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WO2025212745A1 - Protéine de capside de virus adéno-associé (aav) modifiée et ses utilisations - Google Patents

Protéine de capside de virus adéno-associé (aav) modifiée et ses utilisations

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Publication number
WO2025212745A1
WO2025212745A1 PCT/US2025/022705 US2025022705W WO2025212745A1 WO 2025212745 A1 WO2025212745 A1 WO 2025212745A1 US 2025022705 W US2025022705 W US 2025022705W WO 2025212745 A1 WO2025212745 A1 WO 2025212745A1
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WIPO (PCT)
Prior art keywords
aav
capsid protein
raav
gfp
retinal
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PCT/US2025/022705
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English (en)
Inventor
Guangping Gao
Mengtian CUI
Jianghui Wang
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University of Massachusetts Amherst
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University of Massachusetts Amherst
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Publication of WO2025212745A1 publication Critical patent/WO2025212745A1/fr
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    • 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
    • A61K48/0008—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • 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
    • C12N2750/14141—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 at least in part, relates to the discovery that grafting certain cell penetrating peptides (CPP) (e.g., CPPs described in Table 1) into an AAV2 capsid protein produces capsid proteins that have enhanced transduction efficiency in retinal cells (e.g., photoreceptors and retinal pigment epithelium (RPE) cells).
  • CPP cell penetrating peptides
  • RPE retinal pigment epithelium
  • capsid proteins described herein a useful in treating retinal diseases (e.g., AMD, diabetic retinopathy, Leber congenital amaurosis, or other inherited retinal diseases (IRDs)).
  • an AAV capsid protein described herein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1, and comprises one or more amino acid insertion between position 587 and 588 of SEQ ID NO: 1.
  • a recombinant adeno-associated virus (rAAV) comprising a capsid protein described herein transduces retinal cells (e.g., photoreceptors and retinal pigment epithelium (RPE) cells) via a less invasive route (e.g., intravitreal injection (IVT)) relative to other more invasive route (e.g., subretinal injections).
  • retinal cells e.g., photoreceptors and retinal pigment epithelium (RPE) cells
  • RPE retinal pigment epithelium
  • injection e.g., intravitreal injection
  • injection e.g., intravitreal injection
  • a transgene e.g., a transgene encoding a therapeutic protein
  • pan-retinal transduction e.g., relative to rAAV comprising wildtype AAV2 capsid or an AAV2.7m8 capsid protein
  • a rAAV comprising a capsid protein described herein is less immunogenic (e.g., relative to rAAV comprising wildtype AAV2 capsid or an AAV2.7m8 capsid protein).
  • the present disclosure provides an adeno-associated virus (AAV) capsid protein comprising an amino acid sequence having at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1, and an insertion of a cell penetrating peptide that comprises 5 to 18 amino acids between amino acid residues 587 and 588 of SEQ ID NO:1.
  • AAV adeno-associated virus
  • the CPP is selected from a CPP set forth in Table 1. In some embodiments, the CPP is selected from any one of SEQ ID NOs: 2-39. In some embodiments, the CPP comprises the amino acid sequence of KLGVM (SEQ ID NO: 2).
  • the AAV capsid protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 40-77. In some embodiments, the AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 40.
  • the AAV capsid protein has tropism for retinal cells. In some embodiments, the AAV capsid protein has tropism for photoreceptors or retinal pigment epithelium (RPE) cells.
  • RPE retinal pigment epithelium
  • the capsid protein has reduced immunogenicity relative to wildtype AAV2 capsid protein or an AAV2.7m8 capsid protein.
  • the disclosure provides a nucleic acid encoding any one of the AAV capsid proteins described herein.
  • the nucleic acid sequence comprises any one of SEQ ID NOs: 80-117.
  • the disclosure provides a vector comprising the nucleic acid encoding any one of the AAV capsid proteins described herein.
  • the vector is a plasmid.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) comprising: (i) an AAV capsid protein described herein; and (ii) an isolated nucleic acid comprising a transgene encoding a gene product (e.g., a therapeutic gene product, such as a therapeutic protein or interfering nucleic acid), flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • a gene product e.g., a therapeutic gene product, such as a therapeutic protein or interfering nucleic acid
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the therapeutic transgene is associated with a retinal disease.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) comprising: (i) an AAV capsid protein comprising the amino acid sequence of SEQ ID NO: 40; and (ii) an isolated nucleic acid comprising a transgene encoding a gene product (e.g., a therapeutic gene product), flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • the transgene further comprises a promoter operably linked the nucleic acid sequence encoding the gene product.
  • the promotor is a CB6 promoter or a GRK1 promoter.
  • the disclosure provides a host cell comprising an isolated nucleic acid, vector, or rAAV as described herein.
  • the disclosure provides a method of delivering a transgene to a retinal cell, the method comprising contacting the cell with an rAAV or composition as described herein.
  • the retinal cells comprise photoreceptor cells.
  • FIGs. 3A-3D show the retinal transduction profile of CPP1 capsid in adult mice.
  • FIG. 3A shows representative fundus images of adult C57BL/6 mice 4 weeks post-IVT injection of low (2.0X10 8 vgs/eye) or high (2.0X10 9 vgs/eye) dose of single- stranded (ss) AAVs via IVT injection.
  • FIG. 3B shows relative mRNA expression of GFP in the mouse retina 4 weeks post-IVT injection of low or high dose of ssAAVs.
  • FIG. 3C shows representative fundus images of adult C57BL/6 mice 4 weeks post-IVT injection of low dose of self-complementary (sc) AAVs.
  • FIG. 3D shows relative mRNA expression of GFP in the mouse retina 4 weeks post-IVT injection of low dose of scAAV s.
  • 4D shows immunolabelled cross sections of mouse retina 4 weeks post-IVT injection of high dose of ssAAV2.GRKl.H2BGFP, ssAAV2.7m8.GRKl.H2BGFP and ssAAV2.CPPl.GRKl.H2BGFP, respectively.
  • the dots indicate nuclear GFP expression.
  • PNA indicates peanut agglutinin.
  • ONE indicates outer nuclear layer - photoreceptor nuclei.
  • FIG. 5 shows the relative mRNA levels of TNF-a, IE-ip, IFN-y and IE-6 by ddPCR in the retinas of adult mice 4 weeks post-IVT injection of indicated AAV vectors at the dose of 2.0X10 9 vgs/eye.
  • FIGs. 6A-6F show the identification of AAV2 capsid variant enriched in the retina of mice.
  • FIG. 6A shows the schematic representation of the backbone plasmid construct containing the cellpenetrating peptide (CPP) library insert.
  • FIG. 6B shows the workflow of the screening process in mice, involving two rounds of selection.
  • FIG. 6C shows representative RT-PCR results of recovered RNA of capsid variants from pooled retina/RPE tissues 28 days after injection of the GRK1 -driven AAV library.
  • FIG. 6D The reduction and enrichment of CPP variants during each round of selection.
  • FIG. 6E shows the identification of AAV2.CPP1 as the leading capsid variant after the second round of selection based on enrichment and yield scores.
  • FIG. 7C Immunostaining of retinal cross sections 4 weeks after high-dose intravitreal injection of ssAAV2.CB6.GFP, ss7m8.CB6.GFP, and sAAV2.CPPl.CB6.GFP.
