[go: up one dir, main page]

WO2025217214A2 - Recombinant adeno-associated viruses and uses thereof - Google Patents

Recombinant adeno-associated viruses and uses thereof

Info

Publication number
WO2025217214A2
WO2025217214A2 PCT/US2025/023741 US2025023741W WO2025217214A2 WO 2025217214 A2 WO2025217214 A2 WO 2025217214A2 US 2025023741 W US2025023741 W US 2025023741W WO 2025217214 A2 WO2025217214 A2 WO 2025217214A2
Authority
WO
WIPO (PCT)
Prior art keywords
raav
capsid protein
capsid
amino acid
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023741
Other languages
French (fr)
Other versions
WO2025217214A3 (en
Inventor
Elad FIRNBERG
Andrew Mercer
April R. TEPE
Samantha YOST
Ye Liu
Ting Chen
Sigmund TEJADA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Regenxbio Inc
Original Assignee
Regenxbio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Regenxbio Inc filed Critical Regenxbio Inc
Publication of WO2025217214A2 publication Critical patent/WO2025217214A2/en
Publication of WO2025217214A3 publication Critical patent/WO2025217214A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—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
    • 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
    • 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
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source

Definitions

  • the present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties when incorporated into a rAAV capsid.
  • rAAVs adeno-associated viruses
  • the invention provides engineered capsid proteins comprising peptide insertions inserted within or near variable region IV (VR-IV) of the virus capsid, such that the insertion is surface exposed on the AAV particle.
  • VR-IV variable region IV
  • the invention also provides capsid proteins that direct rAAVs to target tissues, in particular, capsid proteins derived from rAAV libraries, and provides such libraries constructed to reduce the parental vector production and thus overrepresentation of the parental capsid in the library and comprising random peptides inserted into surface-exposed variable regions to target rAAVs to and/or improve transduction of tissues of interest, including the ocular tissue, and deliver therapeutics for treating ocular disorders.
  • AAV adeno-associated viruses
  • AAV vectors When administered in the suprachoroidal space, for example, AAV vectors encounter barriers that prevent efficient transduction of ocular tissues. To reach retinal and RPE target tissues, rAAVs must pass around the choriocapillaris, cross the Bruch’s membrane and transit across the RPE.
  • Several approaches can be taken to overcome these challenges, including the engineering of novel capsids with improved ocular transduction profiles that can provide improved transgene expression to the back of the eye.
  • rAAV vectors with enhanced tropism to particular tissues and properties for use, e.g., in high transduction to ocular tissues to delivery' therapies in treating disorders such as ocular disorders, including those amenable to suprachoroidal administration of rAAV.
  • rAAV vectors with enhanced tissue-specific targeting and/or enhanced tissue-specific transduction to deliver therapies using lower dosing than is currently available are currently available.
  • rAAVs recombinant adeno-associated viruses
  • capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties such as tissue targeting, transduction or expression of the rAAV genome.
  • engineered capsid proteins comprising peptide insertions, derived from peptide libraries, inserted within or near variable region IV (VR-IV) of the virus capsid, or, in certain embodiments, within or near variable region VIII (VR-VIII), such that the peptide insertion is surface exposed on the AAV particle when the engineered capsid protein is incorporated into an rAAV particle.
  • the peptide is 7 or 9 amino acids of one of the peptides having an amino acid sequence of SEQ ID NOs: 1-32 (Tables 3A and 3B). In embodiments, the peptide is a 7 amino acid peptide within consensus sequences of SEQ ID NOs: 115-131 (see Table 6). In embodiments, the insertion is immediately after an amino acid residue corresponding to one of the amino acids 451 to 461 or 585 to 593 of the AAV8 capsid protein (SEQ ID NO: 33 and as numbered, e.g., in FIG.
  • the capsid protein may be an AAV8 capsid protein but may also be any AAV capsid protein, such as AAV type 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype hu.31 (AAVhu.31), serotype hu.32 (AAVhu.32).
  • AAV1 AAV1
  • AAV2 serotype 2
  • AAV3 serotype 3
  • AAV4 serotype 4
  • serotype 5 AAV5
  • serotype 6 AAV6
  • serotype 7 AAV7
  • serotype 8 AAV8
  • serotype 9 AAV9
  • serotype hu.31 AAVhu.31
  • serotype rhlO AAVrhlO
  • serotype rh20 AAVrh20
  • serotype hu.37 AAVVhu.37
  • serotype rh39 AAVrh39
  • serotype rh74 AAVrh74
  • AAV8.AAA AAV8.AAA (496NNN/AAA498 amino acid substitutions)
  • an engineered capsid protein comprising a peptide insertion from a heterologous protein (i.e., not an AAV capsid protein) inserted immediately after or near an amino acid corresponding to the amino acid residue at position 455 or at position 589 of AAV8, as numbered in FIG. 1.
  • capsid proteins that direct rAAVs to target tissues
  • capsid proteins comprising peptides (derived from peptide libraries) or a peptide that promotes tissue targeting and/or cellular uptake and/or expression of the rAAV genome, that are inserted into surface-exposed variable regions and that target rAAVs to ocular tissue, including retina, RPE-choroid, and/or sclera tissue, and deliver therapeutics for treating ocular disorders.
  • peptides including 7 or 9 contiguous amino acids of one of the peptides in Tables 3A and 3B (SEQ ID NOs: 1-32), are advantageously inserted into the amino acid sequence of the capsid protein (VP1, VP2 and/or VP3) such that, when the capsid protein is incorporated into the AAV particle, the inserted peptide is surface exposed.
  • consensus sequences for the 7 mer to 9 mer peptides based upon the peptides identified as described in Example 5. Consensus sequences are provided in Table 6 (SEQ ID NOs: 1 ISIS 1).
  • These peptides are inserted immediately after one of the amino acid residues of, or after one of the amino acids corresponding to the amino acid, 585-593 of VR-VIII of the AAV8 capsid (SEQ ID NO: 33) or an AAV8 capsid with substitutions e.g. AAV8.AAA capsid (SEQ ID NO: 114, e.g. see FIG. 1 for alignment), or immediately after one of the amino acid residues of, or after one of the amino acids corresponding to, 451-461 of VR-IV of the AAV8 capsid or AAV8.AAA capsid and amino acids corresponding to any one of positions 451-461 of the AAV8 capsid (SEQ ID NO: 33. see, e.g. FIG.
  • capsid proteins having amino acid sequence of one of SEQ ID NOs: 50 to 113.
  • capsid proteins and AAV particles incorporating capsid proteins comprising an amino acid sequence of SEQ ID NO: 33 (AAV8 parental capsid).
  • capsid proteins and AAV particles incorporating capsid proteins comprising an amino acid sequence of SEQ ID NO: 114 (AAV8.AAA parental capsid).
  • engineered capsid proteins comprising peptides that target specific tissues, including, when incorporated into an rAAV vector as a capsid to promote or increase cellular uptake and/or integration of an rAAV genome and/or expression of a transgene within the rAAV genome wherein the peptides are inserted into surface-exposed variable regions of the capsid protein.
  • the peptides target and/or promote transduction or genome integration in cells of the eye, including the retina, RPE-choroid, the sclera or other ocular tissues, for example, at least 7 contiguous amino acids or at least 9 contiguous amino acids of any of the peptides in Tables 3A and 3B, and capsids containing one of these peptides, for example, immediately after one of the positions 451 -461, including after position 455 or 589, of AAV8 or AAV8.AAA, preferentially target the rAAV with the capsid to ocular tissue, including retina, RPE-choroid and/or sclera tissue, and, in embodiments detargeting peripheral tissues and/or the liver when administered to the eye.
  • the capsid having the peptide insert targets and transduces ocular tissue.
