Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/76—Viruses; Subviral particles; Bacteriophages
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
  • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
• • 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
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2207/00—Modified animals
  • A01K2207/15—Humanized animals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • 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
  • C12N2510/00—Genetically modified 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
  • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14123—Virus like particles [VLP]
• • 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
  • C12N2750/14133—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
• • 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
  • 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
  • C12N2750/14145—Special targeting system for viral vectors

Definitions

• the disclosure herein relates to methods of making and using recombinant viral particles, e.g., recombinant AAV particles, comprising capsid proteins retargeted to a muscle-specific surface protein, e.g., Calcium Voltage-Gaged Auxiliary Subunit Gamma 1 (CACNG1) or Cadherin 15 (CADI 5), useful for modification of muscle cells, such as skeletal muscle cells, in vitro or in vivo.
• a muscle-specific surface protein e.g., Calcium Voltage-Gaged Auxiliary Subunit Gamma 1 (CACNG1) or Cadherin 15 (CADI 5
• a gene delivery vehicle is able to stably introduce genetic material into desired cells and avoid introducing genetic material into non-target cells.
• Viral particles particularly those based on adeno-associated virus (AAV), as a gene delivery vehicles have been the focus of much research since AAVs are capable of transducing a wide range of primate species and tissues in vivo with no evidence of toxicity or pathogenicity.
• AAV safely transduces postmitotic tissues.
• the virus can occasionally integrate into host chromosomes, it does so very infrequently into a safe-harbor locus in human chromosome 19, and only when the replication (Rep) proteins are supplied in trans.
• AAV genomes rapidly circularize and concatemerize in infected cells, and exist in a stable, episomal state in infected cells to provide long-term stable expression of their payloads.
• manipulating and redirecting AAV infection to specific cells has been achieved in recent years.
• Many of the advances in targeted gene therapy using viral particles may be summarized as non-recombinatorial (non-genetic) or recombinatorial (genetic) modification of the viral particle, which result in the pseudotyping, expanding, and/or retargeting of the natural tropism of the viral particle. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012: 1-15).
• a targeting ligand is directly inserted into, or coupled to, a viral capsid, i.e., protein viral capsid genes are modified to express capsid proteins comprising a heterologous targeting ligand.
• the targeting ligand then redirects, e.g., binds, a receptor or marker preferentially or exclusively expressed on a target cell.
• a viral capsid is modified with a heterologous “scaffold”, which then links to an adaptor that includes a targeting ligand.
• the adaptor binds to the scaffold and the target cell.
• Scaffolds such as (1) Fc binding molecules (e.g., Fc receptors, Protein A, etc.), which bind to the Fc of antibody adaptors, (2) (strept)avidin, which binds to biotinylated adaptors, (3) biotin, which binds to adaptors fused with (strept)avidin, (4) a detectable label, which is useful for detection and/or isolation of viral particles, bound by a bispecific adaptor able to non- covalently bind the detectable label and target molecule, and recently (5) protein: protein binding pairs that form isopeptide bonds have been described for a variety of viral particles.
• Fc binding molecules e.g., Fc receptors, Protein A, etc.
• streptavidin which binds to biotinylated adaptors
• biotin which binds to adaptors fused with (strept)avidin
• (4) a detectable label which is useful for detection and/or isolation of
• an AAV capsid protein may be modified to allow for the targeted introduction of a nucleotide of interest into mammalian skeletal muscle cells.
• Viral particles as described herein are particularly suited for the targeted introduction of a nucleotide of interest specifically to a muscle cell since the viral capsid or viral capsid protein(s) described herein comprise a targeting ligand that binds a muscle-cell specific surface protein.
• a viral capsid or viral capsid protein comprises a first member of a binding pair, associated with its cognate second member of the binding pair, wherein the second member is linked (e.g., fused to) a targeting ligand that binds a muscle-cell specific surface protein.
• the targeting ligand is operably linked to the second member, e.g., fused to the second member, optionally via a linker.
• a targeting ligand may be a binding moiety, e.g., a natural ligand, antibody, a multispecific binding molecule, etc.
• the targeting ligand is an antibody or portion thereof.
• the targeting ligand is an antibody comprising a variable domain that binds a muscle-specific surface protein on a muscle cell and a heavy chain constant domain.
• the targeting ligand is an antibody comprising a variable domain that binds a muscle-specific surface protein on a target cell and an IgG heavy chain constant domain.
• the targeting ligand is an antibody comprising a variable domain that binds a muscle-specific surface protein on a target cell and an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked, e.g., via a linker, to a protein (e.g., second member of a protein: protein binding pair) that forms an isopeptide covalent bond with the first member.
• a protein e.g., second member of a protein: protein binding pair
• a capsid protein described herein comprises a first member comprising SpyTag operably linked to the viral capsid protein, and covalently linked to the SpyTag, an second member comprising SpyCatcher linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, e.g., GSGESG (SEQ ID NO:253).
• the muscle-specific surfrase protein comprises CACNG1.
• the targeting ligand binds CACNG1, e.g., human CACNG1.
• the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3- LCDR1-LCDR2-LCDR3 comprising an amino acid sequence of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in any one of SEQ ID NOs: 1-240.
• Figure 1 shows AAV2-based retargeted virus infections delivering GFP to 293 cell lines genetically modified to express ASGR1 or CACNG1.
• the scatter plots are obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by cells positive (+) for hASGRl expression after infection with AAV2 WT particles, AAV2 HBM de-targeted mutant particles, AAV2 SpyTag anti-ASGRl particles, or AAV2 SpyTag anti- CACNG1 particles.
• GFP green fluorescent protein
• GFP green fluorescent protein
• AAV2 WT particles AAV2 HBM de-targeted mutant particles
• AAV2 SpyTag anti- ASGRl particles AAV2 SpyTag anti-CACNGl particles.
• Viruses express GFP as a marker of transduction.
• B The graph quantifies the percentage of GFP+ cells from the flow cytometry plots in (A).
• Figure 2 shows AAV9-based retargeted virus infections delivering GFP to 293 cell lines genetically modified to express ASGR1 or CACNG1.
• A The scatter plots are obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by cells positive (+) for hASGRl expression after infection with “AAV9 wt” particles, “AAV9 detargeted mutant” particles, “AAV9 SpyTag anti-ASGRl” particles, or “AAV9 SpyTag anti- CACNGl” particles.
• GFP green fluorescent protein
• GFP green fluorescent protein
• Figure 3 shows AAV2- and AAV9-based retargeted virus infections (MOI IxlO 6 ) delivering Luciferase to 293 cell lines genetically modified to express ASGR1 or CACNG1.
• MOI IxlO 6 retargeted virus infections
• a luciferase assay was performed to evaluate Firefly luciferase expression by cells positive (+) for hCACNGl after infection with AAV2 WT, AAV2 HBM+ anti- hASGRl, and AAV2 HBM+ anti-hCACNGl mAb#l particles.
• results of a luciferase assay evaluating Firefly luciferase expression by cells positive (+) for hASGRl after infection with AAV2 WT, AAV2 HBM+ anti-hASGRl, and AAV2 HBM+ anti- hCACNGl mAb#l particles.
• a luciferase assay was performed to evaluate Firefly luciferase expression by cells positive (+) for hCACNGl after infection with AAV9 WT, AAV9 N272A, AAV9 N272A+ anti-hASGRl full antibody, AAV9 N272A+ anti-hASGRl Fab, AAV9 N272A+ anti-CACNGl mAb#l full antibody, and AAV9 N272A+ anti- hCACNGl mAb#l Fab.
• a luciferase assay evaluating Firefly luciferase expression by cells positive (+) for hCACNGl after infection with AAV9 WT, AAV9 N272A, AAV9 N272A+ anti-hASGRl full antibody, AAV9 N272A+ anti-hASGRl Fab, AAV9 N272A+ anti-CACNGl mAb#l full antibody, and AAV9 N272A+ anti- hCACNGl mAb#l Fab.
• Figure 4 shows AAV2-based retargeted virus transduction of human skeletal myotubes.
• A Representative immunofluorescence images and
• B transduction efficiency assessed by quantifying average GFP expression in myosin heavy chain (MyHC) positive areas of human skeletal myotubes after transduction for 3 days with 2E+5vg/cell of the indicated AAV expressing eGFP under the control of the CAG promoter.
• MyHC myosin heavy chain
• Transduction efficiency was assessed in by quantifying the average GFP fluorescence intensity within the myosin heavy chain (MyHC) positive myotube areas.
• Figure 5 shows AAV9-based retargeted virus transduction of human skeletal myotubes.
• A representative immunofluorescence images and
• B transduction efficiency assessed by quantifying average GFP expression in myosin heavy chain (MyHC) positive areas of human skeletal myotubes after transduction for 3 days with 2E+5vg/cell of the indicated AAV expressing eGFP under the control of the CAG promoter.
• MyHC myosin heavy chain
• Figure 6 shows AAV9-based retargeted virus transduction of differentiated mouse C2C12 myotubes.
• A representative immunofluorescence images and (B) transduction efficiency assessed by quantifying average GFP expression in myosin heavy chain (MyHC) positive areas of differentiated mouse C2C12 myotubes transduced for 3 days with 2E+5vg/cell of the indicated AAV expressing eGFP under the control of the CAG promoter.