  • the arrows show the GFP expression in the inner segments (ISs) or outer segments (OSs) of photoreceptors successfully transduced.
  • the arrowheads indicate the transduced Muller cells.
  • FIG. 7D shows quantitative analysis of the number of transduced photoreceptors in retinas treated with different viral vectors.
  • FIGs. 8A-8D show retinal transduction profile of self-complementary AAV2.CPP1 vector in adult mice.
  • FIG. 8A shows representative fundus images showing retinal transduction 4 weeks after intravitreal (IVT) injection of a high dose (2.0x10 9 vg/eye) of self-complementary (sc) AAVs in adult mice.
  • FIG. 8B shows the quantification of genomic DNA and mRNA expression levels of GFP in the mouse retina 4 weeks post-injection of high-dose sc AAVs.
  • FIG. 8C shows representative immunostained retinal cross sections from mice injected with high-dose scAAV2.CB6.GFP, sc7m8.CB6.GFP, and scAAV2.CPPl.CB6.GFP.
  • FIG. 8D shows the quantitative analysis of transduced photoreceptors in retinas treated with different viral vectors.
  • FIGs. 9A-9D show that AAV2.CPP1 vector induces a minimal immune response comparted to AAV2.7m8.
  • FIGs. 9A-9B show representative immunostaining of retinal microglia using the IBA1 marker in eyes treated with ssAAV2.CB6.GFP, ss7m8.CB6.GFP, and ssAAV2.CPPl.CB6.GFP.
  • FIG. 9C shows the quantification of IB Al -positive cells in different retinal layers following IVT injection of the indicated viral vectors.
  • FIG. 10D shows the quantitative analysis of GFP expression indicating that the Matrigel-HS -based Transwell model can effectively distinguish between AAV2, AAV2.7m8, and AAV2.CPP1 capsids, based on their different affinities for HS binding.
  • FIGs. 10E- 10F show representative molecular models of AAV2 containing the KLGVM (SEQ ID NO: 2) insertion (highlighted) at amino acid N587. The interaction between the inserted loop and other surface loops of the capsid may contribute to the novel properties observed with this vector.
  • KLGVM SEQ ID NO: 2
  • FIGs. 12A-12C show the identification of the leading AAV2 capsid variant enriched in the retina of mice using CMV/CB-AAV2-CPP library.
  • FIG. 12A shows a representative schematic of the backbone plasmid construct containing the CPP library insert.
  • FIG. 12B shows a representative RT- PCR results of recovered RNA capsid variants from pooled retina/RPE tissues 28 days after injection of the CMV/CBOdriven AAV library.
  • FIG. 12C shows the reduction and enrichment of CPP variants during each round of selection and the identification of AAV2.CPP1 as the leading capsid variant after the second round of selection based on enrichment and yield scores.
  • FIGs. 13A-13C show engineered AAV2.CPP1 capsid can efficiently package AAV vectors with iodixanol gradient purification.
  • FIG. 13A shows the titers of each AAV vectors packaged by AAV2, AAV2.7m8 and AAV2.CPPl capsids.
  • FIG. 13B shows representative transmission electron microscopy images of scAAV2.CB6.GFP and scAAV2.CPPl.CB6.GFP. Light and black arrows indicate full virions and completely or partially empty virions, respectively.
  • FIG. 13C shows the quantification analysis of full/empty capsid ratio of each vector.
  • FIGs. 14A-14B show the retinal transduction profile of the single-stranded AAV2.CPP1 vector in adult mice at low dose.
  • FIG. 14A shows representative fluorescence funds images of adult C57BL/65 mice four weeks after intravitreal injection with a low dose (2.0xl0 8 vg/eye) of singlestranded AAVs.
  • FIG. 14B shows the quantification of genomic DNA levels of GFP in mouse retinas four weeks post-injection of low dose ssAAVs.
  • FIGs. 15A-15B show retinal transduction profile of the self-complementary AAV2.CPP1 vector in adult mice at low dose.
  • FIG. 15A shows representative fluorescence fundus images of adult C57BL/6 mice four weeks after intravitreal injection with a low dose (2.0xl0 8 vg/eye) of singlestranded AAVs.
  • FIG. 15B shows quantification of genomic DNA levels of GFP in mouse retinas four weeks postinjection of low dose sc AAVs. DETAILED DESCRIPTION
  • aspects of the disclosure relate to grafting of cell penetrating peptide (CPP) (e.g., CPPs described in Table 1) into AAV2 capsid protein to produce AAV capsid proteins that have enhanced transduction efficiency in retina cells (e.g., photoreceptors and retinal pigment epithelium (RPE) cells).
  • CPP cell penetrating peptide
  • RPE retinal pigment epithelium
  • capsid proteins described herein a useful in treating retinal diseases (e.g., AMD, diabetic retinopathy, Leber congenital amaurosis, or other inherited retinal diseases (IRDs)).
  • injection e.g., intravitreal injection
  • injection of a rAAV comprising a capsid protein described herein and a transgene to a subject results in enhanced pan-retinal transduction (e.g., relative to rAAV comprising wildtype AAV2 capsid or an AAV2.7m8 capsid protein).
  • a rAAV comprising a capsid protein described herein is less immunogenic (e.g., relative to (e.g., relative to rAAV comprising wild-type AAV2 capsid or an AAV2.7m8 capsid protein).
  • the disclosure relates to AAV capsid proteins grafted with a cell penetrating peptide (CPP) (e.g., any one of the CPPs described in Table 1) in a wild-type capsid protein.
  • CPP cell penetrating peptide
  • “Graft” or “grafting” or “grafted, as used herein, refers to insertion of a peptide between two amino acid residues in an AAV capsid protein.
  • the cap gene encodes the three structural proteins of the AAV capsid. Differential splicing yields major and minor spliced products.
  • the AAV VP1, VP2, and VP3 are translated from the same mRNA transcribed from the p40 promoter, and the three capsid proteins differ only in their N-terminal region.
  • VP3 capsid protein is the most abundant in an AAV capsid.
  • a CPP is grafted into a wild type AAV2 capsid protein.
  • a wild type AAV2 VP1 protein comprises the amino acid sequence of SEQ ID NO: 1.
  • a wild type AAV2 VP2 protein comprises the amino acids 138- 735 of the amino acid sequence of SEQ ID NO: 1.
  • a wild type AAV2 VP1 protein comprises the amino acids 138- 735 of the amino acid sequence of SEQ ID NO: 1.
  • a wild type AAV2 VP2 protein comprises the amino acids 138- 735 of the amino acid sequence of SEQ ID NO: 1.
  • VP3 protein comprises amino acids 203-735 of the amino acid sequence of SEQ ID NO: 1.
  • a CPP is a peptide having a net positive charge, which enables it to penetrate cells (see., e.g., Herce et al., Cell Penetrating Peptides: How Do They Do It? J Biol Phys. 2007 Dec; 33(5-6): 345-356).
  • a CPP penetrates cells while carrying a cargo (e.g., proteins, oligonucleotide, or drugs).