  • the inserted peptide is at least 7 contiguous amino acids and may be 7 contiguous amino acids, 8 contiguous amino acids or 9 contiguous amino acids, and, in addition, the capsid is engineered to have one or more amino acid substitutions which may improve tropism, transduction or reduce immune neutralizing activity.
  • amino acid modifications may include amino acid substitutions of the NNN (asparagines) at 496 to 498 with AAA (alanines) in the AAV8 capsid or corresponding substitutions in other AAV type capsids.
  • Still other parental capsids having the peptide insertion may also have substitutions of S263F/S269T/A273T in AAV8. and corresponding substitutions in other AAV type capsids.
  • W530R or Q474A in AAV8 and corresponding substitutions in other AAV type capsids, and/or A269S in AAV8, and corresponding substitutions in other AAV type capsids.
  • capsid proteins that promote transduction of the rAAV in one or more ocular tissues, including one or more ocular cell types, upon ocular administration, including suprachoroidal administration (into the suprachoroidal space), wherein the capsid proteins comprise a peptide of Tables 3A and 3B (SEQ ID NOs: 1 to 32) or Table 6 (SEQ ID NOs: 115-131) that is inserted into a surface-exposed variable region (VR) of the capsid, e.g.
  • Tables 3A and 3B SEQ ID NOs: 1 to 32
  • Table 6 SEQ ID NOs: 115-131
  • VR-I, VR-IV or VR-VIII or after the first amino acid of VP2, e.g, immediately after residue 138 of the AAV8 capsid (amino acid sequence of SEQ ID NO: 33) or immediately after the corresponding residue of another AAV capsid, or alternatively is engineered with one or more of the amino acid substitutions described herein, and transduction of the AAV having the engineered capsid in the ocular tissue is increased upon said administration compared (for example, 1 fold, 2 fold, 5 fold, 10 fold or 20 fold greater) to the transduction of the AAV having the corresponding unengineered capsid (parental capsid) or a reference capsid such as AAV8 or AAV8.AAA.
  • Such capsids may also exhibit reduced transduction of one or more tissues, including peripheral tissue and/or liver upon administration compared (for example, 1 fold, 2 fold, 5 fold, 10 fold or 20 fold greater) to the transduction of the AAV in ocular tissue or having the corresponding unengineered capsid (parental capsid) or a reference capsid such as AAV8 or AAV8.AAA when administered to the eye.
  • transduction is measured by detection of transgene, such as DNA, RNA transcript or expressed protein in the cell, e.g. reporter transgenes may be utilized and measured such as GFP fluorescence.
  • rAAVs incorporating the engineered capsids described herein including rAAVs with genomes comprising a transgene of therapeutic interest, including for an ocular disease or disorder. Plasmids and cells for production of a pool (stock) of plasmids for the production of the rAAVs are described herein. Packaging cells and methods for the production of the rAAVs comprising the engineered capsids are also provided herein. Method of treatment by delivery of, and pharmaceutical compositions comprising, the engineered rAAVs described herein are provided. Also provided are methods of manufacturing the rAAVs with the engineered capsids described herein.
  • ocular indications including Age-Related Macular Degeneration (AMD) and associated geographic atrophy (GA)
  • ALD Age-Related Macular Degeneration
  • GA geographic atrophy
  • a pharmaceutical composition comprising the rAAV capsid protein comprising a peptide insertion of at least 4 and up to 9 contiguous amino acids and a transgene.
  • pharmaceutical compositions formulated for administration to the suprachoroidal space with a microneedle or microinjector for treatment of dry AMD including dry AMD with geographic atrophy.
  • the method comprises providing a starting plasmid that contains a gene expression cassette encoding a capsid gene, wherein a stop codon is placed at a target insertion site within the capsid gene, randomizing a repertoire of nucleic acids encoding randomized peptides to produce a peptide library; creating individual plasmids based on the starting plasmid 1) each having a nucleic acid encoding a random peptide from the peptide library inserted at the target insertion site of the capsid gene thus replacing the stop codon, and 2) each encoding a barcode for identification of the capsid gene having the insert placed before the 5'- or after the 3'-end of the capsid gene; collecting the individual plasmids to form a population or collection of plasmids encoding the capsids with peptide inserts and transfecting with the collection of plasmid
  • the parental AAV is AAV8, AAV8.AAA, or any other suitable AAV serotype, for example, as in Table 7.
  • the insertion site may be in VR-IV. including immediately after one of amino acids 451 -461 of AAV8 or corresponding to one of those residues or in VR-IV or VR-VIII, including immediately after amino acid 455 or 589 of AAV8 or corresponding to that position in a different AAV capsid type (see FIG. 1 for alignment).
  • the library of modified capsids is harvested from these cells.
  • the rAAV library population produced has high levels of capsids having the peptide inserts, including 85%, 90%, 95% or 98%, 99% or even 100%.
  • the invention is illustrated by way of examples infra describing the construction of engineered rAAV 8 capsids having peptide inserts designed from rAAV libraries enabling the detection of desirable properties such as tissue targeting.
  • a recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insertion of at least 4 and up to 9 contiguous amino acids, wherein the peptide insertion is immediately after an amino acid residue corresponding to one of amino acids 451 to 461 or amino acids 585-593 of a parental capsid protein, wherein the parental capsid protein is an AAV8 capsid protein having an amino acid sequence of SEQ ID NO: 33 or a capsid protein that has 90%, 95%, or 99% sequence identity thereto, wherein said peptide insertion has an amino acid sequence of one of SEQ ID NOs: 1-32, and wherein an rAAV vector comprising the capsid protein comprising the peptide insertion has enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein.
  • rAAV adeno-associated virus
  • adeno-associated virus (rAAV) capsid protein comprising a peptide insertion of at least 4 and up to 9 contiguous amino acids, wherein the peptide insertion is immediately after an amino acid residue corresponding to one of amino acids 451 to 461 or amino acids 585-593 of a parental capsid protein, wherein the parental capsid protein is an AAV8 capsid protein having an amino acid sequence of SEQ ID NO: 33 or a capsid protein that has 90%, 95%, or 99% sequence identity thereto, wherein said peptide insertion has an amino acid sequence of one of SEQ ID NOs: 115-131, and wherein an rAAV vector comprising the capsid protein comprising the peptide insertion has enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein.
  • rAAV adeno-associated virus
  • he rAAV capsid protein of embodiment 1 or 2 wherein said parental capsid protein is serotype 8 having 496NNN/AAA498 substitutions (AAV8.AAA) and has an amino acid sequence of SEQ ID NO: 114.
  • he rAAV capsid protein of embodiment 3 wherein said peptide insertion occurs immediately after one of amino acids Q451, T452, T453, G454, G455, T456, A457, N458, T459, Q460, or T461 of the parental capsid.
  • he rAAV capsid protein of embodiment 4 wherein said peptide insertion occurs immediately after amino acid G455 of the parental capsid.
  • he rAAV capsid protein of embodiment 6 which has an amino acid sequence of one of
  • he rAAV capsid protein of embodiment 1 or 2 wherein the peptide insertion occurs immediately after one of amino acids 585 to 593 of the parental capsid.
  • he rAAV capsid protein of embodiment 8. wherein the peptide insertion occurs immediately after amino acid Q589 of the parental capsid.
  • he rAAV capsid protein of embodiments 8 or 9 wherein the peptide insertion is 7 to 9 amino acids of one of the amino acid sequences of SEQ ID NO: 13-32 or 123-131.
  • he rAAV capsid protein of embodiment 10 which has an amino acid sequence of one of SEQ ID NOs: 62 to 113.
  • he rAAV capsid protein of any one of the preceding embodiments which has enhanced tropism for retina or RPE choroid tissue relative to the parental capsid protein.