• MyHC myosin heavy chain
• FIG. 7 shows systemically delivered AAV2 retargeted to CACNG1 demonstrates antibody-dependent transduction of skeletal muscles in vivo.
• the graphs provide average radiance values (photons/sec/cm2/sr) from luminescence images of (A) liver, (B) tongue, (C) diaphragm, or (D) quadriceps (quad) tissue imaged ex vivo and isolated from mice genetically modified to express human CACNG1 on skeletal muscle cells (CACNG1 Humanized mice) and wildtype 50500 mice that were injected intravenously with phosphate buffered saline (PBS) or with 5el 1 viral genomes (vg)/ animal of wildtype (wt) AAV2 particles, AAV2 detargeted particles or SpyTagged AAV2 particles carrying firefly luciferase nucleotides of interest and modified by (1) SpyCatcher-anti-human ASGR1 antibody or (2) Spy Catcher-anti -human CACNG1 antibody.
• AAV2 viral particles are mosaic viral particles comprised of a 1 :7 ratio between (a) “SpyTag” capsids proteins wherein the SpyTag is inserted directly following residue G453 flanked on either side by a 10 amino acid linker and (b) capsids without SpyTag but containing R585A and R588A mutation, which reduces natural receptor binding.
• Viruses express Firefly luciferase as a marker of transduction. Five weeks post IV injection, mice were anesthetized using isoflurane, injected with a Luciferin substrate and euthanized 7-10 minutes later. Organs were harvested and imaged using IVIS Spectrum in vivo Imaging System (PerkinElmer). The raw data was analyzed using living image software to determine average radiance (photons/sec/cm2/sr).
• FIG 8 shows systematically delivered AAV9 retargeted to CACNG1 demonstrates antibody-dependent transduction of skeletal muscles in vivo.
• the graphs provide average radiance values (photons/sec/cm2/sr) from luminescence images of (A) liver, (B) hindlimb, (C) quadriceps (quad), or (D) tongue tissue imaged ex vivo and isolated from mice genetically modified to express human CACNG1 (CACNG1 humanized mice) injected intravenously with phosphate buffered saline (PBS) or with 5el0 viral genomes (vg)/ animal of wildtype (wt) AAV9 particles, AAV9 detargeted particles or SpyTagged AAV9 particles carrying firefly luciferase nucleotide of interest and modified by (1) Spy Catcher-anti -human ASGR1 full antibody, (2) SpyCatcher-anti-human ASGR1 Fab, (3) SpyCatcher-anti-human CACNG1 mAb
• AAV9 viral particles are mosaic viral particles comprised of a 1 :7 ratio between (a) “SpyTag” capsids proteins wherein the SpyTag is inserted directly following residue G453 flanked on both sides by a 10 amino acid linker and (b) capsids without SpyTag but containing an N272A mutation which reduces natural receptor binding.
• Viruses express Firefly luciferase as a marker of transduction.
• mice were anesthetized using isoflurane, injected with a Luciferin substrate and euthanized 7-10 minutes later.
• the following organs were harvested for ex vivo imaging: liver, hindlimb, quad, and tongue.
• the organs were imaged using IVIS Spectrum in vivo Imaging System (PerkinElmer). The raw data was analyzed using living image software to determine average radiance (photons/sec/cm2/sr).
• Figure 9 displays GFP gene expression analysis in the liver and quadriceps muscle of (A) CACNGl hu/hu , (B) WT C57BL/6, and (C) D2-mdx mice 3 weeks after tail vein injection of 1E+11 vg/mouse of wildtype AAV9, de-targeted AAV9 N272A, and AAV9 conjugated to antibodies targeting CACNG1 (mAb#l and mAb #2) or hASGRl as a nontargeting control.
• GFP expression was quantified via a Taqman-based qPCR assay and normalized to RplpO as an endogenous control.
• GFP mRNA expression is displayed relative to WT AAV9 for each tissue/mouse strain.
• Figure 10 displays representative immunofluorescence images of the tibialis anterior and gastrocnemius/plantaris/soleus muscles of D2-mdx mice 3 weeks after tail vein injection of 1E+11 vg/mouse of wildtype AAV9, de-targeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 (mAb#l, mAb#2, and mAb#3) or hASGRl as a non-targeting control.
• CACNG1 mAb#l, mAb#2, and mAb#3
• FIG 11 shows immunohistochemistry staining for eGFP expression in the liver and quadriceps of D2-mdx mice following injection of wildtype AAV9, de-targeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 or hASGRl as a non-targeting control.
• AAV9 wildtype particles can transduce the liver of D2-mdx mice, while AAV9 N272A particles are detargeted from the liver and do not express GFP in the liver.
• AAV9 N272A particles conjugated to antibodies that bind to both human and mouse CACNG1 (mAb#2 and mAb#3) show very little GFP staining in the liver and strong GFP staining in the quadriceps.
• Figure 12 shows immunohistochemistry staining for eGFP expression in the gastrocnemius/plantaris/soleus of D2-mdx mice following injection of wildtype AAV9, detargeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 or hASGRl as a non-targeting control.
• AAV9 wildtype particles can transduce the gastrocnemius/plantaris/soleus of D2-mdx mice at a low level, while AAV9 N272A particles transduce the gastrocnemius/plantaris/soleus with limited efficiency.
• D2-mdx mice injected with AAV9 N272A conjugated to an irrelevant antibody (hASGRl) or to a CACNG1- targeting antibody specific for human CACNG1 that does not bind mouse CACNG1 (mAb#l) show very little staining in the gastrocnemius/plantaris/soleus.
• AAV9 N272A particles conjugated to antibodies that bind to both human and mouse CACNG1 show very strong GFP staining in the gastrocnemius/plantaris/soleus.
• Figure 13 shows immunohistochemistry staining for eGFP expression in the tibialis anterior of D2-mdx mice following injection of wildtype AAV9, de-targeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 or hASGRl as a non-targeting control.
• AAV9 wildtype particles can transduce the tibialis anterior of D2-mdx mice at a low level.
• AAV9 N272A particles conjugated to antibodies that bind to both human and mouse CACNG1 show very strong GFP staining around the periphery of the tibialis anterior.
• Figure 14 shows immunohistochemistry staining for eGFP expression in the heart and tongue of D2-mdx mice following injection of wildtype AAV9, de-targeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 or hASGRl as a non-targeting control.
• AAV9 wildtype particles can transduce the tongue of D2-mdx mice at a low level, but can transduce the heart efficiently.
• AAV9 N272A particles conjugated to antibodies that bind to both human and mouse CACNG1 (mAb#2 and mAb#3) show very strong GFP staining in the tongue, with low but detectable levels of staining in the heart.
• Figure 15 shows immunohistochemistry staining for eGFP expression in the spleen and diaphragm of D2-mdx mice following injection of wildtype AAV9, de-targeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 or hASGRl as a non-targeting control.
• AAV9 wildtype particles can transduce the diaphragm of D2-mdx mice at a low level.
• D2-mdx mice injected with AAV9 N272A particles alone, or AAV9 N272A conjugated to an irrelevant antibody (hASGRl, or to a CACNG1 -targeting antibody that binds human and monkey CACNG1 but not mouse CACNG1 (mAb#l) show very little staining in the diaphragm.
• AAV9 N272A particles conjugated to antibodies that bind to both human and mouse CACNG1 (mAb#2 and mAb#3) show very strong GFP staining in the diaphragm. Very little transduction of spleen is observed with any of the tested AAVs, as expected.
• Figure 16 provides an illustrative schematic (not to scale) of the single stranded (ss) viral genome comprising from 5’ to 3’ : a 141 base pair inverted terminal repeat (ITR), a CAGG promoter, a sequence encoding enhanced green fluorescent protein (GFP), Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a 32 base pair barcode, human (h) grown hormone (GH) poly A tail, and the 141 base pair ITR.
• ITR inverted terminal repeat
• GFP enhanced green fluorescent protein
• WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
• GH grown hormone
• Figure 17 provides bar graphs that demonstrate enhanced transduction to various muscles in vivo in non-human primates (cynomolgus monkey) after administration of AAV9 viral particles comprising the viral genome depicted in Figure 16, each with a unique barcode, and retargeted with anti-CACNGl antibodies, compared to wildtype AAV9 viral particles (AAV) comprising the viral genome depicted in Figure 16.
• AAV9 viral particles comprising the viral genome depicted in Figure 16
• Each candidate AAV was packaged with a unique barcoded genome as described in Fig 16. Following IV dosing of the 12 candidate barcoded pool, the indicated tissues were collected and relative abundance of each barcode in the total RNA purified from each tissue was assessed using next generation sequencing (NGS).
• NGS next generation sequencing
• the data represented here is the mean of two animals in the study.
• AAV9 alone and AAV9 W503A or N272A conjugated to an ASGR1 mAb represent the majority of all barcodes present in the tissue, as expected.
• detargeted AAV9 (N272A or W503A) capsids conjugated to CACNG1 targeting antibodies represent the majority of all barcodes present in the tissue, outperforming AAV9 alone, which accounted for a small percentage of total barcodes.
• Figure 18 shows AAV9-based retargeted virus transduction of human skeletal myotubes and C2C12 mouse myotubes using a vector genome construct expressing uDys5.
• the graphs provide transduction efficiency assessed by quantifying relative uDys5 mRNA expression in (A) human myotubes and (B) C2C12 mouse myotubes after transduction for 3 days with 2E+5vg/cell of either AAV9 WT or a de-targeted AAV9 (N272A) conjugated to an antibody targeting CACNG1 (mAb#3) that express uDys5 under the control of the CK8 promoter.
• uDys5 expression was quantified via a Taqman-based qPCR assay and normalized to Hprt as an endogenous control.
• uDys5 mRNA expression is displayed relative to WT AAV9 for each cell type.
• AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 (mAb#3) produce higher levels of uDys5 mRNA relative to AAV9 WT in both human and mouse myotubes.
• Figure 19 displays uDys5 gene expression in multiple tissues from D2-mdx mice 5 weeks after tail vein injection of 1E+12 vg/mouse of either AAV9 WT or a detargeted AAV9 (N272A) conjugated to an antibody targeting CACNG1 (mAb#3) that express uDys5 under the control of the CK8 promoter.