  • grafting a CPP into an AAV capsid protein e.g., AAV2 capsid protein
  • RPE retinal pigment epithelium
  • a CPP described herein is between 3 and 42 amino acids in length (e.g., 3-42 amino acids, 3-40 amino acids, 3-35 amino acids, 3-30 amino acids, 3-25 amino acids, 3-20 amino acids, 3-15 amino acids, 3-10 amino acids, 3-5 amino acids, 5-42 amino acids, 5-40 amino acids, 5-35 amino acids, 5-30 amino acids, 5-25 amino acids, 5-20 amino acids, 5-18 amino acids, 5-15 amino acids, 5-12 amino acids, 5-10 amino acids, 5-8 amino acids, 8-18 amino acids, 8-15 amino acids, 8-12 amino acids, 8-10 amino acids, 10-18 amino acids, 10-16 amino acids, 10-15 amino acids, 10-12 amino acids, 12-18 amino acids, 12-16 amino acids, 12-15 amino acids, 15-18 amino acids, 15- 16 amino acids, 10-42 amino acids, 10-40 amino acids, 10-35 amino acids, 10-30 amino acids, 10-25 amino acids, 10-20 amino acids, 15-42 amino acids, 15-40 amino acids, 15-35 amino acids, 15-30 amino acids, 15-25 amino acids, 15-20 amino acids, 20-
  • a CPP has an overall net positive charge, which can be calculated by suitable methods (e.g., by determining the total number of photos and the total number of electrons from all amino acids in the CPP, and subtracting the total number of electrons from the total number of photons to get the net charge).
  • a CPP comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of the CPPs set forth in Table 1. In some embodiments, a CPP comprises the amino acid sequence of any one of the CPPs set forth in Table 1.
  • a CPP comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 2-39.
  • a CPP comprises the amino acid sequence of any one of the CPPs set forth in Table 1.
  • a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a wild type AAV2 capsid protein (e.g., VP1 protein, VP2 protein, or VP3 protein) and comprises an insertion of a CPP between amino acid residue 587 and 588 of SEQ ID NO: 1.
  • a wild type AAV2 capsid protein e.g., VP1 protein, VP2 protein, or VP3 protein
  • a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a wild type AAV2 capsid protein (e.g., VP1 protein, VP2 protein, or VP3 protein) and comprises an insertion of a CPP comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 2-39 between amino acid residue 587 and 588 of SEQ ID NO: 1.
  • a wild type AAV2 capsid protein e.g., VP1 protein, VP2 protein, or VP3
  • a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a wild type AAV2 capsid protein (e.g., VP1 protein, VP2 protein, or VP3 protein) and comprises an insertion of a CPP consists of the amino acid sequence of any one of SEQ ID NOs: 2-39 between amino acid residue 587 and 588 of SEQ ID NO: 1.
  • a capsid protein described herein comprises an insertion of a CPP comprises or consists of KLGVM (SEQ ID NO: 2) between amino acid residue 587 and 588 of SEQ ID NO: 1.
  • a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of the AAV capsid protein (e.g., VP1, VP2, or VP3) in Table 1.
  • AAV capsid protein e.g., VP1, VP2, or VP3
  • a capsid protein described herein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 40-77.
  • a capsid protein described herein comprises the amino acid sequence of SEQ ID NO: 40.
  • a capsid protein described herein is not a capsid protein described in WO2018160686 (e.g., AAV2.7m8).
  • An AAV2.7m8 capsid protein has an insertion of a peptide consists of amino acids LALGETTRPA (SEQ ID NO: 134) between amino acids residues 587 and 588 of a wild-type AAV2 VP1 protein as set forth in SEQ ID NO: 1 (see, e.g., AAV2.7m8 capsid protein as described in WO2018160686).
  • an AAV2.7m8 capsid VP1 protein is set forth in SEQ ID NO: 78.
  • a capsid protein described herein does not comprise or consist of the amino acid sequence of SEQ ID NO: 78.
  • MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEP VNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEP LGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPP
  • AAP S GLGTNTMA TGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVI TTS TR TWALP TYNNHL YKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLT
  • a capsid protein described herein can be produced by inserting the coding sequence for the CPP to between the codon encoding the asparagine at amino acid 587 (e.g., codon AAC as shown below in SEQ ID NO: 73) and the arginine at amino acid 588 (e.g., codon AGA as shown below in SEQ ID NO: 73) in a nucleic acid sequence encoding a wild type AAV2 protein.
  • a capsid protein described herein can be produced by inserting the coding sequence for the CPP to between nucleotides 1761 and 1762 of SEQ ID NO: 79.
  • a nucleic acid sequence encoding the wild type AAV2 protein is set forth in SEQ ID NO: 79 (Codon AAC encoding N587 and codon AGA encoding R588 underlined).
  • a capsid protein described herein is encoded by a nucleic acid sequence set forth in Table 2.
  • a nucleic acid encoding a capsid protein described herein comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 80-117.
  • a nucleic acid encoding a capsid protein comprising the amino acid sequence of SEQ ID NO: 1 comprises the nucleic acid sequence of SEQ ID NO: 80.
  • Adeno-associated virus is a small ( ⁇ 26 nm) replication-defective, non-enveloped virus that generally depends on the presence of a second virus, such as adenovirus or herpes virus, for its growth in cells.
  • AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell.
  • the disclosure provides rAAVs comprising a capsid protein described that show enhanced tissue targeting (e.g., to retinal cells such as photoreceptors and RPE cells) capabilities for gene therapy (e.g., for treating IRDs) and research applications.
  • an rAAV comprises a capsid protein described herein (e.g., any one of the AAV capsid protein described in Table 1 or a variant thereof) and an isolated nucleic acid encoding a transgene (e.g., any one of the transgenes associated with a retinal disease such as an IRD).
  • a capsid protein described herein e.g., any one of the AAV capsid protein described in Table 1 or a variant thereof
  • an isolated nucleic acid encoding a transgene e.g., any one of the transgenes associated with a retinal disease such as an IRD.
  • an rAAV comprises a capsid protein comprising the amino acid sequence of any one of SEQ ID NOs: 40-77, and an isolated nucleic acid encoding a transgene (e.g., a therapeutic transgene, for example a transgene encoding a gene product associated with a retinal disease such as an IRD).
  • a transgene e.g., a therapeutic transgene, for example a transgene encoding a gene product associated with a retinal disease such as an IRD.
  • an rAAV described herein has enhanced cell penetration capability (e.g., enhanced cell penetration by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or more) to retinal cells (e.g., photoreceptors and/or retinal pigment epithelium (RPE) cells) relative to an rAAV comprising a wild tyle AAV2 capsid or an AAV2.7m8 capsid.
  • retinal cells e.g., photoreceptors and
  • an rAAV described herein has enhanced cell penetration capability (e.g., enhanced cell penetration by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or more) to retinal cells (e.g., photoreceptors and/or retinal pigment epithelium (RPE) cells) when administered to a subject via a less invasive route (e.g., intravitreal injection as opposed to subretinal injection) relative to an rAAV comprising a wild t
  • an rAAV described herein has enhanced transduction efficiency (e.g., enhanced transduction efficiency by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or more) to retinal cells (e.g., photoreceptors and/or retinal pigment epithelium (RPE) cells) relative to an rAAV comprising a wild tyle AAV2 capsid or an AAV2.7m8 capsid.