  • he rAAV capsid protein of embodiment 12 wherein an rAAV vector comprising the capsid protein exhibits at least about 2 fold, 5 fold, 10 fold, 15 fold. 20 fold, 25 fold or 100 fold greater transduction of retina and/or RPE choroid tissue than an rAAV vector comprising the parental capsid protein.
  • he rAAV capsid protein of any one of the preceding embodiments wherein, upon administration to an eye, a rAAV vector comprising the capsid protein has reduced transduction of peripheral tissues than a rAAV vector comprising the parental capsid protein.
  • the rAAV vector comprising the capsid protein has an at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 25 fold or 40 fold lower transduction of liver than an rAAV vector comprising the parental capsid protein.
  • nucleic acid comprising a nucleotide sequence encoding the rAAV capsid protein of any one of the preceding embodiments, or encoding an amino acid sequence having at least 80% identity therewith, wherein an rAAV vector comprising the rAAV capsid protein of any of the preceding claims retains enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein.
  • he nucleic acid of embodiment 16 which encodes the rAAV capsid protein of any one of embodiments 1-15.
  • packaging cell capable of expressing the nucleic acid of embodiment 16 or embodiment 17 to produce AAV vectors comprising the capsid protein encoded by said nucleotide sequence.
  • rAAV vector comprising the rAAV capsid protein of any one of embodiments 1-15.
  • pharmaceutical composition comprising the rAAV vector of embodiment 19 or 20 and a pharmaceutically acceptable carrier.
  • he pharmaceutical composition of embodiment 21 formulated for administration to the suprachoroidal space.
  • method of delivering a transgene to a cell comprising contacting said cell with the rAAV vector of embodiment 19 or 20.
  • method of delivering a transgene to ocular tissue of a subject in need thereof comprising administering to said subject the rAAV vector of embodiment 19 or 20.
  • he method according to embodiment 25 wherein said rAAV vector is administered to the suprachoroidal space in the eye.
  • RPE choroidal tissue pharmaceutical composition for use in delivering a transgene to a cell in a subject in need thereof, wherein the pharmaceutical composition comprises the rAAV vector of embodiment 19 or 20.
  • pharmaceutical composition for use in treating Age-Related Macular Degeneration pharmaceutical composition for use in treating Age-Related Macular Degeneration
  • AMD AMD in a subject or a method of treating AMD in a human subject in need thereof comprising administering a therapeutically effective amount of recombinant adeno- associated virus (rAAV) vector comprising the recombinant adeno-associated virus (rAAV) capsid protein of any one of embodiments 1 to 15.
  • rAAV recombinant adeno- associated virus
  • rAAV recombinant adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • FIG. 1 depicts alignment of AAVs 1-8, hu31. hu32, AAV9, and rhlO capsid sequences (VP1) highlighting the VR-IV insertion site for these capsids (see VR4 corresponding to amino acids 451 to 461 of the AAV9 capsid protein and VR8 corresponding to amino acids 585 to 593 of the AAV9 capsid protein).
  • VP1 rhlO capsid sequences
  • AAV1 is SEQ ID NO: 34
  • AAV2 is SEQ ID NO: 35
  • AAV3 is SEQ ID NO: 36
  • AAV4 is SEQ ID NO: 37
  • AAV5 is SEQ ID NO: 38
  • AAV6 is SEQ ID NO: 39
  • AAV7 is SEQ ID NO: 40
  • AAV8 is SEQ ID NO: 33
  • AAV9 is SEQ ID NO: 43
  • AAVrhlO is SEQ ID NO: 44
  • hu31 is SEQ ID NO: 41
  • hu32 is SEQ ID NO: 42.
  • FIG. 2 illustrates a protein model of variable region four and eight of the adeno- associated virus type 9 (AAV9 VR-IV and AAV9 VR-VIII, respectively) indicating the NNN sequence at amino acids 498-500 of the AAV9 capsid sequence and the point of peptide insertion in VR-IV.
  • AAV9 VR-IV adeno- associated virus type 9
  • FIG. 3 depicts a representative genome construct of the capsid gene for use in construction of rAAV libraries having from 5’ to 3’: 5'-inverted terminal repeat (ITR), CMV enhancer-promoter, Rep intron, the AAV Cap gene of interest. polyA sequence, and 3 ’-ITR.
  • ITR 5'-inverted terminal repeat
  • CMV enhancer-promoter CMV enhancer-promoter
  • Rep intron the AAV Cap gene of interest.
  • polyA sequence polyA sequence
  • 3 ’-ITR 3
  • the illustration depicts insertion of a random peptide library in the place of a stop codon (see arrow) that was inserted into the Cap gene variable region before construction of the library (to reduce expression of wildtype sequence in the library).
  • FIG. 4 demonstrates the PCR amplicon obtained from representative libraries Al to G1 used for NGS analysis of library diversity.
  • FIGs. 5A-5F show-s (A) a mass spectrometry VP3 profile for a representative AAV5 library produced without a stop in the capsid sequence template and (B) with a stop codon in the template. (C) A representative AAV9 round 1 library adequately produced with the stop codon template. (D) NGS analysis of non-mutated sequence in the plasmid and vector library shows a 5-fold increase in non-stop codon sequence between the plasmid and vector and a 22- fold decrease in stop codon sequence, indicating selection against cross-packaging.
  • FIGs. 6A-6B Presents results of biodistribution after dosing NHPs in retina.
  • A Relative abundance (RA) and coefficient of variance (CV) of AAV8.VR4 capsids library capsids as indicated in retina relative to parental (AAV8) spiked-in vector control
  • B Relative abundance (RA) and coefficient of variance (CV) of AAV8.VR8 capsids library capsids as indicated in retina relative to parental (AAV8) spiked-in vector control.
  • FIGs. 7A-7B Presents abundance of each vector in the pool measured by NGS in retina and RPE-choroid tissue for each insertion capsid compared to parental capsid, represented as fold change RA relative to the control parental vector for both the 80% (A) and 20% (B) input pools.
  • FIGs. 8A-8F are bar graphs depicting the fold increase in mRNA transgene expression relative to the transgene expression from parental AAV8 when pooled capsids are administered into the suprachoroidal space in the 80% DF pool in (A) RPE-Choroid (CHR), (B) retina (RER), and (C) sclera (SCR) and in the 20% DF pool in (D) RPE-Choroid (CHR), (E) retina (RER), and (F) sclera (SCR).
  • A RPE-Choroid
  • RER retina
  • SCR sclera
  • FIGs. 9A-9B are plots illustrating engineered capsids from the AAV8.VR-IV library all having a higher abundance than parental AAV8 in both retina and RPE-C, with all performing more than lOx better than AAV8 in retina and nearly all in RPE-C.
  • AAV8 capsids 8.1 and 8.2 were selected for further evaluation.
  • FIGs. 10A-10B illustrate the fold-change difference of round 3 engineered capsids with increased transduction of (A) retina and (B) RPE-choroid compared to parental AAV8 following an SCS dose of 3x10 12 GC/eye to NHPs.
  • FIGs 11A-11D represent (A) aqueous humor (AH) transgene product (TP) levels measured by ELISA following single vector SCS administration in mini-pigs at a dose of 3xl0 n and 3xl0 12 vg/eye (AAV vectors carry a CAG.scFvOl transgene). (B) TP expression level following transduction of iRPE cells at an MOI of 3xl0 5 vg/cell. TP levels following single vector SCS administration in NHPs at a dose of 3xl0 12 vg/eye in (C) aqueous humor and in D) retina tissue punches.
  • AH aqueous humor
  • TP transgene product
  • FIGs 12A-12B are bar graphs showing transgene product expression (A) and biodistribution (B) of PEPIN3.1, PEPIN8.1, PEPIN8.2 and PEPIN8.4 in the retina at 3el2 GC/eye (solid) and 3el 1 GC/eye (brick).