• uDys5 mRNA expression was quantified via a Taqman-based qPCR assay and normalized to RplpO as an endogenous control.
• uDys5 mRNA expression is displayed relative to WT AAV9 for each tissue.
• AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 show reduced transduction of the liver relative to AAV9 WT as expected, and produce extremely low levels of uDys5 mRNA relative to AAV9 WT in the liver.
• AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 produce lower levels of uDys5 mRNA relative to AAV9 WT in the heart, but the levels are detectable.
• AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 (mAb#3) produce higher levels of uDys5 mRNA relative to AAV9 WT in all skeletal muscles examined.
• Figure 20 displays representative immunofluorescence images of the gastrocnemius muscle and heart of wildtype DB A2/J and D2-mdx mice 5 weeks after tail vein injection of 1E+12 vg/mouse of wildtype AAV9 or de-targeted AAV9 N272A particles conjugated to antibodies targeting CACNG1 (mAb#3) expressing uDys5 under the control of the CK8 promoter.
• mice injected with WT AAV9 show a low level of expression of dystrophin at the myofiber membrane in the gastrocnemius, and robust expression in the heart; whereas mice injected with de-targeted AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 (mAb#3) display robust dystrophin expression at the myofiber membrane in the gastrocnemius, and mild expression in the heart.
• Figure 21 displays protein abundance of uDys5 in the quadriceps muscle of D2-mdx mice 5 weeks after tail vein injection of 1E+12 vg/mouse of wildtype AAV9 or detargeted AAV9 N272A particles conjugated to antibodies targeting CACNG1 (mAb#3) expressing uDys5 under the control of the CK8 promoter.
• P-actin was used as a protein loading control, and protein abundance was quantified and plotted as arbitrary densitometry units (A.U.).
• Mice injected with de-targeted AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 (mAb#3) display substantially more uDys5 protein compared to mice injected with wildtype AAV9.
• Figure 22 displays serum creatine kinase (CK) levels in D2-mdx mice 4 weeks after tail vein injection of 1E+12 vg/mouse of wildtype AAV9 or de-targeted AAV9 N272A particles conjugated to antibodies targeting CACNG1 (mAb#3) expressing uDys5 under the control of the CK8 promoter.
• PBS treated and wildtype AAV9 treated mice did not display a decrease in serum CK following treatment, while all mice that were treated with detargeted AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 (mAb#3) displayed a reduction of serum CK, indicating reduced muscle damage.
• Figure 23 displays maximal forelimb grip strength measurements in D2-mdx mice 12 weeks after tail vein injection of 1E+12 vg/mouse of wildtype AAV9 or de-targeted AAV9 N272A particles conjugated to antibodies targeting CACNG1 (mAb#3) expressing uDys5 under the control of the CK8 promoter.
• Dotted line represents grip strength of age- matched control DBA/2J mice.
• Mice injected with AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 (mAb#3) display enhanced maximum grip strength relative to the mice injected with WT AAV9 particles.
• Figure 24 shows serum levels of liver enzymes and complement pathway biomarkers in non-human primates (cynomolgous monkey) at the indicated timepoints following injection of wildtype AAV9 or AAV9 N272A conjugated to antibodies targeting CACNG1 (mAb#3) expressing eGFP under the control of a CAG promoter.
• AAV9 wildtype particles resulted in an elevation of (A) ALT, (B) AST, (C) Bb and (D) C3a 48 hours post-dosing, as expected, while administration of AAV9 N272A conjugated to antibodies targeting CACNG1 (mAb#3) did not result in an elevation of these markers, suggesting that liver-detargeted AAV9 N272A particles conjugated to antibodies targeting CACNG1 provide a safety advantage over liver-tropic wildtype AAV serotypes.
• WT refers to wildtype, e.g., AAV capsid proteins with no mutations or modifications.
• HBM capsid proteins comprising R585A and R588A.
• All “reference’7“detargeted” AAV2 capsid proteins, e.g., AAV capsid proteins without a SpyTag modification comprise only R585A and R588A.
• All AAV2 capsid proteins in the figures described above that display SpyTag bound to a SpyCatcher fused antibody further comprise R484A, R487A, R585A, R588A, and K532A mutations.
• N272A refers capsids comprising an N272A mutations.
• all “reference’7“detargeted” AAV9 capsid proteins e.g., AAV9 capsid proteins without a SpyTag modification, comprise an N272A mutation.
• all AAV9 capsid proteins with a SpyTag modification further comprise a W503 A mutation but not the N272A mutation.
• anti CACNG1 in association with an AAV refers to AAV viral capsids comprising an insertion of the SpyTag peptide directly following residue G453 flanked on both sides by a 10 amino acid linker, where the SpyTag peptide is bound by an isopeptide bond to a SpyCatcher fused to an anti- CACNG1 antibody (or Fab fragment thereof) that specifically binds CACNG1.
• All AAV particles displaying the SpyTag peptide bound by an isopeptide bond to a SpyCatcher fused to an anti-CACNGl antibody (or Fab fragment thereof) that specifically binds CACNG1 in in the figures described above comprise a mosaic viral capsid comprising a 1 :7 ratio of SpyTag modified viral capsid proteins to “detargeted” viral proteins (denoted “1/8”).
• CACNG1 mAb#l refers to an anti-CACNGl antibody that binds human and monkey CACNG1, but does not bind mouse CACNG1.
• CACNG1 mAb#2 refers to an anti-CACNGl antibody that binds human, monkey, and mouse CACNG1.
• CACNG1 mAb#3 refers to an anti-CACNGl antibody that binds human, monkey, and mouse CACNG1.
• CACNG1 mAb#4 refers to an anti-CACNGl antibody that binds human, monkey, and mouse CACNG1.
• CACNG1 mAb#5 refers to an anti-CACNGl antibody that binds human and monkey, but not mouse CACNG1.
• CACNG1 in association with 293 cells or mice respectively refer to 293 cells or mice genetically modified to express human C ACNG1.
• CACNGl hu/hu Mice comprising a homozygous replacement of endogenous Cacngl with human Cacngl sequences are referred to as “CACNGl hu/hu ”.
• mice refers to a strain-matched control for CACNG1 mice.
• anti ASGR1 in association with an AAV refers to AAV viral capsids comprising an insertion of the SpyTag peptide directly following residue G453 flanked on both sides by a 10 amino acid linker, where the SpyTag peptide is bound by an isopeptide bond to a SpyCatcher fused to an anti-ASGRl antibody or Fab fragment thereof that specifically binds ASGR1.
• All AAV particles displaying the SpyTag peptide bound by an isopeptide bond to a SpyCatcher fused to an anti-ASGRl antibody (or Fab fragment thereof) that specifically binds ASGR1 in the figures described above comprise a mosaic viral capsid comprising a 1 :7 ratio of SpyTag modified viral capsid proteins to “detargeted” viral capsid proteins, (denoted “1/8”).
• ASGR1 in association with 293 cells or mice respectively refer to 293 cells or mice genetically modified to express human ASGR1.
• Vk refers to an antibody light chain
• D2-mdx refers to a mouse model for Duchenne muscular dystrophy
• Skeletal muscle is the largest organ in the body, comprising -40% of total body mass. Skeletal muscle is one of the three significant muscle tissues in the human body. Each skeletal muscle consists of thousands of muscle fibers wrapped together by connective tissue sheaths.
• the primary functions of the skeletal muscle take place via its intrinsic excitation-contraction coupling process. As the muscle is attached to the bone tendons, the contraction of the muscle leads to movement of that bone that allows for the performance of specific movements.
• the skeletal muscle also provides structural support and helps in maintaining the posture of the body.
• the skeletal muscle also acts as a storage source for amino acids that can be used by different organs of the body for synthesizing organ-specific proteins.
• the skeletal muscle also acts as a storage source of glucose in the form of glycogen.
• the skeletal muscle also plays a central role in maintaining thermostasis and acts as an energy source during starvation. Thus, skeletal muscle plays key roles in locomotion, thermoregulation, and in controlling whole body metabolism.
• the size and function of skeletal muscle tissue is reduced, resulting in impaired functional mobility; and in the case of severe muscle diseases, long-term disability and early mortality.
• Treatments for muscle wasting and genetic muscle diseases typically consist of broad-acting therapies, such as testosterone therapy for muscle wasting, glucocorticoids for muscular dystrophies, and systemic AAV delivery for treatment of muscle diseases (e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), Facioscapulohumeral muscular dystrophy Type 1 (FSHD), congenital muscular dystrophy type 1 A (MDC1 A), Limb girdle muscular dystrophy, and dystroglycanopathy, etc.).
• XLMTM X-linked myotubular myopathy
• DMD Duchenne muscular dystrophy
• DM1 myotonic dystrophy
• FSHD Facioscapulohumeral muscular dystrophy Type 1
• MDC1 A congenital muscular dystrophy type 1 A
• Limb girdle muscular dystrophy and dystroglycanopathy, etc.
• viral particles e.g., AAV viral particles
• target musclespecific surface proteins such as Calcium Voltage-Gaged Auxiliary Subunit Gamma 1 (CACNG1) or mammalian Cadherin 15 (CAD15).
• CACNG1 Calcium Voltage-Gaged Auxiliary Subunit Gamma 1
• CAD15 mammalian Cadherin 15
• Voltage-dependent calcium channels are generally composed of five subunits.
• the protein encoded by the CACNG1 gene represents one of these subunits.
• the protein encoded by the CACNG1 gene, gamma is one of two known gamma subunit proteins. This particular gamma subunit is part of skeletal muscle 1,4-dihydropyridine-sensitive calcium channels and is an integral membrane protein that plays a role in excitationcontraction coupling.
• This gene is part of a functionally diverse eight-member protein subfamily of the PMP-22/EMP/MP20 family and is located in a cluster with two family members that function as transmembrane AMPA receptor regulatory proteins (TARPs).
• CACNG1 is highly and specifically expressed in skeletal muscle.