  • retinal cells e.g., photoreceptors
  • an rAAV described herein has enhanced transduction efficiency (e.g., enhanced transduction efficiency by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 300 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or more) to retinal cells (e.g., photoreceptors and/or retinal pigment epithelium (RPE) cells) when administered to a subject via a less invasive route (e.g., intravitreal injection as opposed to subretinal injection) relative to an rAAV comprising a wild
  • an rAAV described herein has reduced immunogenicity (e.g., reduced immunogenicity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) administered to a subject as compared to an rAAV comprising a wild tyle AAV2 capsid or an AAV2.7m8 capsid.
  • reduced immunogenicity e.g., reduced immunogenicity by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at
  • the disclosure provides isolated AAVs.
  • isolated refers to an AAV that has been artificially obtained or produced. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs”.
  • Recombinant AAVs preferably have tissue- specific targeting capabilities, such that a transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s) (e.g., retinal cells such as photoreceptors and/or RPE cells).
  • tissue(s) e.g., retinal cells such as photoreceptors and/or RPE cells.
  • the AAV capsid is an important element in determining these tissue-specific targeting abilities. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected.
  • Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003/0138772), the contents of which are incorporated herein by reference in their entirety).
  • the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein (e.g., a nucleic acid encoding a capsid protein having a sequence as set forth in any one of SEQ ID NOs: 80-117) or fragment thereof; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins.
  • a nucleic acid sequence encoding an AAV capsid protein e.g., a nucleic acid encoding a capsid protein having a sequence as set forth in any one of SEQ ID NO
  • capsid proteins are structural proteins encoded by a cap gene of an AAV.
  • AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which may be expressed from a single cap gene.
  • the VP1, VP2 and VP3 proteins share a common core sequence.
  • the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa.
  • capsid proteins upon translation, form a spherical 60-mer protein shell around the viral genome.
  • the protein shell is primarily comprised of a VP3 capsid protein.
  • the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host.
  • capsid proteins deliver the viral genome to a host in a tissue specific manner.
  • VP1 and/or VP2 capsid proteins may contribute to the tissue tropism of the packaged AAV.
  • the tissue tropism of the packaged AAV is determined by the VP3 capsid protein.
  • the tissue tropism of an AAV is enhanced or changed by modification (e.g., insertion of a CPP as described herein) occurring in the capsid proteins.
  • AAV variants described herein may be useful for delivering gene therapy to tissue or cells of the eye (e.g., retinal cells). Accordingly, in some embodiments, AAV variants described herein may be useful for the treatment of disorders affecting the eye (e.g. retinal disorder).
  • a disorder of the eye e.g., retinal disorder
  • a disorder of the eye may affect the retina.
  • a disorder of the eye e.g., retinal disorder
  • Non-limiting examples of disorders and diseases affecting the eye include, but are not limited to: age-related macular degeneration, diabetic retinopathy, Leber congenital amaurosis (LCA), X-linked retinitis pigmentosa (RP), achromatopsia, Stargardt disease, or cone-rod dystrophy (CRD).
  • age-related macular degeneration e.g., diabetic retinopathy
  • LCA Leber congenital amaurosis
  • RP X-linked retinitis pigmentosa
  • achromatopsia e.g., Stargardt disease
  • CCD cone-rod dystrophy
  • the components to be cultured in the host cell to package a rAAV may be provided to the host cell in trans.
  • any one or more of the required components e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions
  • a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
  • a stable host cell will contain the required component(s) under the control of an inducible promoter.
  • the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene.
  • a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters.
  • a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.
  • the recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector).
  • a single nucleic acid encoding all three capsid proteins e.g., VP1, VP2 and VP3 is delivered into the packaging host cell in a single vector.
  • the selected genetic element may be delivered by any suitable method, including those described herein.
  • the methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques.
  • rAAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650).
  • the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector.
  • An AAV helper function vector encodes the "AAV helper function" sequences (e.g., rep and cap), which function in trans for productive AAV replication and encapsidation.
  • the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes).
  • AAV virions e.g., AAV virions containing functional rep and cap genes.
  • vectors suitable for use with the present disclosure include pHLP19, described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No. 6,156,303, the entirety of both incorporated by reference herein.
  • the accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (e.g., "accessory functions").
  • the accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly.
  • Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.
  • the disclosure provides transfected host cells.
  • transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell (e.g., across the cell membrane).
  • a number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells.
  • a “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV vector, an accessory function vector (e.g., rep/cap vector), or other transfer DNA associated with the production of rAAVs. The term includes the progeny of the original cell that has been transfected. Thus, a “host cell” as used herein may refer to a cell that has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
  • the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.
  • Cells may also be transfected with a vector (e.g., helper vector) that provides helper functions to the AAV.
  • the vector providing helper functions may provide adenovirus functions, including, e.g., Ela, Elb, E2a, and E4ORF6.
  • the sequences of adenovirus gene providing these functions may be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 8, 9, 12 and 40, and further including any of the presently identified human types known in the art.
  • the methods involve transfecting the cell with a vector expressing one or more genes necessary for AAV replication, AAV gene transcription, and/or AAV packaging.
  • vector includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., that is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • useful vectors are contemplated to be those vectors in which the nucleic acid segment (e.g., nucleic acid sequence) to be transcribed is positioned under the transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene.
  • the phrases “operatively positioned,” “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitor) from a transcribed gene.
  • inhibitory RNA e.g., shRNA, miRNA, miRNA inhibitor
  • an isolated capsid gene can be used to construct and package recombinant AAVs, using methods well known in the art, to determine functional characteristics associated with the capsid protein encoded by the gene.
  • isolated capsid genes can be used to construct and package a recombinant AAV (rAAV) comprising a reporter gene (e.g., B- Galactosidase, GFP, Luciferase, etc.).
  • the rAAV can then be delivered to an animal (e.g., mouse) and the tissue targeting properties of the novel isolated capsid gene can be determined by examining the expression of the reporter gene in various tissues (e.g., heart, liver, kidneys) of the animal.
  • Other methods for characterizing the novel isolated capsid genes are disclosed herein and still others are well known in the art.
  • a rAAV described herein also comprises an isolated nucleic acid encoding a transgene.
  • a recombinant AAV vector comprising the isolated nucleic acid encoding the transgene can be used to packaging the rAAV for transgene expression in a cell.
  • “Recombinant AAV (rAAV) vectors” of the disclosure are typically composed of, at a minimum, a transgene (e.g., a transgene encoding one or more gene products) and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell.
  • the transgene is a nucleic acid sequence, heterologous to the vector sequences, that encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest.
  • the nucleic acid coding sequence is operatively linked to regulatory components in a manner that permits transgene transcription, translation, and/or expression in a cell of a target tissue.
  • the isolated nucleic acid comprises inverted terminal repeats.
  • the isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors).
  • AAV adeno-associated virus
  • an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof.
  • the isolated nucleic acid e.g., the recombinant AAV vector
  • Recombinant AAV (rAAV) vectors are typically composed of, at a minimum, a transgene, and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).
  • ITR sequences are about 145 bp in length.
  • substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible.
  • the ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K.
  • the isolated nucleic acid further comprises a region (e.g., a second region, a third region, a fourth region, etc.) comprising a second AAV ITR.
  • an isolated nucleic acid encoding a transgene is flanked by AAV ITRs (e.g., in the orientation 5’-ITR-transgene-ITR-3’).