  • FIGs 14A-14B are bar graphs showing in vitro transduction efficiency of rAAV comprising various capsids (AAV8. AAV3B. 3.1, 3.2, 8.1. 8.2, 8.3, and 8.4) in iRPE cells at day 21 (A) and ARPE cells at 48 hours (B).
  • FIG. 15 illustrates productivity in a NAVXpress® manufacturing platform, showing harvest yields (titer GC/mL) for AAV8 variants 8.1, 8.2, 8.3, and 8.4 were mostly comparable to AAV8.
  • rAAVs recombinant adeno-associated viruses
  • capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties, such as tissue targeting, transduction and integration of the rAAV genome.
  • engineered capsid proteins comprising peptide insertions of 7 or 9 contiguous amino acids, from random peptide libraries, inserted within or near variable region IV (VR-IV) or VR-V1I1 of the virus capsid, such that the peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle.
  • capsid proteins and libraries of the individual rAAVs comprising them, that have inserted peptides that target specific tissues and/or promote rAAV cellular uptake, transduction and/or transgene expression, for example, see Table 7.
  • capsids having 7 to 9 amino acid peptides which enhance targeting, transduction and/or integration of the rAAV genome in ocular tissue with peptides inserted in VR-IV of AAV 8 capsid protein with peptides having or comprising the amino acid sequences of SEQ ID NOs: 1-32 or 115-131.
  • novel AAV capsids which harbor peptide insertions that have undergone selection in non-human primates to identify variants with improved biodistribution to retina and RPE-choroid following suprachoroidal administration.
  • AAV or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses.
  • the AAV can be an AAV derived from a naturally occurring “wild-type” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and/or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene.
  • An example of the latter includes a rAAV having a capsid protein comprising a peptide insertion into the amino acid sequence of the naturally-occurring capsid.
  • rAAV refers to a “recombinant AAV.”
  • a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.
  • rep-cap helper plasmid refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from rAAV genomes lacking functional rep and/or the cap gene sequences.
  • cap gene refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus.
  • the capsid protein may be VP1, VP2, or VP3.
  • replica gene refers to the nucleic acid sequences that encode the non- structural protein needed for replication and production of virus.
  • nucleic acids and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules.
  • Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tri tylated bases.
  • Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes.
  • nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.
  • a subject is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), or, in certain embodiments, a human.
  • a primate e.g., monkey and human
  • '‘Library” or '‘libraries” generally refer to a repertoire of capsid genes (each unique and usually placed recombinantly into a vehicle, such as in a plasmid) or rAAV vectors produced from the unique capsids.
  • conservative amino acid substitution means substitutions made in accordance with Tables A and B.
  • variants and derivatives of the disclosed capsid proteins herein are to define them in terms of homology /identity to specific known sequences.
  • variants of capsids herein disclosed which have at least, 70% or at least 75% or at least 80% or at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% identity to the capsid sequences specifically recited herein.
  • Variants of capsids described herein may have 1. 2, 3.
  • a “therapeutic agent” refers to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene.
  • a “therapeutically effective amount” refers to the amount of agent, (e.g.. an amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder, when administered to a subject suffering therefrom.
  • a therapeutically effective amount w ith respect to an agent of the invention means that amount of agent alone, or when in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of the disease or disorder.
  • prophylactic agent refers to any agent which can be used in the prevention, delay, or slowing down of the progression of a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene.
  • a “prophylactically effective amount” refers to the amount of the prophylactic agent (e.g., an amount of product expressed by the transgene) that provides at least one prophylactic benefit in the prevention or delay of the target disease or disorder, when administered to a subject predisposed thereto.
  • a prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the occurrence of the target disease or disorder; or slow the progression of the target disease or disorder; the amount sufficient to delay or minimize the onset of the target disease or disorder; or the amount sufficient to prevent or delay the recurrence or spread thereof.
  • a prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the exacerbation of symptoms of a target disease or disorder.
  • a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or when in combination with other agents, that provides at least one prophylactic benefit in the prevention or delay of the disease or disorder.
  • a prophylactic agent of the invention can be administered to a subj ect “pre-disposed” to a target disease or disorder.
  • a subject that is “pre-disposed” to a disease or disorder is one that show's symptoms associated with the development of the disease or disorder, or that has a genetic makeup, environmental exposure, or other risk factor for such a disease or disorder, but where the symptoms are not yet at the level to be diagnosed as the disease or disorder.
  • a patient with a family history of a disease associated with a missing gene may qualify as one predisposed thereto.
  • One aspect relates to capsid protein libraries and the recombinant adeno-associated virus (rAAV) vectors thereof, the capsid proteins within the library engineered to comprise a peptide insertion from a random peptide library, wherein the peptide is not an AAV protein or peptide fragment thereof, where the peptide insertion is surface exposed when packaged as an AAV particle.
  • the peptide insertion occurs within (i.e., between two amino acids without deleting any capsid amino acids) variable region IV (VR-1V) of an AAV8 capsid or AAV8.AAA capsid, or a corresponding region for another type AAV capsid (see alignment in FIG. 1).
  • the peptide insertion occurs within (/. ⁇ ?., between two amino acids without deleting any capsid amino acids) variable region VIII (VR-VIII) of an AAV8 capsid or AAV8.AAA, or a corresponding region of a capsid for another AAV type (see alignment in FIG. 1).
  • the peptide insertion is from a heterologous protein or domain (that is not an AAV capsid protein or domain), which directs the rAAV particles to target tissues and/or promote rAAV uptake, transduction and/or transgene expression.
  • nucleic acids encoding the engineered capsid proteins and variants thereof, packaging cells for expressing the nucleic acids to produce rAAV vectors, rAAV vectors further comprising a transgene, and pharmaceutical compositions of the rAAV vectors, as well as methods of using the rAAV vectors to deliver the transgene to a target cell type or target tissue of a subject in need thereof.
  • the target tissue may be ey e/retina tissue (including RPE- choroid or sclera tissue), and the capsid with the peptide insertion specifically recognizes and/or binds to and/or homes to that tissue, or for example, one or more specific cell types, such as within the target tissue, or cellular matrix thereof.
  • peptides that can target rAAVs to ocular tissue can be useful for delivering therapeutics for treating ocular disorders and may be delivered by ocular, including, suprachoroidal administration.
  • rAAV capsids containing the engineered capsid protein with the peptide insert have increased uptake, transduction, and/or transgene expression than the parental capsid or a reference capsid, including AAV8 or AAV8.AAA.
  • the engineered capsids may further have reduced transduction in tissues such as peripheral tissues and/or liver relative to the targeted tissue or relative to transduction with the parental capsid or a reference capsid, including AAV8 or AAV8.AAA when administered to the eye.
  • the target tissue may be ocular tissue, and particularly retina, RPE-choroid and/or sclera tissue.
  • the parental capsid and/or the peptide insert detarget the rAAV vector comprising the parental capsid and/or the engineered capsid protein (having the peptide insert) from one or more tissue types, including liver and/or peripheral tissues.
  • a peptide insertion described as inserted "at" a given site refers to insertion immediately after that is having a peptide bond to the carboxy group of, the residue normally found at that site in the wild type virus.
  • insertion at G455 in AAV8 means that the peptide insertion appears between G455 and the consecutive amino acid (G456) in the AAV8 wildtype capsid protein sequence (SEQ ID NO: 33).
  • insertion at Q589 in AAV8 means that the peptide insertion appears between Q589 and the consecutive amino acid (N560) in the AAV8 wildtype capsid protein sequence (SEQ ID NO: 33).
  • the capsid protein is an AAV8 capsid protein (SEQ ID NO: 33, or a capsid protein having an amino acid sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 33) or an AAV8.AAA capsid protein and the insertion occurs immediately after at least one of the amino acid residues 451 to 461.