• CACNGJ The gene encoding human CACNG1 (CACNGJ) is located on the long arm of chromosome 17.
• CACNG1 comprises 4 exons and is approximately 12,244 bases long.
• An exemplary sequence for human CACNG1 gene is assigned NCBI Accession Number NM_0007582.2 (SEQ ID NO:241).
• An exemplary human CACNG1 protein is assigned UniProt Accession No. 070578 (SEQ ID NO:242).
• the "percent (%) identity” or the like may be readily determined for amino acid or nucleotide sequences, over the full-length of a protein, or a portion thereof. A portion may be at least about 5 amino acids or 24 nucleotides, respectively, in length, and may be up to about 700 amino acids or 2100 nucleotides, respectively. Generally, when referring to “identity”, “homology”, or “similarity” between two different adeno-associated viruses, “identity”, “homology” or “similarity” is determined in reference to “aligned” sequences. "Aligned" sequences or “alignments” refer to multiple nucleic acid sequences or protein (amino acids) sequences, often containing corrections for missing or additional bases or amino acids as compared to a reference sequence.
• Alignments may be performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Sequence alignment programs are available for amino acid sequences, e.g., the "Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box” programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See, e.g., J. D. Thomson et al, Nucl. Acids.
• FastaTM provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTATM with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference.
• “Significant identity” encompasses amino acid or nucleic acid sequences alignments that are at least 90%, e.g., at least 93%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, or e.g., at least 100% identical.
• chimeric encompasses a functional gene or polypeptide comprising nucleic acid sequences or amino acid sequences, respectively, from at least two different AAV serotype, e.g., portions of a gene or polypeptide of at least a first and second AAV, wherein the at least first and second portions are operably linked to form a functional chimeric AAV nucleic acid that encodes a functional amino acid.
• nucleotide sequences, genes, polypeptides, and amino acids are considered nonchimeric in that the nucleotide sequences, genes, polypeptides, and amino acids comprise a nucleic acid sequence or amino acid sequence having significant identity to a nucleic acid sequence or amino acid sequence, respectively, of a single AAV serotype.
• antibody includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
• Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant region (CH).
• the heavy chain constant region comprises at least three domains, CHI, CH2, CH3 and optionally CEU.
• Each light chain comprises a light chain variable domain (CH) and a light chain constant region (CL).
• CDR complementarity determining regions
• FR framework regions
• Each heavy and light chain variable domain comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3.
• Typical tetrameric antibody structures comprise two identical antigen-binding domains, each of which formed by association of the VH and VL domains, and each of which together with respective CH and CL domains form the antibody Fv region.
• Single domain antibodies comprise a single antigen-binding domain, e.g., a VH or a VL.
• the antigen-binding domain of an antibody e.g., the part of an antibody that recognizes and binds to the first member of a specific binding pair of an antigen, is also referred to as a “paratope.” It is a small region (of 5 to 10 amino acids) of an antibody's Fv region, part of the fragment antigen-binding (Fab region), and may contain parts of the antibody's heavy and/or light chains.
• a paratope specifically binds a first member of a specific binding pair when the paratope binds the first member of a specific binding pair with a high affinity.
• high affinity antibody refers to an antibody that has a KD with respect to its target first member of a specific binding pair about of 10' 9 M or lower (e.g., about 1 x 10' 9 M, l x IO' 10 M, 1 x 10' 11 M, or about 1 x 10' 12 M).
• KD is measured by surface plasmon resonance, e.g., BIACORETM; in another embodiment, KD is measured by ELISA.
• CDR complementarity determining region
• a CDR includes an amino acid sequence encoded by a nucleic acid sequence of an organism’s immunoglobulin genes that normally (i.e., in a wild-type animal) appears between two framework regions in a variable region of a light or a heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor).
• a CDR can be encoded by, for example, a germ line sequence or a rearranged or unrearranged sequence, and, for example, by a naive or a mature B cell or a T cell.
• a CDR can be somatically mutated (e.g., vary from a sequence encoded in an animal’s germ line), humanized, and/or modified with amino acid substitutions, additions, or deletions.
• CDRs can be encoded by two or more sequences (e.g., germ line sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in a B cell nucleic acid sequence, e.g., as the result of splicing or connecting the sequences (e.g., V-D-J recombination to form a heavy chain CDR3).
• light chain includes an immunoglobulin light chain sequence from any organism, and unless otherwise specified includes human K and X light chains and a VpreB, as well as surrogate light chains.
• Light chain variable domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified.
• FR framework
• a full-length light chain includes, from amino terminus to carboxyl terminus, a variable domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region.
• a light chain variable domain is encoded by a light chain variable region gene sequence, which generally comprises VL and JL segments, derived from a repertoire of V and J segments present in the germ line.
• Light chains include those, e.g., that do not selectively bind either a first or a second first member of a specific binding pair selectively bound by the first member of a specific binding pairbinding protein in which they appear. Light chains also include those that bind and recognize, or assist the heavy chain or another light chain with binding and recognizing, one or more first member of a specific binding pairs selectively bound by the first member of a specific binding pair-binding protein in which they appear.
• Common or universal light chains include those derived from a human VK1-39JK gene or a human VK3-20JK gene, and include somatically mutated (e.g., affinity matured) versions of the same.
• Exemplary human VL segments include a human VK1-39 gene segment, a human VK3-20 gene segment, a human V/ -40 gene segment, a human V/J -44 gene segment, a human V/.2-8 gene segment, a human V/.2- I4 gene segment, and human V/3 -21 gene segment, and include somatically mutated (e.g., affinity matured) versions of the same.
• Light chains can be made that comprise a variable domain from one organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken) and a constant region from the same or a different organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken).
• one organism e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken
• a constant region from the same or a different organism
• the term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range.
• the allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.
• immunoglobulin heavy chain includes an immunoglobulin heavy chain sequence, including immunoglobulin heavy chain constant region sequence, from any organism.
• Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof.
• a typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CHI domain, a hinge, a CH2 domain, and a CH3 domain.
• a functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an first member of a specific binding pair (e.g., recognizing the first member of a specific binding pair with a KD in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.
• Heavy chain variable domains are encoded by variable region nucleotide sequence, which generally comprises VH, DH, and JH segments derived from a repertoire of VH, DH, and JH segments present in the germline. Sequences, locations and nomenclature for V, D, and J heavy chain segments for various organisms can be found in IMGT database, which is accessible via the internet on the world wide web (www) at the URL “imgt.org.”
• the term "heavy chain only antibody,” “heavy chain only antigen binding protein,” “single domain antigen binding protein,” “single domain binding protein” or the like refers to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, that is unable to associate with a light chain because the heavy chain constant region typically lacks a functional CHI domain.
• the term "heavy chain only antibody,” “heavy chain only antigen binding protein,” “single domain antigen binding protein,” “single domain binding protein” or the like encompasses a both (i) a monomeric single domain antigen binding protein comprising one of the immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region lacking a functional CHI domain, or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each of which comprising a variable domain operably linked to a heavy chain constant region lacking a functional CHI domain.
• a homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which comprising an identical variable domain operably linked to an identical heavy chain constant region lacking a functional CHI domain.
• each immunoglobulin-like chain of a single domain antigen binding protein comprises a variable domain, which may be derived from heavy chain variable region gene segments (e.g., VH, DH, JH), light chain gene segments (e.g., VL, JL), or a combination thereof, linked to a heavy chain constant region (CH) gene sequence comprising a deletion or inactivating mutation in a CH 1 encoding sequence (and, optionally, a hinge region) of a heavy chain constant region gene, e.g., IgG, IgA, IgE, IgD, or a combination thereof.
• CH heavy chain constant region
• a single domain antigen binding protein comprising a variable domain derived from heavy chain gene segments may be referred to as a " VH- single domain antibody” or "VH-single domain antigen binding protein”, see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 20140289876; 20150197553; 20150197554; 20150197555; 20150196015; 20150197556 and 20150197557, each of which is incorporated in its entirety by reference.