  • the AAV ITRs are selected from the group consisting of AAV 1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, and AAV6 ITR.
  • the second ITR is a mutant ITR that lacks a functional terminal resolution site (TRS).
  • lacking a terminal resolution site can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non-synonymous mutation, or missense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ATRS ITR, or AITR).
  • TRS terminal resolution site
  • a rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16(10): 1648- 1656.
  • vectors described herein comprise one or more AAV ITRs, and at least one ITR is an ITR variant of a known AAV serotype ITR.
  • the AAV ITR variant is a synthetic AAV ITR (e.g., AAV ITRs that do not occur naturally).
  • the AAV ITR variant is a hybrid ITR (e.g., a hybrid ITR comprises sequences derived from ITRs of two or more different AAV serotypes).
  • the rAAVs of the present disclosure are pseudotyped rAAVs.
  • Pseudotyping is the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. The result is a pseudotyped virus particle.
  • the foreign viral envelope proteins can be used to alter host tropism or an increased/decreased stability of the virus particles.
  • a pseudotyped rAAV comprises nucleic acids from two or more different AAVs, wherein the nucleic acid from one AAV encodes a capsid protein and the nucleic acid of at least one other AAV encodes other viral proteins and/or the viral genome.
  • a pseudotyped rAAV refers to an AAV comprising an inverted terminal repeat (ITR) of one AAV serotype and a capsid protein of a different AAV serotype.
  • ITR inverted terminal repeat
  • a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y will be designated as AAVX/Y (e.g., AAV2/1 has the ITRs of AAV2 and the capsid of AAV1).
  • pseudotyped rAAVs may be useful for combining the tissue-specific targeting capabilities of a capsid protein from one AAV serotype with the viral DNA from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue.
  • the vector also includes conventional control elements necessary which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the disclosure.
  • "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • RNA processing signals such as splicing and polyadenylation (poly A) signals
  • sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • nucleic acid sequence e.g., coding sequence
  • regulatory sequences are said to be “operably” linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences.
  • two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame.
  • operably linked coding sequences yield a fusion protein.
  • operably linked coding sequences yield a functional RNA e.g., shRNA, miRNA, miRNA inhibitor).
  • a poly adenylation sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence.
  • a rAAV construct useful in the present disclosure may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene.
  • One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence.
  • Another vector element that may be used is an internal ribosome entry site (IRES).
  • An IRES sequence is used to produce more than one polypeptide from a single gene transcript.
  • An IRES sequence would be used to produce a protein that contains more than one polypeptide chains. Selection of these and other common vector elements are conventional, and many such sequences are available.
  • the precise nature of the regulatory sequences needed for gene expression in host cells may vary between species, tissues, or cell types, but shall in general include, as necessary, 5’ non-transcribed and 5’ non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like.
  • 5’ non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the disclosure may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter [Invitrogen].
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art.
  • inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline -repressible system (Gossen et al, Proc. Natl. Acad. Sci.
  • MT zinc-inducible sheep metallothionine
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system WO 98/10088
  • ecdysone insect promoter No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)
  • the regulatory sequences impart tissue-specific gene expression capabilities.
  • the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner.
  • tissue-specific regulatory sequences e.g., promoters, enhancers, etc.
  • tissue-specific regulatory sequences are well known in the art.
  • tissue-specific regulatory sequences include, but are not limited to the following tissue specific promoters: a liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a-myosin heavy chain (a-MHC) promoter, a gastrointestinal-specific mucin-2 promoter, an eye-specific retinoschisin promoter, an eye-specific K12 promoter, a respiratory tissue-specific CC10 promoter, a respiratory tissue-specific surfactant protein C (SP-C) promoter, a breast tissue-specific PRC1 promoter, a breast tissue-specific RRM2 promoter, a urinary tract tissue-specific uroplakin 2 (
  • Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron- specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among others which will be apparent to the skilled artisan.
  • NSE neuron-specific enolase
  • the transgene may be designed such that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites.
  • the presence of multiple miRNA binding sites may result in the cooperative action of multiple RISCs and provide highly efficient inhibition of expression.
  • the target site sequence may comprise a total of 5-100, 10-60, or more nucleotides.
  • the target site sequence may comprise at least 5 nucleotides of the sequence of a target gene binding site.
  • Reporter sequences that may be provided in a transgene include, without limitation, DNA sequences encoding P-lactamase, P -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP, EGFP), chloramphenicol acetyltransferase (CAT), luciferase (e.g., Firefly luciferase), and others well known in the art.
  • DNA sequences encoding P-lactamase, P -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP, EGFP), chloramphenicol acetyltransferase (CAT), luciferase (e.g., Firefly luciferase), and others well known in the art.
  • the method involves administration of an rAAV that encodes one or more gene products (e.g., therapeutic peptides, polypeptides, siRNAs, microRNAs, antisense nucleotides, etc.) in a pharmaceutically-acceptable carrier to the subject in an amount and for a period of time sufficient to treat the deficiency or disorder in the subject suffering from such a disorder.
  • gene products e.g., therapeutic peptides, polypeptides, siRNAs, microRNAs, antisense nucleotides, etc.
  • transgenes encoding proteins or polypeptides
  • mutations that result in conservative amino acid substitutions may be made in a transgene to provide functionally equivalent variants or homologs of a protein or polypeptide.
  • the disclosure embraces sequence alterations that result in conservative amino acid substitution of a transgene.
  • the transgene comprises a gene having a dominant negative mutation.
  • a transgene may express a mutant protein that interacts with the same elements as a wild-type protein, and thereby blocks some aspect of the function of the wild-type protein.
  • Useful transgene products also include miRNAs.
  • miRNAs and other small interfering nucleic acids regulate gene expression via target RNA transcript cleavage/degradation or translational repression of the target messenger RNA (mRNA).
  • miRNAs are natively expressed, typically as final 19-25 non-translated RNA products. miRNAs exhibit their activity through sequence-specific interactions with the 3' untranslated regions (UTR) of target mRNAs. These endogenously expressed miRNAs form hairpin precursors that are subsequently processed into a miRNA duplex, and further into a “mature” single stranded miRNA molecule.
  • a small interfering nucleic acid that is substantially complementary to a miRNA is a small interfering nucleic acid that is complementary to the miRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bases.
  • a small interfering nucleic acid sequence that is substantially complementary to a miRNA or is a small interfering nucleic acid sequence that is complementary to the miRNA with at least one base.
  • a “miRNA Inhibitor” is an agent that blocks miRNA function, expression and/or processing.
  • these molecules include, but are not limited to, microRNA specific antisense oligonucleotides, microRNA sponges, tough decoy RNAs (TuD RNAs), and microRNA oligonucleotides (double-stranded, hairpin, short oligonucleotides) that inhibit miRNA interaction with a Drosha complex.
  • MicroRNA inhibitors can be expressed in cells from a transgene of a rAAV vector, as discussed above.
  • MicroRNA sponges specifically inhibit miRNAs through a complementary heptameric seed sequence. Other methods for silencing miRNA function (derepression of miRNA targets) in cells will be apparent to one of ordinary skill in the art.
  • the cloning capacity of the recombinant RNA vector may limit a desired coding sequence and may require the complete replacement of the virus's 4.8 kilobase genome. Large genes may, therefore, not be suitable for use in a standard recombinant AAV vector, in some cases.