  • the peptide insertion occurs immediately after amino acid T451, T452, G453, G454, T455, A456, N457, T458. Q459, T460, or L461 of the AAV8 capsid (ammo acid sequence SEQ ID NO: 33).
  • the peptide is inserted between residues G455 and G456 of AAV8 capsid protein or between the residues corresponding to G455 and G456 of an AAV capsid protein other than an AAV8 capsid protein (amino acid sequence SEQ ID NO: 33).
  • the peptide is inserted immediately after one of the amino acid residues of 585 to 593 in VR-VIII of the AAV8 capsid protein (SEQ ID NO: 33), including between residues Q589 and N560 of AAV8 capsid protein or immediately after one of the amino acid residues corresponding to 585 to 593 of the AAV8 capsid protein or between the residues corresponding to Q589 and N560 of an AAV capsid protein other than an AAV8 capsid protein (amino acid sequence SEQ ID NO: 33) (see Fig. 1 for alignment).
  • the capsid protein is an AAV8.AAA capsid protein and the insertion occurs immediately after at least one of the amino acid residues 451 to 461.
  • the peptide insertion occurs immediately after amino acid T451, T452, G453, G454, T455, A456, N457. T458, Q459, T460. or L461 of the AAV8.AAA capsid (amino acid sequence SEQ ID NO: 114).
  • the peptide is inserted between residues G455 and G456 of AAV8.AAA capsid protein or between the residues corresponding to G455 and G456 of an AAV8.AAA capsid protein other than an AAV8 capsid protein (amino acid sequence SEQ ID NO: 114).
  • the peptide is inserted immediately after one of the amino acid residues of 585 to 593 in VR-VIII of the AAV8.AAA capsid protein (SEQ ID NO: 114), including between residues Q589 and N560 of AAV8.AAA capsid protein or immediately after one of the amino acid residues corresponding to 585 to 593 of the AAV8.AAA capsid protein or between the residues corresponding to Q589 and N560 of an AAV capsid protein other than an AAV8.AAA capsid protein (amino acid sequence SEQ ID NO: 33) (see Fig. 1 for alignment).
  • the capsid protein has an amino acid sequence of one of SEQ ID NOs: 50-113).
  • the capsid protein is from at least one AAV type selected from AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6). serotype 7 (AAV7), serotype rh8 (AAVrh8).
  • AAV1 AAV serotype 1
  • AAV2 AAV2
  • AAV3 serotype 3
  • AAV4 serotype 4
  • serotype 5 AAV5
  • serotype 6 AAV6
  • serotype 7 AAV7
  • serotype rh8 AAVrh8
  • serotype 9 AAV9
  • serotype 9e AAV9e
  • serotype rhlO AAVrhlO
  • serotype rh20 AAVrh20
  • serotype rh39 AAVrh39
  • seroty pe hu.37 AAVhu.37
  • seroty pe rh74 AAVrh74, versions 1 and 2 (see FIG. 1 for alignment with AAV l-AAV9, hu31.
  • Hu32, and rhlO capsid sequences or a capsid that is at least 90%, 95% or 99% identical in amino acid sequence to the amino acid sequence of the VP1, VP2, or VP3 of the foregoing capsid proteins, and the insertion occurs immediately after an amino acid residue corresponding to at least one of the amino acid residues 451 to 461 of AAV8.
  • the alignments of these different AAV serotypes, as show n in FIG. 1, indicates “corresponding” amino acid residues in the different capsid amino acid sequences such that a “corresponding” amino acid residue is lined up at the same position in the alignment as the residue in the reference sequence.
  • the peptide insertion occurs immediately after one of the amino acid residues within: 450-459 of AAV1 capsid (SEQ ID NO: 34); 449-458 of AAV2 capsid (SEQ ID NO: 35); 449-459 of AAV3 capsid (SEQ ID NO: 36); 443-453 of AAV4 capsid (SEQ ID NO: 37); 442-445 of AAV5 capsid (SEQ ID NO: 38); 450-459 of AAV6 capsid (SEQ ID NO: 39); 451-461 of AAV7 capsid (SEQ ID NO: 40); 451-461 of AAV8 capsid (SEQ ID NO: 33); 451-461 of AAV9 capsid (SEQ ID NO: 43); 451-461 of AAVhu.32 capsid (SEQ ID NO: 42); 452-461 of AAVrhlO capsid (SEQ ID NO: 44); 452-461 of AAVrh20 capsid (SEQ ID NO: 47); 452-461
  • the rAAV capsid protein comprises a peptide insertion immediately after (i.e., C-terminal to) amino acid 589 of AAV8 capsid protein (having the amino acid sequence of SEQ ID NO: 33 and see FIG. 1) or AAV8.AAA (having SEQ ID NO: 114), where said peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle.
  • the rAAV capsid protein has a peptide insertion that is not immediately after amino acid 589 of AAV8 or AAV8.AAA. or corresponding to amino acid 589 of AAV8.
  • AAV vectors comprising the engineered capsids.
  • the AAV vectors are non-replicating and do not include the nucleotide sequences encoding the rep or cap proteins (these are supplied by the packaging cells in the manufacture of the rAAV vectors).
  • AAV-based vectors comprise components from one or more serotypes of AAV.
  • AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15.
  • AAV based vectors provided herein comprise components from one or more of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B.
  • rAAV particles comprise a capsid protein at least 80% or more identical, e.g. 85%. 86%.
  • AAV8, AAV9, AAV 10 AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8.
  • These engineered AAV vectors may comprise a genome comprising a transgene encoding a therapeutic protein.
  • the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety ).
  • the recombinant AAV for use in compositions and methods herein is AAV.7m8 (including variants thereof) (see, e.g., US 9,193,956; US 9,458,517; US 9,587,282; US 2016/0376323, and WO 2018/075798, each of which is incorporated herein by reference in its entirety)-
  • the AAV for use in compositions and methods herein is any AAV disclosed inUS 9,585,971, such as AAV-PHP.B.
  • the AAV for use in compositions and methods herein is an AAV2/Rec2 or AAV2/Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4): e60361, which is incorporated by reference herein for these vectors).
  • the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7.282,199; US 7,906.111; US 8,524,446; US 8,999,678; US 8,628,966; US 8,927,514; US 8,734,809; US 9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015/0374803; US 2015/0126588; US 2017/0067908; US 2013/0224836; US 2016/0215024; US 2017/0051257; PCT/US2015/034799; and PCT/EP2015/053335.
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos.
  • rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al.. 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety.
  • rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo etal.. 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety.
  • rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1.
  • rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No.
  • WO 2003/052051 see, e.g., SEQ ID NO: 2 of '051 publication
  • WO 2005/033321 see, e.g, SEQ ID NOs: 123 and 88 of '321 publication
  • WO 03/042397 see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication
  • WO 2006/068888 see, e.g.. SEQ ID NOs: 1 and 3-6 of '888 publication.
  • WO 2006/110689 see, e.g...
  • SEQ ID NOs: 5-38 of '689 publication W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010/127097 (see, e.g., SEQ ID NOs: 5-38 of '097 publication), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294 of '508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of '924 publication), the contents of each of which is herein incorporated by reference in its entirety.
  • rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g, SEQ ID NO: 2 of '051 publication). WO 2005/033321 (see, e.g.
  • rAAV particles comprise a pseudo typed AAV capsid.
  • the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids.
  • Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan etal., J. Virol., 75:7662-7671 (2001); Halbert etal., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).
  • a single-stranded AAV may be used.
  • a self-complementary vector e.g., scAAV
  • scAAV single-stranded AAV
  • the peptide insertion is sequence of contiguous amino acids from a heterologous protein or domain thereof.
  • the peptide to be inserted typically is long enough to retain a particular biological function, characteristic, or feature of the protein or domain from which it is derived.