• a single domain antigen binding protein comprising a variable domain derived from light chain gene segments may be referred to as a or "VL-single domain antigen binding protein," see, e.g., U.S. Publication No. 20150289489, incorporated in its entirety by reference.
• light chain includes an immunoglobulin light chain sequence from any organism, and unless otherwise specified includes human kappa (K) and lambda (X) light chains and a VpreB, as well as surrogate light chains.
• Light chain variable domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified.
• FR framework
• a full-length light chain includes, from amino terminus to carboxyl terminus, a variable domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence.
• Light chain variable domains are encoded by the light chain variable region nucleotide sequence, which generally comprises light chain VL and light chain JL gene segments, derived from a repertoire of light chain V and J gene segments present in the germline. Sequences, locations and nomenclature for light chain V and J gene segments for various organisms can be found in IMGT database, which is accessible via the internet on the world wide web (www) at the URL “imgt.org.” Light chains include those, e.g., that do not selectively bind either a first or a second first member of a specific binding pair selectively bound by the first member of a specific binding pair-binding protein in which they appear.
• Light chains also include those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more first member of a specific binding pairs selectively bound by the first member of a specific binding pair-binding protein in which they appear.
• Light chains also include those that bind and recognize, or assist the heavy chain with binding and recognizing, one or more first member of a specific binding pairs selectively bound by the first member of a specific binding pair-binding protein in which they appear.
• Common or universal light chains include those derived from a human VK1-39JK5 gene or a human VK3-20JK1 gene, and include somatically mutated e.g., affinity matured) versions of the same.
• operably linked includes a physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact, directly or indirectly with one another, or otherwise coordinate with each other to participate in a biological event, which juxtaposition achieves or permits such interaction and/or coordination.
• a regulatory element e.g., an expression control sequence
• operably linked involves covalent linkage of relevant components or elements with one another.
• covalent linkage is not required to achieve effective operable linkage.
• proteins operably linked together may be associated with each other, e.g., via a covalent bond or a non-covalent bond.
• a capsid protein as describd herein may be operably linked to a targeting ligand, where the capsid protein is non-covalently bound to the targeting ligand, or covalently bound to the targeting ligand, optionally with or without a scaffold and/or adaptor between the capsid protein and the targeting ligand.
• nucleic acid regulatory elements that are operably linked with coding sequences that they control are contiguous with the nucleotide of interest.
• one or more such regulatory elements acts in trans or at a distance to control a coding sequence of interest.
• regulatory element refers to polynucleotide sequences which are necessary and/or sufficient to effect the expression and processing of coding sequences to which they are ligated.
• a regulatory element may be or comprise appropriate transcription initiation, termination, promoter and/or enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and/or, in some embodiments, sequences that enhance protein secretion.
• one or more regulatory elements are preferentially or exclusively active in a particular host cell or organism, or type thereof.
• regulatory elements may typically include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, in many embodiments, regulatory elements may typically include promoters, enhancers, and/or transcription termination sequences.
• regulatory elments refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and/or fusion partner sequences).
• “Retargeting” or “redirecting” may include a scenario in which the wildtype particle targets several cells within a tissue and/or several organs within an organism, and general targeting of the tissue or organs is reduced or abolished by insertion of the heterologous amino acid, and retargeting to more a specific cell in the tissue or a specific organ in the organism is achieved with the targeting ligand (e.g., via a targeting ligand) that binds a marker expressed by the specific cell.
• the targeting ligand e.g., via a targeting ligand
• Such retargeting or redirecting may also include a scenario in which the wildtype particle targets a tissue, and targeting of the tissue is reduced to or abolished by insertion of the heterologous amino acid, and retargeting to a completely different tissue is achieved with the targeting ligand.
• “Specific binding pair,” “binding pair,” “protein: protein binding pair” and the like includes two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a non- covalent bond between a first member epitope and a second member antigen-binding portion of an antibody that recognizes the epitope; a covalent bond between e.g., proteins capable of forming isopeptide bonds; split inteins that recognize each other and, through the process of protein trans-splicing, mediate ligation of the flanking proteins and their own removal).
• a bond e.g., a non- covalent bond between a first member epitope and a second member antigen-binding portion of an antibody that recognizes the epitope
• a covalent bond between e.g., proteins capable of forming isopeptide bonds split intein
• cognate refers to components that function together.
• Epitopes and cognate antibodies thereto, particularly epitopes that may also act as a detectable label (e.g., c-myc) are well-known in the art.
• Specific protein: protein binding pairs capable of interacting to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol.
• a first member of a protein: protein binding pair refers to member of a protein: protein binding pair, which is generally less than 30 amino acids in length, and which forms a spontaneous covalent isopeptide bond with the second cognate protein, wherein the second cognate protein is generally larger, but may also be less than 30 amino acids in length such as in the SpyTag:KTag system.
• isopeptide bond refers to an amide bond between a carboxyl or carboxamide group and an amino group at least one of which is not derived from a protein main chain or alternatively viewed is not part of the protein backbone.
• An isopeptide bond may form within a single protein or may occur between two peptides or a peptide and a protein.
• an isopeptide bond may form intramolecularly within a single protein or intermolecularly i.e. between two peptide/protein molecules, e.g. between two peptide linkers.
• an isopeptide bond may occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue or the terminal carboxyl group of the protein or peptide chain or may occur between the alpha-amino terminus of the protein or peptide chain and an asparagine, aspartic acid, glutamine or glutamic acid.
• Each residue of the pair involved in the isopeptide bond is referred to herein as a reactive residue.
• an isopeptide bond may form between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue.
• isopeptide bonds can occur between the side chain amine of lysine and carboxamide group of asparagine or carboxyl group of an aspartate.
• Zakeri et al. obtained a peptide “SpyTag” having the sequence AHIVMVDAYKPTK (SEQ ID NO:243) which forms an amide bond to its cognate protein “SpyCatcher,” an 112 amino acid polypeptide having the amino acid sequence set forth in SEQ ID NO:244. (Zakeri (2012), supra).
• SpyTag:KTag An additional specific binding pair derived from CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase.
• SpyLigase was engineered by excising the P strand from SpyCatcher that contains a reactive lysine, resulting in KTag, 10-residue first member of a protein: protein binding pair having the amino acid sequence ATHIKFSKRD (SEQ ID NO:245).
• the SpyTag002:SpyCatcher002 system is described in Keeble et al (2017) Angew Chem Int Ed Engl 56: 16521-25, incorporated herein in its entirety by reference.
• SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, set forth as SEQ ID NO:255, and binds SpyCatcher002.
• SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK, set forth as SEQ ID NO:259, and binds SpyCatcher003.
• SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113: 1202-07.
• the D4 Ig-like domain of RrgA an adhesion from Streptococcus pneumoniae, was split to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860).
• SnoopTag an adhesion from Streptococcus pneumoniae
• the isopeptag:pilin-C specific binding pair was derived from the major pilin protein Spy0128 from Streptococcus pyogenes. (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). Isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, set forth as SEQ ID NO:254, and binds pilin-C (residues 18-299 of Spy0128). Incubation of SnoopTag and SnoopCatcher results in a spontaneous isopeptide bond that is specific between the complementary proteins. Zakeir and Howarth (2010), supra.
• detectable label includes a polypeptide sequence that is a member of a specific binding pair, e.g., that specifically binds via a non-covalent bond with another polypeptide sequence, e.g., an antibody paratope, with high affinity.
• detectable labels include hexahistidine tag, FLAG tag, Strep II tag, streptavidin- binding peptide (SBP) tag, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and the myc tag from c-myc (SEQ ID NO:246).
• a common detectable label for primate AAV is the Bl epitope (SEQ ID NO:247).
• Some AAV capsid proteins describedherein, which do not naturally comprise the Bl epitope, may be modified herein to comprise a Bl epitope.
• AAV capsid proteins described herein may comprise a sequence with substantial homology to the Bl epitope within the last 10 amino acids of the capsid protein.