  • the skilled artisan will appreciate that options are available in the art for overcoming a limited coding capacity. For example, the AAV ITRs of two genomes can anneal to form head to tail concatemers, almost doubling the capacity of the vector. Insertion of splice sites allows for the removal of the ITRs from the transcript. Other options for overcoming a limited cloning capacity will be apparent to the skilled artisan.
  • IRD are divided into two sub-types: stationary and progressive.
  • a stationary IRD e.g., cone and rod dysfunction syndromes
  • a progressive IRD e.g., progressive cone dystrophy (COD), cone -rod dystrophy (CORD), and rod-cone dystrophy (RCD)
  • COD progressive cone dystrophy
  • CORD cone -rod dystrophy
  • RCD rod-cone dystrophy
  • a progressive IRD is congenital onset (e.g., Leber congenital amaurosis/early onset severe retinal dystrophy (LCA/EOSRD)).
  • a subject having LCA/EOSRD is blind from birth or early infancy.
  • IRD include macular dystrophies (e.g., Stargardt disease (STGD), Best disease (BD), X-linked retinoschisis (XLRS), Pattern dystrophy (PD), Sorsby fundus dystrophy (SFD), etc.), cone dysfunction syndromes (e.g., Achromatopsia, Blue cone monochromatism (BCM), Bornholm eye disease (BED), etc.), cone and cone-rod dystrophies (e.g., AD GUCAlA-associated COD/CORD, AD GUCY2D-associated COD/CORD, Autosomal dominant PRPH2-associated CORD, Autosomal recessive ABCA4-associated COD/CORD, X-linked RPGR-associated COD/CORD, etc.), ROD dysfunction syndromes (e.g., Complete and incomplete congenital stationary night blindness (cCSNB/iCSNB), Fundus Alb
  • a retinal disease is not an IRD.
  • a non-IRD regina disease include but are not limited to Age-related macular degeneration (AMD), or diabetic retinopathy.
  • the disclosure provides methods for treating IRD (e.g., any of the IRD described herein), AMD or diabetic retinopathy, the method comprising administering the subject an effective amount of a rAAV or a composition comprising a rAAV described herein.
  • the rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art.
  • the rAAV preferably suspended in a physiologically compatible carrier (e.g., in a composition) may be administered to a subject, e.g., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque).
  • a host animal does not include a human.
  • Delivery of the rAAVs to a mammalian subject may be by, for example, intravitreal, subretinal, intramuscular injection or by administration into the bloodstream of the mammalian subject such as intravenous injection.
  • the rAAV is delivered to the eye.
  • the rAAV is delivered to the eye via intravitreal injection.
  • the rAAV is delivered to the eye via subretinal injection.
  • Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit.
  • the rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions.
  • isolated limb perfusion technique described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline).
  • Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.
  • compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.
  • suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • the dose of rAAV virions required to achieve a particular "therapeutic effect,” e.g., the units of dose in genome copies/per kilogram of body weight (GC/kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product.
  • a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.
  • an effective amount of an rAAV is an amount sufficient to target infect an animal or target a desired tissue (e.g., retinal cells such as photoreceptors or RPE cells).
  • a desired tissue e.g., retinal cells such as photoreceptors or RPE cells.
  • an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model.
  • the effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary between animals or tissues.
  • an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 10 7 to 10 16 genome copies.
  • the rAAV is administered at a dose of 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies per subject. In some embodiments the rAAV is administered at a dose of 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies per kg. In some cases, a dosage between about 10 11 to 10 12 rAAV genome copies is appropriate.
  • the rAAV is administered to the retinal cells (e.g., photoreceptors or RPE cells) e.g., by intravitreal injection at a dose of about 10 9 to 10 16 genome copies per subject. In some embodiments, the rAAV is administered to the retina (e.g., by intravitreal injection) at a dose of about 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , or 10 16 genome copies per subject. In some embodiments, the rAAV is administered to the retina (e.g., by intravitreal injection) at a dose of IxlO 9 genome copies per subject.
  • the retinal cells e.g., photoreceptors or RPE cells
  • the rAAV is administered to the retina (e.g., by intravitreal injection) at a dose of 3.6xl0 9 genome copies per subject.
  • stable transgenic animals are produced by multiple doses of an rAAV.
  • rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ⁇ 10 13 gc/mL or more).
  • high rAAV concentrations e.g., ⁇ 10 13 gc/mL or more.
  • Methods for reducing aggregation of rAAVs include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.)
  • Formulation of pharmaceutically acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.
  • these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
  • the amount of active compound in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraopancreatically, intranasally, parenterally, intravenously, , intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation.
  • the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 may be used to deliver rAAVs.
  • a preferred mode of administration is by portal vein injection.
  • a preferred mode of administration is by facial vein injection.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., vegetable oils
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • a sterile aqueous medium that can be employed will be known to those of skill in the art.
  • one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.
  • Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various other ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the rAAV compositions disclosed herein may also be formulated in a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.
  • Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells.
  • the rAAV delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500.
  • ANG. containing an aqueous solution in the core.
  • Nanocapsule formulations of the rAAV may be used.
  • Nanocapsules can generally entrap substances in a stable and reproducible way.
  • ultrafine particles sized around 0.1 pm
  • Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.
  • Sonophoresis z.e., ultrasound
  • U.S. Pat. No. 5,656,016 has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system.
  • Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback- controlled delivery (U.S. Pat. No. 5,697,899).
  • Example 1 Engineering adeno-associated virus 2 (AAV2) capsid proteins with cell-penetrating peptides for enhanced retinal transduction via intravitreal injection
  • AAV2 adeno-associated virus 2
  • rAAV Recombinant adeno-associated virus
  • FDA US Food and Drug Administration
  • LCA2 Leber congenital amaurosis type 2
  • RPE retinal pigment epithelium
  • the major limitation for IVT injection of rAAV2-based vectors is its inability to transduce photoreceptors and RPEs due to strong binding of enriched heparan sulfate proteoglycans (HSPGs) in the inner limiting membrane (ILM), a major structural barrier for rAAV2- based ocular gene therapy through IVT route.
  • HSPGs heparan sulfate proteoglycans
  • ILM inner limiting membrane
  • One promising development is the engineered AAV2 capsid 7m8 which has demonstrated the capability to transduce photoreceptors via ITV route in mice. 7m8 was discovered through direct evolution-based screening of a seven-amino-acid library inserted at the 3-fold spike region of AAV2, specifically N587 of VP1.
  • CPPs Cell-penetrating peptides
  • AAV2.CPP1 a 5-mer insertion
  • AAV2.CPP1 demonstrated superior photoreceptor transduction.
  • the present disclosure demonstrates that the CPP1 insertion reduces HS binding, likely facilitating greater vector penetration through the ILM and more effective retinal cell transduction.
  • the capsid with insertion of KLGVM (SEQ ID NO: 2), termed CPP1
  • CPP1 demonstrated pan-retinal and enhanced photoreceptor transduction with reduced immunogenicity via IVT injection compared to the AAV2 and 7m8.
  • a CPP library (comprising 1090 CPPs) was synthesized as the DNA fragments and the library was cloned into the backbone encoding AAV2 Cap gene at between amino acid residues 587 and 588 of AAV2 (SEQ ID NO:1) under the regulation of the CB6 promoter (FIG. 1 and FIG. 2A).
