector. 4.
  • AAV vectors (AAVpo1.A1, AAV8, AAV9, AAVrh10) expressing MTM1 under the control of Desmin promoter were injected intravenously at 2x10 13 vg/kg in 5 rats at 3 weeks of age. Sera were collected 3 weeks post-injection and neutralizing antibody titers against AAV8, AAV9, AAVrh10 and AAVpo1.A1 were determined.
  • Rats injected with AAVpo1.A1 develop high levels of neutralizing antibodies against the injected serotype (AAVpo1.A1) (titers above 1:1000).
  • Neutralizing titers against AAV9 were lower at a level of 1:100. In contrast, neutralizing titers against AAV8 and AAVrh10, were much lower (for AAV8: titer of 1:1 in 3/5 rats, titer of 1:3.16 and 1:10 for 2/5 rats; for AAVrh10: undetectable in 2/5 rats; titer of 1:1, 1:3,16 and 1:10 in 3/5 rats; Figure 4). 5. Neutralization of rAAV from porcine AAV serotype 1 and various human or NHP serotypes by pooled human immunoglobulin [000163] Normal human immunoglobulin (TEGELINE; 50 mg/mL) was tested in neutralizing antibody assay.
  • TEGELINE Normal human immunoglobulin
  • All AAVpo1 (AAVpo1WT or AAVpo1A1) positive donors are AAV8 and AAV9 positive.
  • the neutralizing antibody titers against AAVpo1 (AAVpo1WT or AAVpo1A1) are lower than the neutralizing antibody titers against AAV8 and AAV9 ( Figure 5B).
  • AAVpo1A1 vector administered at a dose of 1x10 13 vg/kg, effectively transduced skeletal muscles following a first administration of AAV8, AAV9 or AAVrh10 vectors at a dose of 2x10 13 vg/kg, achieving results comparable to a single dose of AAVpo1A1.
  • the second administration of AAVpo1.A1 is neutralized by antibodies produced from the first injection of AAVpo1.A1, preventing transduction of the skeletal muscles by the second vector.

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Abstract

L'invention concerne une combinaison d'un vecteur AAVpo1 recombinant et d'un vecteur AAV9, AAVrh10 ou AAVrh74 recombinant destinée à être utilisée dans la thérapie génique de troubles musculaires et/ou neurologiques chez un individu en ayant besoin.
PCT/EP2024/088101 2023-12-21 2024-12-20 Procédés de ré-administration de vecteur aav Pending WO2025133247A1 (fr)

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