• a non-primate AAV capsid protein of the invention may be modified with one but less than five point mutations within the last 10 amino acids of the capsid protein such that the AAV capsid protein comprises a Bl epitope.
• target cells includes any cells in which expression of a nucleotide of interest is desired.
• target cells exhibit a receptor on their surface that allows the cell to be targeted with a targeting ligand, as described below.
• transduction or “infection” or the like refers to the introduction of a nucleic acid into a target cell nucleus by a viral particle.
• efficiency in relation to transduction or the like e.g., “transduction efficiency” refers to the fraction (e.g., percentage) of cells expressing a nucleotide of interest after incubation with a set number of viral particles comprising the nucleotide of interest.
• Well-known methods of determining transduction efficiency include flow cytometry of cells transduced with a fluorescent reporter gene, RT- PCR for expression of the nucleotide of interest, etc.
• “reference” viral capsid protein/capsid/particle are identical to test viral capsid protein/capsid/particle but for the change for which the effect is to be tested. For example, to determine the effect, e.g., on transduction efficiency, of inserting a first member of a specific binding pair into a test viral particle, the transduction efficiencies of the test viral particle (in the absence or presence of an appropriate targeting ligand) can be compared to the transduction efficiencies of a reference viral particle (in the absence or presence of an appropriate targeting ligand if necessary) which is identical to the test viral particle in every instance (e.g., additional point mutations, nucleotide of interest, numbers of viral particles and target cells, etc.) except for the presence of a first member of a specific binding pair.
• a reference viral capsid protein is one that is able to form a capsid with a second viral capsid protein modified to comprise at least a first member of a protein: protein binding pair, where the reference viral capsid protein does not comprise the first member of a protein: protein binding pair, preferably wherein the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.
• Adeno-associated viruses AAV
• AAV is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof.
• AAVs are small, non-enveloped, single-stranded DNA viruses.
• a wildtype AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITR) and two open reading frames (ORFs), rep and cap.
• the wildtype rep reading frame encodes four proteins of molecular weight 78 kD (“Rep78”), 68 kD (“Rep68”), 52 kD (“Rep52”) and 40 kD (“Rep 40”).
• Rep78 and Rep68 are transcribed from the p5 promoter
• Rep52 and Rep40 are transcribed from the pl9 promoter.
• the wildtype cap reading frame encodes three structural (capsid) viral proteins (VPs) having molecular weights of 83-85 kD (VP1), 72-73 kD (VP2) and 61-62 kD (VP3). More than 80% of total proteins in an AAV virion (capsid) comprise VP3; in mature virions VP1, VP2 and VP3 are found at relative abundance of approximately 1 : 1 : 10, although ratios of 1 : 1 :8 have been reported. Padron et al. (2005) J. Virology 79:5047-58.
• AAV encompasses all subtypes and both naturally occurring and modified forms that are well-known in the art.
• AAV includes primate AAV (e.g., AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3B), primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV type rhlO (AAV rhlO), AAV type hlO (AAV hlO), AAV type hul l (AAV hul l), AAV type rh32.33 (AAV rh32.33), AAV retro (AAV
• Prime AAV refers to AAV generally isolated from primates.
• non-primate animal AAV refers to AAV isolated from non-primate animals.
• “of a [specified] AAV” in relation to a gene e.g., rep, cap, etc.
• capsid protein e.g., a VP1 capsid protein, a VP2 capsid protein, a VP3 capsid protein, etc.
• region of a capsid protein of a specified AAV e.g., PLA2 region, VPl-u region, VP1/VP2 common region, VP3 region
• nucleotide sequence e.g., ITR sequence
• a cap gene or capsid protein of AAV etc. encompasses, in addition to the gene or the polypeptide respectively comprising a nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, also variants of the gene or polypeptide,
• a variant gene or a variant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the gene or polypeptide of a specified AAV, wherein the difference(s) does not generally alter at least one biological function of the gene or polypeptide, and/or the phylogenetic characterization of the gene or polypeptide, e.g., where the difference(s) may be due to degeneracy of the genetic code, isolate variations, length of the sequence, etc.
• rep gene and the cap gene as used here may encompass rep and cap genes that differ from the wildtype gene in that the genes may encode one or more Rep proteins and Cap proteins, respectively.
• a Rep gene encodes at least Rep78 and/or Rep68.
• cap gene includes those may differ from the wildtype in that one or more alternative start codons or sequences between one or more alternative start codons are removed such that the cap gene encodes only a single Cap protein, e.g., wherein the VP2 and/or VP3 start codons are removed or substituted such that the cap gene encodes a functional VP1 capsid protein but not a VP2 capsid protein or a VP3 capsid protein.
• a rep gene encompasses any sequence that encodes a functional Rep protein.
• a cap gene encompasses any sequence that encodes at least one functional cap gene.
• the wildtype cap gene expresses all three VP1, VP2, and VP3 capsid proteins from a single open reading frame of the cap gene under control of the p40 promoter found in the rep ORF.
• the term "capsid protein,” “Cap protein” and the like includes a protein that is part of the capsid of the virus.
• the capsid proteins are generally referred to as VP1, VP2 and/or VP3, and may be encoded by the single cap gene.
• the three AAV capsid proteins are produced in nature an overlapping fashion from the cap ORF alternative translational start codon usage, although all three proteins use a common stop codon.
• the ORF of a wildtype cap gene encodes from 5’ to 3’ three alternative start codons: “the VP1 start codon,” “the VP2 start codon,” and “the VP3 start codon”; and one “common stop codon”.
• the largest viral protein, VP1 is generally encoded from the VP1 start codon to the “common stop codon.”
• VP2 is generally encoded from the VP2 start codon to the common stop codon.
• VP3 is generally encoded from the VP3 start codon to the common stop codon.
• VP1 comprises at its N- terminus sequence that it does not share with the VP2 or VP3, referred to as the VP 1 -unique region (VPl-u).
• the VPl-u region is generally encoded by the sequence of a wildtype cap gene starting from the VP1 start codon to the “VP2 start codon.”
• VPl-u comprises a phospholipase A2 domain (PLA2), which may be important for infection, as well as nuclear localization signals which may aid the virus in targeting to the nucleus for uncoating and genome release.
• PHA2 phospholipase A2 domain
• the VP1, VP2, and VP3 capsid proteins share the same C-terminal sequence that makes up the entirety of VP3, which may also be referred to herein as the VP3 region.
• the VP3 region is encoded from the VP3 start codon to the common stop codon.
• VP2 has an additional ⁇ 60 amino acids that it shares with the VP1. This region is called the VP1/VP2 common region.
• one or more of the Cap proteins of the invention may be encoded by one or more cap genes having one or more ORFs.
• the VP proteins of the invention may be expressed from more than one ORF comprising nucleotide sequence encoding any combination of VP1, VP2, and/or VP3 by use of separate nucleotide sequences operably linked to at least one expression control sequence for expression in packaging cell, each producing one or more of VP1, VP2, and/or VP3 capsid proteins of the invention.
• a VP capsid protein of the invention may be expressed individually from an ORF comprising nucleotide sequence encoding any one of VP1, VP2, or VP3 by use of separate nucleotide sequences operably linked to one expression control sequence for expression in a viral replication cell, each producing only one of VP1, VP2, or VP3 capsid protein.
• VP proteins may be expressed from one ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in a viral replication cell, each producing VP1, VP2, and VP3 capsid protein.
• amino acid positions provided herein may be provided in relation to the VP1 capsid protein of the referenced AAV, a skilled artisan would be able to respectively and readily determine the position of that same amino acid within the VP2 and/or VP3 capsid protein of the AAV, and the corresponding position of amino acids among different AAV.
• ITR Inverted terminal repeat
• the phrase “Inverted terminal repeat” or “ITR” includes symmetrical nucleic acid sequences in the genome of adeno-associated viruses required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as the origins of replication for viral DNA synthesis and are essential cis components for generating AAV particles, e.g., packaging into AAV particles.
• AAV ITR comprise recognition sites for replication proteins Rep78 or Rep68.
• A"D" region of the ITR comprises the DNA nick site where DNA replication initiates and provides directionality to the nucleic acid replication step.
• An AAV replicating in a mammalian cell typically comprises two ITR sequences.
• a single ITR may be engineered with Rep binding sites on both strands of the “A” regions and two symmetrical D regions on each side of the ITR palindrome.
• Such an engineered construct on a double-stranded circular DNA template allows Rep78 or Rep68 initiated nucleic acid replication that proceeds in both directions.
• a single ITR is sufficient for AAV replication of a circular particle.
• the rep encoding sequence encodes a Rep protein or Rep protein equivalent that is capable of binding an ITR comprised on the transfer plasmid.
• the Cap proteins of the invention when expressed with appropriate Rep proteins by a packaging cell, may encapsidate a transfer plasmid comprising a nucleotide of interest and an even number of two or more ITR sequences.
• a transfer plasmid comprises one ITR sequence.