  • the resulting plasmid library 1 (comprising 725 CPPs) was then packaged into AAV library 1 (comprising 565 CPPs) which was intravitreally injected into the eyes of adult C57BL/6 mice.
  • CPPs were retrieved from the retina at mRNA level one-week post-injection and cloned into the plasmid backbone, resulting in plasmid library 2 (comprising 174 CPPs) and subsequent AAV library 2 (comprising 180 CPPs).
  • This AAV library 2 was again intravitreally injected into mouse eyes, and the CPPs (comprising 38 CPPs) were retrieved at mRNA level one-week post-injection and subjected to Next-Generation Sequencing (NGS). Enrichment analysis was performed to rank these CPPs based on the enrichment score (FIG. 2B).
  • KLGVM SEQ ID NO: 2 (CPP1) was selected for further validation due to its higher enrichment score (ranked #2 out of 38 CPPs) and mRNA abundance (-15.7% of entire reads) in the retina after second round screening.
  • rAAV encoding GFP gene controlled by either the CB6 promoter or the photoreceptor-specific promoter GRK1 in both single-stranded (ss) and self- complementary (sc) genome formats, was packaged with the CPP1 capsid as well as with AAV2 and 7m8 capsid.
  • the titer of the resulting AAVs indicated that CPP1 can be more efficiently packaged compared to AAV2 and 7m8, indicating the positive effects of CPP1 insertion on rAAV production.
  • Quantitative analysis revealed that ssAAV2.CPPl.CB6.GFP achieved ⁇ 2-fold increase in transduction in mice receiving a high dose of viral vectors compared to other vectors.
  • GFP expression was uniform in the mouse eyes receiving low dose of scAAV2.CPPl.CB6.GFP (FIG. 3C), with no significant changes in GFP mRNA expression observed among groups.
  • viral vectors packaged with AAV2 capsid, 7m8 capsid, and CPP1 capsid, individually, all controlled by the photoreceptorspecific promoter, rhodopsin kinase (GRK1), and encoding nuclear-expressing GFP (H2BGFP) were utilized.
  • These vectors, including ssAAV2.GRKl.H2BGFP, ss7m8.GRKl.H2BGFP, and ssAAV2.CPPl.GRKl.H2BGFP were administered via IVT injection in mice at a dose of 2.0X10 9 vgs/eye and retinas were collected 4 weeks post-injection.
  • 3), interferon gamma (IFN-y), and interleukin-6 (IL-6) in the retina were further evaluated by the fourth week following IVT injection of different viral vectors encoding GFR Among these inflammatory cytokines, the expression level of IL-6 was increased in the retina receiving ss7m8.CB6.GFP compared to both uninjected retinas and those injected with ssAAV2.CB6.GFP (FIG. 5), indicating a robust immune response induced by ss7m8.CB6.GFP. Increased expression level of IL-6 in the retina receiving ssAAV2.CPPl.CB6.GFP was not observed (FIG. 5), indicating a lower degree of immunogenicity associated with this vector.
  • TNF-a tumor necrosis factor alpha
  • Example 2 Development and in vivo evaluation ofAAV2 capsid libraries featuring CPPs and photoreceptor-targeted vectors
  • CPPs were grafted onto AAV2 capsids to determine if they could enhance retinal cell transduction following ITV injection.
  • a CPP library was curated by electing peptides with unique amino acid sequences from CPPsite2.0 database (Agrawal et al., CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides. Nucleic Acids Res. 44, D1098-D1103.) which contains approximately 1,700 unique CPPs.
  • a diverse capsid library was constructed by grafting the CPP sequences between residues 587 and 588 in the hypervariable surface loop VIII of AAV2 capsid genes by Gibson assembly, which was combined with AAV2 ITRs and a photoreceptor- specific G-protein-coupled receptor kinase 1 (GRK1) promoter (FIG. 6A). This site permits genetic modifications without disrupting capsid assembly or genome packaging.
  • the resulting plasmid library retained 66.4% (724/1,090) of the CPPs from the initial library. Rep proteins were provided via a separate plasmid during virus production.
  • Viral libraries were generated in HEK293T cells under low-DNA-input conditions to minimize capsid mosaicism and cross -packaging.
  • rAAVs were packaged using AAV2, AAV2.7m8, and AAV2.CPP1 capsids. These carried either single-stranded (ss) genomes encoding cytoplasmic green fluorescent protein (GFP) driven by the CB6 promoter (ssAAV2.CB6.GFP, ssAAV2.7m8.CB6.GFP, and ssAAV2.CPPl.CB6.
  • ss single-stranded genomes encoding cytoplasmic green fluorescent protein driven by the CB6 promoter
  • ssAAV2.CB6.GFP ssAAV2.7m8.CB6.GFP
  • ssAAV2.CPPl.CB6 ssAAV2.CPPl.CB6.
  • H2BGFP Upon intravitreal injection, H2BGFP was homogeneously expressed in the photoreceptor nuclei of eyes treated with ssAAV2.7m8.GRKl.H2BGFP and ssAAV2.CPPl.GRKl.H2BGFP, but not in those treated with ssAAV2.GRKl.H2BGFP. Quantitative analysis showed that ssAAV2.CPPl.GRKl.H2BGFP increased photoreceptor transduction nearly 4-fold compared to ssAAV2.7m8.GRKl.H2BGFP.
  • scAAV2.CPPl.CB6 showed a 5- and 2.9-fold increase in GFP expression at the RNA level compared to scAAV2.CB6.GFP and scAAV2.7m8.CB6. GFP, respectively.
  • the increase in RNA/DNA ratios of scAAV2.CPPl.CB6. GFP was consistent with the results observed for the single-stranded vectors.
  • GFP (FIG. 8C). GFP expression was also assessed at a lower dose (2.0xl0 8 vg/eye). Although fundus imaging showed an increase in GFP expression with both scAAV2.7m8.CB6. GFP and scAAV2.CPPl.CB6. GFP compared to that with scAAV2.CB6.GFP, no significant differences at the RNA level were observed among the vectors (FIGs. 15A-15B).
  • Example 5 Decreased immune responses by AAV2.CPP1 vector
  • AAV vectors typically elicit stronger immune responses compared to subretinal injections.
  • Engineered vectors such as AAV2.7m8, appear to cause increased immune responses compared to their wild-type parent, AAV2, likely due to the higher degree of heterogeneity in vector genomes packaged by AAV2.7m8.
  • AAV2.CPP1 induces immune responses in the retina
  • ssAAV2.CB6.GFP, ssAAV2.7m8.CB6.GFP, and ssAAV2.CPPl.CB6.GFP at a dose of 2.0xl0 9 vg/eye were injected ITV in mice.
  • IPL inner plexiform layer
  • Activated microglia may upregulate proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukin- 1 beta (IL- lb), interferongamma (IFN-g), and interleukin-6 (IL-6).