  • the peptide to be inserted typically is short enough to allow the capsid protein to form a coat, similarly or substantially similarly to the native capsid protein without the insertion.
  • the peptide insertion is from about 4 to about 30 amino acid residues in length, about 4 to about 20, about 4 to about 15, about 5 to about 10, or about 7 amino acids in length.
  • the peptide sequences for insertion are at least 4 amino acids in length and may be 5, 6, 7, 8, 9, 10, 11, 12, 13. 14. or 15 amino acids in length.
  • the peptide sequences are 16, 17, 18, 19, or 20 amino acids in length.
  • the peptide is no more than 7 amino acids, 10 amino acids or 12 amino acids in length.
  • a “peptide insertion from a heterologous protein” in an AAV capsid protein refers to an amino acid sequence that has been introduced into the capsid protein and that is not native to the AAV serotype capsid into which it is inserted.
  • peptides may also or alternatively promote rAAV cell uptake, transduction and/or genome integration in cells of the target tissue.
  • Examples of peptides for use as peptide insertions at any of the AAV capsid sites described herein are presented in Tables 3A and 3B and Table 6 below in the Examples (including the peptides having amino acid sequences of SEQ ID NOs: 1-32 and SEQ ID NOs: 115-131), and include at least 7 amino acid contiguous portions thereof, or 9 amino acid contiguous portions thereof (and includes variants having 1, 2 or 3 amino acid substitutions, including conservative amino acid substitutions) and have the functional attribute of the peptide being inserted alters the properties of the capsid, particularly its tropism.
  • the recombinant AAV capsids and AAV vectors are engineered to include a peptide, or at least 7 or 9 amino acid contiguous portion thereof, from any of Tables 3A and 3B or Table 6 below (including peptides of amino acid sequences of SEQ ID NOs: 1-32 or SEQ ID NOs: 115-131), inserted into the AAV capsid sequence in such a way that the peptide insertion is displayed.
  • the peptides are inserted after an amino acid residue at positions 455 or 589 of the amino acid sequence of the AAV8 capsid (SEQ ID NO: 33) or of the AAV8.AAA capsid (SEQ ID NO: 114) a position corresponding thereto in any other AAV serotype (see FIG. 1 for capsid sequence alignments).
  • Capsid protein (VP3) sequences of AAV8 capsid proteins having these peptide inserts are provided as amino acid sequences SEQ ID NOs: 50 to 1 13.
  • heterologous peptide insertion libraries refers to a collection of rAAV vectors that carry' the same random peptide insertion at the same insertion site in the virus capsid to make the particular library, e.g, at a position within a given variable region of the capsid.
  • the capsid proteins used comprise AAV genomes that contain modified rep and cap sequences to prevent the replication of the virus under conditions in which it could normally replicate (co-infection of a mammalian cell along with a helper virus such as adenovirus).
  • the members of the peptide insertion libraries may then be assayed for functional display of the peptide on the rAAV surface, tissue targeting and/or gene transduction. Enhanced properties or desirable properties may be assessed upon comparison with the parental capsid from which the insertion library was made.
  • peptide insertion libraries and methods of making these libraries.
  • An exemplary method of producing a capsid library with peptide inserts is descnbed herein in Example 1 and such libraries are screened herein as described in Examples 2 and 3.
  • the nucleic acid encoding the parental capsid has a stop codon at the target insertion site for the population of nucleic acids encoding the peptides such that a capsid protein will only be expressed if the peptide insert is present, otherwise, translation will terminate prematurely.
  • Production of the populations of rAAVs from the expression constructs results in an rAAV population with a high percentage of capsids with peptide inserts (including 80%, 85%, 90%, 95%, 98%, 99% or even 100%).
  • the method comprises (1) providing a starting plasmid that contains a gene expression cassette comprising a nucleic acid sequence encoding an AAV capsid, wherein a stop codon is placed at the target insertion site within the capsid gene, (2) providing a repertoire of nucleic acids encoding randomized peptides to produce a peptide library; (3) creating individual plasmids based on the starting plasmid a) each having a nucleic acid encoding a random peptide from the peptide library inserted at the target insertion site of the capsid gene thus replacing the stop codon, and b) each encoding a barcode for identification of the capsid gene having the insert placed before the 5'- or after the 3'-end of the capsid gene; (4) collecting the individual plasmids to form a population or collection of plasmids encoding the capsids with
  • the parental AAV is AAV8. AAV8.AAA, or any other suitable AAV serotype.
  • the insertion site may be in VR-IV, including immediately after one of amino acids 455 of AAV8 or corresponding to one of those residues or in VR-VIII, including immediately after amino acid 589 of AAV8 or corresponding to that position in a different AAV capsid type (see FIG. 1 for alignment).
  • the peptides may be 7 or 9 amino acids in length.
  • the rep gene is on a separate expression plasmid from the plasmid encoding the cap gene with the inserts.
  • the library of modified capsids is harvested from these cells.
  • the rAAV library population produced has high levels of capsids having the peptide inserts, including 85%. 90%. 95% or 98%, 99% or even 100%.
  • the present inventors also have surprisingly discovered peptides that inserted into rAAV vectors “re-target” or enhance targeting properties of such AAV vectors to specific tissues, organs, or cells; in particular, providing peptides that cause rAAV vectors to target ocular tissue and/or other target tissues of interest, such as retinal tissue. RPE-choroid or scleral tissue. This can provide enhanced transport of rAAV particles encapsidating a transgene for optimizing distribution of the vector upon administration to the body, including by suprachoroidal administration.
  • Such peptides, and modified vectors are described below.
  • capsid proteins selected from the random peptide insertion libraries comprising peptide insertions selected to confer or enhance ocular tissue-homing properties, or “ocular tropism”, including homing to ocular tissue.
  • capsids and rAAV vectors having capsids comprising these peptide-containing capsid proteins.
  • the peptides may target other tissues, such as, retinal, and other ocular tissues and may also detarget tissues such as liver and/or peripheral tissues when administered to the eye.
  • the peptide insertion consists of 7 or 9 contiguous amino acids of a peptide sequence of Tables 3A and 3B (SEQ ID NOs: 1-32) or may be a peptide within a consensus sequence of Table 6 (SEQ ID NOs: 115-131).
  • the peptides disclosed herein were identified by screening libraries of peptides inserted in AAV capsids, which are screened for properties such as tropism for ocular tissue, retinal tissue, and other ocular tissues, such as RPE-choroid or scleral tissue, for example, through mouse and NHP biodistribution studies as described in the examples.
  • the capsids having the peptide insertions have increased tropism for a target tissue, including human retinal cells, photoreceptor cells, retina, RPE-choroid, or other ocular tissue, relative to the parental capsid (that is having the capsid protein without the peptide insert) or a reference capsid, such as AAV8 or AAV8.AAA.
  • the capsids with the peptide inserts may also distribute to liver and/or peripheral tissue at levels less than target tissues such as eye, retina, or other target tissue and/or less than a reference capsid, such as AAV8, including when administered to the eye.
  • the peptides that may be inserted into capsid proteins include those listed in Tables 3A and 3B and include peptides having amino acid sequences of 7 or 9 contiguous amino acids of one of the amino acid sequences of SEQ ID NOs: 1-32 or the consensus sequences of Table 6 (SEQ ID NOs: 115-131).
  • the peptide is a 7 contiguous amino acid sequence of one of KPKPQQV (SEQ ID NO: 1), RTLKPQA (SEQ ID NO: 2), RKQVQSP (SEQ ID NO: 3), LQRASVM (SEQ ID NO: 4), RQKNAMV (SEQ ID NO: 5), RIMQTKT (SEQ ID NO: 6), RKTMAAV (SEQ ID NO: 7), RLIQGKP (SEQ ID NO: 8), TKLQAKP (SEQ ID NO: 9), RMKTVQT (SEQ ID NO: 10), RIQMGTK (SEQ ID NO: 11), or RPKSTMV (SEQ ID NO: 12).