• a transfer plasmid comprises two ITR sequences.
• Rep proteins may be expressed from more than one ORF comprising nucleotide sequence encoding any combination of Rep78, Rep68, Rep 52 and/or Rep40 by use of separate nucleotide sequences operably linked to at least one expression control sequence for expression in a viral replication cell, each producing one or more of Rep78, Rep68, Rep 52 and/or Rep40 Rep proteins.
• Rep proteins may be expressed individually from an ORF comprising a nucleotide sequence encoding any one of Rep78, Rep68, Rep 52, or Rep40 by use of separate nucleotide sequences operably linked to one expression control sequence for expression in a packaging cell, each producing only one Rep78, Rep68, Rep 52, or Rep40 Rep protein.
• Rep proteins may be expressed from one ORF comprising nucleotide sequences encoding Rep78 and Rep52 Rep proteins operably linked to at least one expression control sequence for expression in a viral replication cell each producing Rep78 and Rep52 Rep protein.
• a rep encoding sequence and a cap gene of the invention may be provided a single packaging plasmid.
• proviso is not necessary.
• viral particles may or may not include a genome.
• a “chimeric AAV capsid protein” includes an AAV capsid protein that comprises amino acid sequences, e.g., portions, from two or more different AAV and that is capable of forming and/or forms an AAV viral capsid/viral particle.
• a chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide comprising a plurality, e.g., at least two, nucleic acid sequences, each of which plurality is identical to a portion of a capsid gene encoding a capsid protein of distinct AAV, and which plurality together encodes a functional chimeric AAV capsid protein.
• a chimeric capsid protein comprises one or more portions from a capsid protein of that AAV and one or more portions from a capsid protein of a different AAV.
• a chimeric AAV2 capsid protein includes a capsid protein comprising one or more portions of a VP1, VP2, and/or VP3 capsid protein of AAV2 and one or more portions of a VP1, VP2, and/or VP3 capsid protein of a different AAV.
• portion refers to at least 5 amino acids or at least 15 nucleotides, but less than the full-length polypeptide or nucleic acid molecule, with 100% identity to a sequence from which the portion is derived, see Penzes (2015) J. General Virol. 2769.
• a “portion” encompasses any contiguous segment of amino acids or nucleotides sufficient to determine that the polypeptide or nucleic acid molecule form which the portion is derived is “of a [specified] AAV” or has “significant identity” to a particular AAV, e.g., a non-primate animal AAV or remote AAV.
• a portion comprises at least 5 amino acids or 15 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 20 amino acids or 60 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 25 amino acids or 75 nucleotides with 100% identity to a sequence associated with the specified AAV.
• a portion comprises at least 30 amino acids or 90 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 35 amino acids or 105 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 40 amino acids or 120 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 45 amino acids or 135 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 50 amino acids or 150 nucleotides with 100% identity to a sequence associated with the specified AAV.
• a portion comprises at least 60 amino acids or 180 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 70 amino acids or 210 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 80 amino acids or 240 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 90 amino acids or 270 nucleotides with 100% identity to a sequence associated with the specified AAV. In some embodiments, a portion comprises at least 100 amino acids or 300 nucleotides with 100% identity to a sequence associated with the specified AAV.
• a Cap protein e.g., a VP1 capsid protein as described herein, a VP2 capsid protein as described herein, and/or a VP3 capsid protein as described herein, is modified to comprise any one or combination of e.g., insertion of a targeting ligand, a chemical modification, a first member of a binding pair, a detectable label, point mutation, etc.
• modification of gene or a polypeptide of a specified AAV results in nucleic acid sequence or an amino acid sequence that differs from the nucleic acid sequence or amino acid sequence set forth herein for the specified AAV, wherein the modification alters, confers, or removes one or more biological functions, but does not change the phylogenetic characterization of, the gene or polypeptide as an AAV gene or AAV polypeptide.
• Modifications may include any one or a combination of substitution of sequences of a first AAV serotype with sequences of a second AAV serotype to create chimerism; chemical modification; an insertion of a first member of a binding pair, and/or a point mutation; etc., such that the natural tropism of the capsid protein is reduced to abolished, the tropism of the capsid protein may be more easily redirected, and/or such that the capsid protein comprises a detectable label.
• Modifications as described herein generally do not alter and preferably decrease the low to no recognition of the modified capsid by preexisting antibodies found in the general population that were produced during the course of infection with another AAV, e.g., infection with serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions from currently used AAV gene therapy modalities, or a combination thereof.
• serotypes such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03
• Modifications described herein may pertain to the association (e.g,. display, operable linkage, binding) of a targeting ligand to a modified capsid protein and/or capsid comrpsing a modified capsid protein.
• a targeting ligand as described herein binds a surface protein expressed by a mammalian muscle cell, e.g., a protein that is expressed on the surface of a mammalian muscle cell, e.g., a mammalian muscle cell-specific surface protein.
• a modified capsid protein and/or modified capsid comprises a targeting ligand that binds mammalian CACNG1, e.g., a human CACNG1.
• Table 1 provides a summary of the SEQ ID NO for each binding portion (e.g., heavy chain variable domain (HCVR), light chain variable domain (LCVR), and CDR1, CDR2, and CDR3) of non-limiting and exemplary anti-human-CACNGl monoclonal antibodies (mAb ID) that may be used to redirect an AAV capsid as described herein.
• HCVR heavy chain variable domain
• LCVR light chain variable domain
• CDR1, CDR2, and CDR3 non-limiting and exemplary anti-human-CACNGl monoclonal antibodies
• an AAV capsid as described herein comprises a targeting ligand that binds human CACNG1, wherein the targeting ligand comprises heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3 amino acid sequence(s) at least 90% identical to, respectively, an amino acid sequence of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 as set forth in any one of SEQ ID NOs: 1-240.
• an AAV capsid as described herein comprises a targeting ligand that binds human CACNG1, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence at least 95% identical to, respectively, amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1- 240.
• an AAV capsid as described herein comprises a targeting ligand that binds human CACNG1, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set ofHCDRl- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence at least 97% identical to amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240.
• an AAV capsid as described herein comprises a targeting ligand that binds human CACNG1, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence(s) at least 98% identical to amino acid sequence(s) of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240.
• an AAV capsid as described herein comprises a targeting ligand that binds human CACNG1, wherein the targeting ligand comprises a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, CDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDRl- HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences 99% identical to amino acid sequences of a heavy chain variable domain, light chain variable domain, heavy chain variable domain/light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and/or set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240.
• antibodies, or antigen-binding fragments thereof comprising a set of six CDRs (/. ⁇ ., HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3) contained within an HCVR/LCVR amino acid sequence pair as defined by any of the exemplary anti-hCACNGl antibodies listed in Table 1.
• a targeting ligand as described herein comprises the HCDR1-HCDR2-HCDR3-LCDR1- LCDR2-LCDR3 amino acid sequences set contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2/10, SEQ ID NOs: 18/26, SEQ ID NOs: 34/42, SEQ ID NOs: 50/58, SEQ ID NOs: 66/74, SEQ ID NOs: 82/90, SEQ ID NOs: 98/106, SEQ ID NOs: 114/122, SEQ ID NOs: 130/138, SEQ ID NOs: 146/154, SEQ ID NOs: 162/170, and SEQ ID NOs: 178/186.
• a targeting ligand as described herein comprises an HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2/10, SEQ ID NOs: 18/26, SEQ ID NOs: 34/42, SEQ ID NOs: 50/58, SEQ ID NOs: 66/74, SEQ ID NOs: 82/90, SEQ ID NOs: 98/106, SEQ ID NOs: 114/122, SEQ ID NOs: 130/138, SEQ ID NOs: 146/154, SEQ ID NOs: 162/170, and SEQ ID NOs: 178/186.
• N *underlined and bolded asparagine (N) may be mutated to a glutamine (Q) for conjugation by transglutaminase, see, e.g., SEQ ID NO:269
• *underlined and bolded asparagine (N) may be mutated to a glutamine
• N *underlined and bolded asparagine (N) may be mutated to a glutamine (Q) for conjugation by transglutaminase, see, e.g., SEQ ID NO:269
• *underlined and bolded asparagine (N) may be mutated to a glutamine
• N *underlined and bolded asparagine (N) may be mutated to a glutamine (Q) for conjugation by transglutaminase, see, e.g., SEQ ID NO:269
• N *underlined and bolded asparagine (N) may be mutated to a glutamine (Q) for conjugation by transglutaminase, see, e.g., SEQ ID NO:269
• LCDR1 Nucleic Acid Sequence (SEQ ID NO: 123) cag agt gtt age age age tac
• HCDR2 Nucleic Acid Sequence (SEQ ID NO: 133) ATA AGA AAT AAG GCT AAT AGG TAC GCG ACA
• GGC AAA GGA GGA TAT TGT AGT AGT AGC GGC TGC CGT CAC TAC GGT ATG

