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  • C12N15/09—Recombinant DNA-technology
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  • 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
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  • 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

• Viral vectors such as AAV vectors
• AAV vectors are efficient vehicles for in vivo nucleic acid delivery, and their use in the clinic is expanding. Improved AAV vectors and AAV production techniques for making effective AAV vector preparations should further expand the use of AAV vectors in the laboratory and clinic.
• an AAV vector e.g., an AAV2 vector
• retinal cells e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells
• an AAV vector e.g., an AAV2 vector
• an AAV capsid polypeptide that includes an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A
• 7m8 AAV2 vector e.g., an AAV2 vector
• Table 1 or a variant thereof
• Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189): 189ra76 (2013) and Bennett et al., J. Struct.
• an AAV vector e.g., an AAV2 vector
• An AAV vector comprising the polypeptide can infect greater than 2 percent of retinal cells within two or more retinal regions when a titer of at least 1 x 10 7 of the vector is administered intravitreally to an eye of a human (or a non-human primate).
• An AAV vector comprising the polypeptide can express more nucleic acid in retinal cells in at least two retinal regions than the level of expression from a comparable AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1 , wherein the at least two retinal regions are selected from the group consisting of a fovea region, a parafovea region, a vascular arcade region, and a periphery region.
• the capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO: 10 or that is an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1) except that the amino acid sequence of SEQ ID NO: 5 is located between amino acid positions 587 and 588 of SEQ ID NO:1 (or the appropriate amino acid positions of the alternative sequence).
• the capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NOTO or that is an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1) except that the amino acids from position 585 to 590 of SEQ ID NO: 1 (or the appropriate amino acid positions of the alternative sequence) are replaced with the amino acid sequence of SEQ ID NO: 5.
• the capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs: 11-42.
• the vector can be an AAV2 vector.
• this document features a host cell comprising an AAV capsid polypeptide comprising the amino acid sequence of any one of SEQ ID NOs:2-5.
• the capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO: 10 or that is an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1) except that the amino acid sequence of any one of SEQ ID NOs:2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or the appropriate amino acid positions of the alternative sequence).
• the retinal condition can be selected from the group consisting of LCA, 0CA1, retinitis pigmentosa, rod/cone dystrophy, cone dystrophy, Stargardt Disease, Usher syndrome, XLRP, and XLRS.
• the capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO: 10 or that is an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1) except that the amino acid sequence of any one of SEQ ID NOs:2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or the appropriate amino acid positions of the alternative sequence).
• An exogenous nucleic acid sequence can be designed to encode any appropriate polypeptide of interest.
• polypeptides of interest that can be encoded by an exogenous nucleic acid sequence designed to be included within an AAV vector provided herein include, without limitation, therapeutic polypeptides, trophic factor polypeptides, gene editing polypeptides (e.g., a Cas9 polypeptide, a TALEN polypeptide, or a zinc finger polypeptide), enzymes, optogenetic tool polypeptides (e.g., a ChR polypeptide, an NhpR polypeptide, or a ReachR polypeptide), antibodies, antibody domains (e.g., VH domains), cytokines, anti- angiogenic polypeptides, and neuroprotective polypeptides.
• therapeutic polypeptides e.g., trophic factor polypeptides, gene editing polypeptides (e.g., a Cas9 polypeptide, a TALEN polypeptide, or
• any appropriate gene editing components can be engineered into one or more AAV vectors provided herein such that those one or more AAV vectors can be used to deliver the gene editing components to target cells (e.g., one or more retinal cells) within a mammal (e g., a human or a non-human primate) in a manner effective to edit the genome of those cells.
• the gene editing components include, without limitation, a component that is capable of cleaving genomic nucleic acid at a desired location and an optional donor nucleic acid designed to be inserted into that desired location once it is cleaved. Any appropriate rare-cutting endonuclease can be used to cleave genomic nucleic acid at a desired location.
• rare-cutting endonucleases include, without limitation, meganucleases, transcription activator-like effector (TALE) nucleases (TALENsTM; Cellectis, Paris, France), zinc-finger- nucleases (ZFNs), and endonucleases of a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system (e.g., endonucleases of a CRISPR/Cas 9 system).
• TALE transcription activator-like effector
• ZFNs zinc-finger- nucleases
• CRISPR clustered regularly interspaced short palindromic repeats
• CRISPR clustered regularly interspaced short palindromic repeats
• endonucleases of a CRISPR/Cas 9 system See, e.g., Baker, Nature Methods, 9:23-26 (2012); International PCT Patent Application Publication No. WO 2004/067736; International PCT Patent Application Publication No.
• an AAV vector e.g., an AAV2 vector
• compositions containing one or more AAV vectors provided herein e.g., one or more AAV2 vectors provided herein.
• one or more AAV vectors provided herein can be formulated as a pharmaceutical composition for administration to a mammal (e.g., a human or a non-human primate) to treat that mammal.