  • KPKPQQV SEQ ID NO: 1
  • RTLKPQA SEQ ID NO: 2
  • RKQVQSP SEQ ID NO: 3
  • LQRASVM SEQ ID NO: 4
  • RQKNAMV SEQ ID NO: 5
  • RIMQTKT SEQ ID NO
  • the peptide is inserted in VR-IV (including immediately after amino acid 455 of AAV8, AAV8.AAA).
  • the peptide is a 7 or 9 contiguous amino acid sequence of one of SENRAQK (SEQ ID NO: 13), DNTTFRR (SEQ ID NO: 14), RTIRGDL (SEQ ID NO: 15), QNRVTAS (SEQ ID NO: 16), QNTIRTQ (SEQ ID NO: 17), ENVNRSK (SEQ ID NO: 18), MAVGGSK (SEQ ID NO: 19), EQAFKRM (SEQ ID NO: 20), HVNGRSS (SEQ ID NO: 21), EFTNKVR (SEQ ID NO: 22).
  • GSENRAQKA (SEQ ID NO: 23), GDNTTFRRA (SEQ ID NO: 24), GRTIRGDLA (SEQ ID NO: 25), GQNRVTASA (SEQ ID NO: 26), GQNTIRTQA (SEQ ID NO: 27), GENVNRSKA (SEQ ID NO: 28), GMAVGGSKA (SEQ ID NO: 29), GEQAFKRMA (SEQ ID NO: 30), GHVNGRSSA (SEQ ID NO: 31). or GEFTNKVRA (SEQ ID NO: 32).
  • the peptide is inserted in VR-VIII (including immediately after amino acid 589 of AAV8, AAV8.AAA).
  • the 7-mer to 9-mer peptide that is inserted into the AAV8 capsid is a peptide which has an amino acid sequence within a consensus sequence (see Example 5 and Table 6). and has or comprises an amino acid sequence of
  • Xi and X2 are each any amino acid; Xs is R or K or Q or H; X4 is any amino acid; X5 is K or Q or S or T; Xe is S or T or Q or A or I or V; and X7 is V or Q or T or P or S (SEQ ID NO: 115); ii) Xi and X2 are each any amino acid; X3 is R or K or Q; X4 is V or K; X5 is K or Q or S or T; Xe is S or T or Q or A or I or V; and X7 is V or Q or T or P or S (SEQ ID NO: 116); iii) Xi is any amino acid; X2 is R or K or F or P or N; X3 and X4 are each any amino acid;
  • capsids having an amino acid sequence of SEQ ID NO 50-113 (see Table 7).
  • the peptide is a variant of one of the amino acid sequences of SEQ ID NOs: 1 to 32 which has 1, 2 or 3 amino acid substitutions, including conservative amino acid substitutions, while the peptide, when inserted into a capsid protein, retains its biological activity.
  • the peptides may be inserted into wild type or variant capsid protein amino acid sequences at positions such that the peptide is surface displayed when the capsid protein is incorporated into an AAV capsid, for example, at sites that allow surface exposure of the peptide, such as within variable surface-exposed loops, and, in more examples, sites described herein corresponding to VR-I, VR-IV, or VR-VIII, or may be inserted after the first amino acid of VP2, e.g. after amino acid 137 (AAV4, AAV4-4, and AAV5) or at amino acid 138 (AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, rh.10.
  • the capsid protein is an AAV8 capsid protein or an AAV8.AAA capsid protein (or a capsid protein with 90%, 95% or 99% amino acid sequence identity to AAV8 or AAV8.AAA) and the peptide insertion occurs immediately after at least one of (or corresponding to) the amino acid residues 451 to 461 of the AAV8 capsid.
  • the capsid protein is from at least one AAV type selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrh20, AAVhu.31, AAVhu.32, AAVhu.37, AAVrh39, and AAVrh74 (versions 1 and 2) (see, for example, FIG. 1) or a capsid protein that has 90%, 95% or 99% amino acid identity to the capsid protein of AAV1.
  • the peptide insertion occurs immediately after an amino acid residue 455 of AAV8 (SEQ ID NO: 33). In embodiments, the peptide insertion occurs immediately after an amino acid residue corresponding to 589 of AAV8 (SEQ ID NO: 33).
  • the alignments of different AAV serotypes, as shown in FIG. 1, indicates corresponding amino acid residues in the different amino acid sequences.
  • the capsid protein having the peptide insert is one of the capsid proteins in Table 7, with amino acid sequence SEQ ID NOs: 50 to 113.
  • the capsid protein is 90%, 95% or 99% identical to SEQ ID NOs: 50 to 113. except that it is identical with respect to the peptide insert and retains its biological activity.
  • the capsids with peptide inserts as described herein have increased tropism for target tissues, such as retina, and other target tissue relative to the parental capsid, i.e., containing the capsid protein that is identical except that it does not have the peptide insert, or a reference capsid, which may include AAV8, AAV8.AAA. or other capsid of interest that does not contain the peptide insert.
  • the engineered capsid may have reduced tropism or is detargeted for tissues such as liver and/or peripheral tissues, including relative to target tissues, eye, retina, or others, and/or relative to the parental capsid or a reference capsid, which may include AAV8 or AAV8.AAA.
  • the reference capsid does not contain the 496NNN/AAA498 amino acid substitution — for example if the parental capsid is AAV8.AAA. then the liver tropism/detargeting may be relative to AAV8 without the peptide insert, including in embodiments, when administered to the eye.
  • the tissue tropism may be assessed by introducing an rAAV vector having the engineered capsid and a genome with a detectable transgene into a test animal, such as a mouse or NHP, for example by systemic, intravenous, intramuscular, intrathecal, subcutaneous, ocular, suprachoroidal administration or other administration, at an appropriate dosage (for example 1E12, 1E13 or 1E14 vg/kg) and then after an appropriate period of time harvesting the tissues of the animal and assessing the presence of the vector genome, mRNA transcribed from the genome, the ratio of the mRNA to the vector DNA, transgene protein product or activity, including relative to the parental or reference capsid.
  • a test animal such as a mouse or NHP
  • an appropriate dosage for example 1E12, 1E13 or 1E14 vg/kg
  • capsids as described herein which, when administered (for example, IV, IM, subcutaneous, suprachoroidal administration) to an animal, including a mouse or NHP, exhibit at least 2-fold, 5-fold, 10-fold, 15 fold, 20-fold, or 25 fold, or greater tropism for ocular tissue, including retinal, RPE-choroidal or scleral tissue, relative to a parental capsid or reference, such as AAV8 or AAV8.AAA, as measured by vector genome DNA, transgene mRNA, the ratio of mRNA to vector genome DNA, trans gene protein product, including protein product activity.
  • the capsid preferentially transduces retinal cells or other ocular tissue in the eye of the animal.
  • capsids as described herein which when administered (for example, IV, IM, subcutaneous, suprachoroidal administration) to an animal, including a mouse or NHP, exhibit at least 2-fold, 5-fold, 10-fold, 15 fold. 20-fold, 25 fold, 40-fold or 50-fold, less tropism for liver, either relative to a target ocular tissue relative to a parental capsid or reference, such as AAV8 or AAV8.AAA, as measured by amount of vector genome DNA, transgene mRNA, the ratio of mRNA to vector genome DNA, transgene protein product, including protein product activity.
  • AAV1 138; 262-272; 450-459; 595-593; and in an embodiment, between 453-454 (SEQ ID NO: 34).
  • AAV2 138; 262-272; 449-458; 584-592; and in an embodiment between 452-453 (SEQ ID NO: 35).