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• Medicinal Chemistry (AREA)
• Zoology (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• Wood Science & Technology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Biophysics (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Virology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Microbiology (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Epidemiology (AREA)
• Immunology (AREA)
• Plant Pathology (AREA)
• Physics & Mathematics (AREA)
• Gastroenterology & Hepatology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Neurology (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Physical Education & Sports Medicine (AREA)
• Cell Biology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Rheumatology (AREA)
• Environmental Sciences (AREA)
• Mycology (AREA)
• Biodiversity & Conservation Biology (AREA)

Priority Applications (11)

Applications Claiming Priority (4)

Publications (1)

Family

ID=84537880

Family Applications (1)

Country Status (13)

Cited By (6)

Citations (19)

• 2022
  • 2022-11-04 JP JP2024525653A patent/JP2024540181A/ja active Pending
  • 2022-11-04 CN CN202280073053.9A patent/CN118201643A/zh active Pending
  • 2022-11-04 MX MX2024005314A patent/MX2024005314A/es unknown
  • 2022-11-04 US US18/707,217 patent/US20250304918A1/en active Pending
  • 2022-11-04 AU AU2022380841A patent/AU2022380841A1/en active Pending
  • 2022-11-04 EP EP22826538.5A patent/EP4426359A1/en active Pending
  • 2022-11-04 KR KR1020247013639A patent/KR20240095211A/ko active Pending
  • 2022-11-04 PE PE2024000991A patent/PE20241212A1/es unknown
  • 2022-11-04 CA CA3233698A patent/CA3233698A1/en active Pending
  • 2022-11-04 WO PCT/US2022/079339 patent/WO2023081850A1/en not_active Ceased
  • 2022-11-04 IL IL312598A patent/IL312598A/en unknown
• 2024
  • 2024-04-25 CO CONC2024/0005350A patent/CO2024005350A2/es unknown
  • 2024-04-29 CL CL2024001307A patent/CL2024001307A1/es unknown
• 2025
  • 2025-03-21 CL CL2025000839A patent/CL2025000839A1/es unknown

Patent Citations (26)

Non-Patent Citations (65)

Also Published As

Similar Documents

Legal Events