• a mammal e.g., a human or a non-human primate
• an AAV vector e.g., an AAV2 vector
• a retinal condition e.g., a retinal disease
• polypeptides that can be encoded by an exogenous nucleic acid sequence designed to treat a retinal condition (e.g., a retinal disease) and designed to be included within an AAV vector provided herein include, without limitation, an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, an NR2E3 polypeptide, a PDE6A polypeptide, a PDE6B polypeptide, a PDE6C polypeptide, a PRPF31 polypeptide, a RPE65 polypeptide, a RPGR polypeptide, a RSI polypeptide, a TYR polypeptide, a USH2A polypeptide, a MY07A polypeptide, an REP1 polypeptide, an 0PN1LW polypeptide, an 0PN1MW polypeptide, a CNGA3 polypeptide, a CNGB3 polypeptide, a GUCY2D polypeptide, a GACA1
• diabetic macular edema can be treated using an AAV vector provided herein that is designed to express a ChR polypeptide, a ChR2 polypeptide, an ArchT polypeptide, a NpHR polypeptide, and/or a ChrimsonR polypeptide.
• an effective amount of an AAV vector provided herein can be from about IxlO 7 viral genomes to about IxlO 14 viral genomes (e.g., from about 1 x 10' viral genomes to about 1 x 10 13 viral genomes, from about 1 x 10 7 viral genomes to about 1 x 10 12 viral genomes, from about 1 x 10 7 viral genomes to about 1 x 10 11 viral genomes, from about 1 x 10 7 viral genomes to about I x lO 10 viral genomes, from about I x lO 8 viral genomes to about I x lO 14 viral genomes, from about 1 x 10 9 viral genomes to about 1 x 10 14 viral genomes, from about 1 x IO 10 viral genomes to about 1 x 10 14 viral genomes, from about 1 x 10 8 viral genomes to about 1 x 10 12 viral genomes, or from about I x l O 9 viral genomes to about I x l O 11 viral genomes).
• AAV vectors that include an AAV capsid polypeptide e.g., an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) can have the ability to deliver nucleic acid to and express nucleic acid in retinal cells in at least two different retinal regions.
• a high-throughput method was used to create AAV vectors with mutated capsid polypeptides and to screen those created AAV vectors for particular AAV vectors having the ability to exhibit high efficiency and/or specificity for infecting retinal cells of the parafovea region of the eye. See, e.g., Ozturk et al., bioRxiv, 2020.10.01.323196 (2020). Briefly, highly complex libraries of AAV mutants were created and injected into the eyes of primates (cynolmolgus macaques or rhesus macaques). These libraries were created such that each AAV vector in the library contained a unique DNA barcode, which allowed for tracking of a mutated AAV capsid polypeptide.
• the AAV vectors were ranked based on overall rankings with +++ indicating those that performed in the top 1/3 of vectors tested, with ++ indicating those that performed in middle 1/3 of vectors tested, and with + indicating those that performed in the bottom 1/3 of vectors tested. These were determined in terms of total levels of gene expression in retinal cells.
• Embodiment 5 The polypeptide of Embodiment 1, wherein said capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10 except that the amino acids from position 585 to 590 of SEQ ID NO: 1 or SEQ ID NO: 10 are replaced with said amino acid sequence of SEQ ID NO: 5.
• Embodiment 6 The polypeptide of any one of Embodiments 1-5, wherein an AAV vector comprising said polypeptide infects greater than 2 percent of retinal cells within two or more retinal regions when a titer of at least 1 x 10 7 of said vector is administered intravitreally to an eye of a human.
• Embodiment 19 The capsid polypeptide of Embodiment 15, wherein said LI is two ammo acids X2-X1.
• Embodiment 20 The capsid polypeptide of Embodiment 19, wherein said XI is selected from the group of amino acid residues consisting of A, V, I, and L.
• X2 is selected from the group of amino acid residues consisting of A, V, I, and L.
• Embodiment 23 The capsid polypeptide of Embodiment 22, wherein said X2 is L.
• X2 is selected from the group of amino acid residues consisting of A, V, I, and L.
• X3 is selected from the group of amino acid residues consisting of A, V, I, and L.
• Embodiment 32 The capsid polypeptide of Embodiment 15, wherein said LI is absent.
• Embodiment 35 The capsid polypeptide of Embodiment 34, wherein said Z1 is A.
• L2 is two amino acids Z1-Z2.
• Embodiment 37 The capsid polypeptide of Embodiment 36, wherein said Z1 is selected from the group of amino acid residues consisting of A, V, I, and L.
• Embodiment 43 The capsid polypeptide of Embodiment 42, wherein said Z1 is selected from the group of amino acid residues consisting of A, V, I, and L.
• Z2 is selected from the group of amino acid residues consisting of A, V, I, and L.
• Embodiment 48 The capsid polypeptide of any one of Embodiments 42-47, wherein said

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