  • TNF-a tumor necrosis factor-alpha
  • IL- lb interleukin- 1 beta
  • IFN-g interferongamma
  • IL-6 interleukin-6
  • IL-6 expression was notably higher in retinas injected with ssAAV2.7m8.CB6.GFP compared to both uninjected controls and retinas injected with ssAAV2.CB6.GFP and ssAAV2.CPPl.CB6.GFP (FIG. 9C). This indicates that ssAAV2.7m8.CB6.GFP likely induces a robust immune response even 4 weeks post-injection. Interestingly, retinas injected with ssAAV2.CPPl.CB6.GFP did not show elevated IL-6 levels, indicating a lower degree of immunogenicity associated with the AAV2.CPP1 vector.
  • AAV2 is known to utilize HSPG as its primary receptor for binding to host cells, with subsequent cell entry facilitated by proteinaceous co-receptors. Studies have shown that modifying the AAV2 capsid can enhance photoreceptor transduction via intravitreal injection by reducing its HS binding affinity, thereby enabling more vectors to cross the ILM, as demonstrated by AAV2.7m8. To investigate the mechanism behind the enhanced retinal transduction of AAV2.CPP1, the HS binding affinity of ssAAV2. CB6.GFP, ssAAV2.7m8.CB6.GFP, and ssAAV2.CPPl.CB6.GFP was assessed.
  • a Matrigel-HS-based Transwell model was developed to partially mimic ILM by coating culture Transwells with a mixture of Matrigel and HS and seeding HeLa cells, a cell line rich in HSPG, underneath the Transwells. It is proposed that AAVs with strong HS binding would be unable to penetrate this in vitro model, thus failing to transduce the HeLa cells. Conversely, AAVs with reduced HS binding would pass through the HS-enriched Transwells and transduce the cells, resulting in detectable GFP expression (FIG. 10B). Using this system, the transduction profile of ssAAV2.CB6.GFP, ssAAV2.7m8.CB6.
  • GFP, ssAAV2.CPPl.CB6. GFP, and ssAAV9.CB6.GFP was compared. After 48 h of infection, it was observed that in the absence of in vitro model, GFP expression was comparable among ssAAV2.CB6.GFP, ssAAV2.7m8.CB6.GFP, and ssAAV2.CPPl.CB6.GFP, except for cells transduced by ssAAV9.CB6.GFP, which is known for poor transduction with cells enriched with HSPG.
  • the AAV2.CPP1 capsid structure was modeled by superimposing it onto the AAV2 capsid (FIGs. 10E and 10F).
  • the KLGVM SEQ ID NO: 2
  • peptide insertion disrupted the VR-VIII region, which forms the top of the second highest of the three protrusions at the three-fold axis of symmetry. This alteration potentially affects the spacing between R585 and R588 and effectively interrupts the HSPG binding motif.
  • Library backbone vectors containing an AAV capsid expression cassette driven by either the GRK1 or the CB6 promoter were constructed using the Gibson assembly method.
  • the backbone plasmid pAAV-Cap2-scl included inverted terminal repeats (ITRs) and a partial Rep2 C-terminal sequence, which is crucial for capsid gene splicing and protein assembly.
  • ITRs inverted terminal repeats
  • Rep2 C-terminal sequence which is crucial for capsid gene splicing and protein assembly.
  • a full-length cap2 gene was inserted between residues N587 and R588 with a unique Aflll restriction site for CPP library cloning.
  • the backbone plasmid pAAV-Cap2-sc2 also contained ITRs and the partial Rep2 C-terminal sequence, but with a cap2 fragment that featured a unique Aflll site for cloning the CPP sequences recovered from the retina.
  • a plasmid, pRep2-3stop was constructed to exclusively express the Rep protein by introducing three stop codons in the Cap2 gene, thereby preventing the expression of the VP 1/2/3 proteins.
  • Plasmid library AAV library construction, and virus production
  • a double- stranded CPP library was synthesized by GenScript, containing overlapping sequences of the Cap2 gene at the 5’ (5’-CCAACCTCCAGAGAGGCAAC-3’) (SEQ ID NO: 128) and 3’ (3’-TCTGCGGTAGCTGCTTGTCT-5’) (SEQ ID NO: 129) ends for Gibson assembly, to create the plasmid library for the first round of screening.
  • the library was cloned into the backbone plasmid pAAV-Cap2-scl using the standard Gibson assembly method.
  • the pAAV-Cap2-scl plasmid (2 mg) was digested with Aflll enzyme and purified using a DNA and gel purification kit (D4008, Zymo Research, USA).
  • the resulting plasmid library was precipitated using isopropanol and concentrated in 5 mL of water.
  • the library ( ⁇ 100 ng/mL in 1 mL) was electroporated into ElectroMAX DH10B cells (18290015, Invitrogen, USA), following the manufacturer’s instructions. Electroporated cells were pooled and cultured in 500 mL of TB medium overnight. The resulting plasmid library was extracted and purified.
  • pAAV-Cap2-sc2 was digested with Aflll and purified in the same manner.
  • the recovered CPP sequences from the retina (after the first round) were cloned into the pAAVCap2-sc2 using Gibson assembly to generate the second plasmid library.
  • AAV library production was conducted in HEK293 cells.
  • Calcium phosphate transfection was performed using 1.5 mg of pAd-DeltaF6 (containing adenovirus helper genes), 1.0 mg of pRep2-3stop, and 100 mg of either the first or the second plasmid library in 10 roller bottles.
  • Cells and culture medium were harvested 72 h post-transfection by scraping, and the cells were pelleted via low-speed centrifugation. The cells were lysed by undergoing three freeze-thaw cycles, and the supernatant was precipitated on ice using 1:0.3 volume of 40% polyethylene glycol solution, followed by centrifugation.
  • the lysate and supernatant were pooled and fractionated through four rounds of iodixanol gradient purification. Buffer exchange was performed on Amicon-100 columns (Millipore) using phosphate-buffered saline (PBS). Final viral preparations were analyzed by ddPCR using a poly(A)-specific primer/probe set (Table 3), and the samples were tested via silver staining of PAGE gels. Full sequences of all the plasmids used in AAV library production can be found in the supplemental information.
  • the resulting visualization prominently displayed AAV empty particles as characteristic donut-like shapes, formed by the accumulation of uranyl acetate stain in the capsid dimples.
  • the animals were euthanized, and the retina/RPE complexes were harvested, pooled, snap-frozen in liquid nitrogen, and stored at -80C.
  • Total retinal RNA was extracted using the DNA/RNA extraction kit (SKU 47700, Norgen Biotek). mRNA was further purified from the total RNA using the Dynabeads mRNA Purification Kit (#61006, Invitrogen). Reverse transcription was performed using 400 ng of purified mRNA and the gene-specific Cap2-RT primer (Table SI) with the SuperScript IV First- Strand Synthesis Kit (Life Technologies).

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Abstract

La présente divulgation concerne, au moins en partie, des protéines de capside d'AAV greffées avec un CPP entre les résidus d'acides aminés 587 et 588 d'une protéine de capside AAV2 de type sauvage. Dans certains modes de réalisation, un rAAV comprenant une protéine de capside décrite ici présente une efficacité de transduction améliorée sur des cellules de rétine (par exemple, des photorécepteurs ou des cellules RPE). Dans certains modes de réalisation, la présente divulgation concerne des compositions et des méthodes de traitement de maladies de la rétine telles que des IRD.
PCT/US2025/022705 2024-04-04 2025-04-02 Protéine de capside de virus adéno-associé (aav) modifiée et ses utilisations Pending WO2025212745A1 (fr)

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