  • AAV3 138; 262-272; 449-459; 585-593; and in an embodiment, between 452-453 (SEQ ID NO: 36).
  • AAV4 137; 256-262; 443-453; 583-591; and in an embodiment, between 446-447 (SEQ ID NO: 37).
  • AAV5 137; 252-262; 442-445; 574-582; and in an embodiment, between 445-446 (SEQ ID NO: 38).
  • AAV6 138; 262-272; 450-459; 585-593; and in an embodiment, between 452-453 (SEQ ID NO: 39).
  • AAV7 138; 263-273; 451-461; 586-594; and in an embodiment, between 453-454 (SEQ ID NO: 40).
  • AAV8 138; 263-274; 451-461; 587-595; and in an embodiment, between 453-454 (SEQ ID NO: 33).
  • AAV9 138; 262-273; 452-461; 585-593; and in an embodiment, between 454-455 (SEQ ID NO: 43).
  • AAV8.AAA 138; 262-273; 452-461; 585-593; and in an embodiment, between 454- 455 (SEQ ID NO: 114).
  • AAVrhl O 138; 263-274; 452-461 ; 587-595; and in an embodiment, between 454-455 (SEQ ID NO: 44).
  • AAVrh20 138; 263-274; 452-461; 587-595; and in an embodiment, between 454-455 (SEQ ID NO: 47).
  • AAVrh74 138; 263-274; 452-461; 587-595; and in an embodiment, between 454-455 (SEQ ID NO: 48 or SEQ ID NO: 49).
  • AAVhu.32 138; 262-273; 452-461; 585-593; and in an embodiment, between 454-455 (SEQ ID NO: 42).
  • the peptide insertion occurs between amino acid residues 588-589 of the AAV8 capsid, or between corresponding residues of another AAV type capsid as determined by an amino acid sequence alignment (for example, as in FIG. 1). In embodiments, the peptide insertion occurs immediately after amino acid residue G455 or Q589 of the AAV8 capsid sequence, or immediately after corresponding residues of another AAV capsid sequence (FIG. 1)
  • the capsid is chosen and/or further modified to reduce recognition of the AAV particles by the subject’s immune system, such as avoiding preexisting antibodies in the subject. In some embodiments. In some embodiments, the capsid is chosen and/or further modified to enhance desired tropism/targeting.
  • AAV capsids were modified by introducing selected single to multiple amino acid substitutions which increase effective gene delivery to ocular tissue, detarget the liver, and/or reduce immune responses of neutralizing antibodies, prior to making the rAAV library with random peptide insertions.
  • Exposure to the AAV capsid can generate an immune response of neutralizing antibodies.
  • One approach to overcome this response is to map the AAV-specific neutralizing epitopes and rationally design an AAV capsid able to evade neutralization.
  • a monoclonal antibody, specific for intact AAV9 capsids, with high neutralizing titer has recently been described (Giles et al, 2018, Mapping an Adeno-associated Virus 9-Specific Neutralizing Epitope To Develop Next-Generation Gene Delivery' Vectors).
  • the epitope was mapped to the 3-fold axis of symmetry on the capsid, specifically to residues 496-NNN-498 and 588- QAQAQT-592 of, e.g., AAV9.
  • capsids which were further modified by substituting asparagines at amino acid positions 498, 499, and 500 (such as AAV8.AAA) or 496, 497, and 498 (herein referred to as AAV8.AAA, SEQ ID NO: 114) with alanines.
  • capsids having three asparagines at amino acid positions 496. 497, and 498 of the AAV8 capsid replaced with alanines and optionally tryptophan at amino acid 503 of the AAV 8 capsid with arginine or capsids with substitutions corresponding to these positions in other AAV types.
  • the rAAVs described herein increase tissue-specific (such as. but not limited to, eye) cell transduction in a subject (a human, non-human-primate, or mouse subject) or in cell culture, compared to the rAAV not comprising the peptide insertion.
  • the increase in tissue specific cell transduction is at least 2, 10, 20, 30, 40, 50, 60. 70, 80, 90, or 100 fold more than that without the peptide insertion.
  • the increase in transduction may be assessed using methods described in the Examples herein and known in the art.
  • a molecule according to the invention is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid protein molecules herein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein.
  • the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%.
  • the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of the AAV8 capsid protein (SEQ ID NO: 33 and see FIG. 1), while retaining (or substantially retaining) biological function of the AAV8 capsid protein and the inserted peptide.
  • the capsid protein, coat, and rAAV particles may be produced by techniques known in the art.
  • the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector.
  • the viral genome further comprises a cap gene and/or a rep gene for expression and splicing of the cap gene.
  • the cap and rep genes are provided by a packaging cell and not present in the viral genome.
  • the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap helper plasmid in place of the existing capsid gene.
  • this plasmid When introduced together into host cells, this plasmid helps package a rAAV genome into the engineered capsid protein as the capsid coat.
  • Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Nonlimiting examples include 293 cells or derivatives thereof, HELA cells, or insect cells.
  • Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g, electroporation, lipofection).
  • Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See. e.g.. Sambrook et al.. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
  • the rAAVs provide transgene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below'.
  • the rAAV vector also includes regulatory control elements known to one skilled in the art to influence the expression of the RNA and/or protein products encoded by nucleic acids (transgenes) within target cells of the subject. Regulator ⁇ ' control elements and may be tissue-specific, that is, active (or substantially more active or significantly more active) only in the target cell/tissue.
  • the AAV vector comprises a regulatory sequence, such as a promoter, operably linked to the transgene that allows for expression in target tissues.
  • the promoter may be a constitutive promoter, for example, the CB7 promoter.
  • Additional promoters include: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter, opsin promoter, the TBG (Thyroxine-binding Globulin) promoter, the APOA2 promoter, SERPINA1 (hAAT) promoter, or MIR122 promoter.
  • an inducible promoter is used, e.g.. hypoxia-inducible or rapamycin-inducible promoter.
  • AAV8 vectors comprising a viral genome comprising an expression cassette for expression of the transgene, under the control of regulatory elements, and flanked by ITRs and an engineered viral capsid as described herein or is at least 95%, 96%, 97%. 98%. 99% or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (see FIG. 1), while retaining the biological function of the engineered AAV8 capsid.
  • the encoded AAV8 capsid has the sequence of wild type AAV8, with the peptide insertion as described herein, with, in addition, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. 25.
  • engineered AAV vectors other than AAV8 vectors such as engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVhu.31, AAVhu.32, AAVhu.37, AAVrh39, or AAVrh74 vectors, with the peptide insert as described herein and 1, 2, 3. 4, 5, 6. 7, 8, 9. 10. 1 1. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions relative to the wild type or unengineered sequence for that AAV type and that retains its biological function.
  • the recombinant adenovirus can be a first-generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region.
  • the recombinant adenovirus can be a second-generation vector, which contains full or partial deletions of the E2 and E4 regions.
  • a helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi).
  • the transgene generally is inserted between the packaging signal and the 3 TTR, with or without stuffer sequences to keep the genome close to wild-type size of approximately 36 kb.
  • the rAAV vector for delivering the transgene to target tissues, cells, or organs has a tropism for that particular target tissue, cell, or organ. Tissue-specific promoters may also be used.
  • the construct further can include expression control elements that enhance expression of the transgene driven by the vector (e.g., introns such as the chicken P-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), P-globin splice donor/immunoglobulin heavy chain spice acceptor intron, adenovirus splice donor /immunoglobulin splice acceptor intron, SV40 late splice donor /splice acceptor (19S/16S) intron, and hybrid adenovirus splice donor/IgG splice acceptor intron and polyA signals such as the rabbit P-globin polyA signal, human growth hormone (hGH)
  • nucleic acids sequences disclosed herein may be codon- optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review 7 by Quax et al., 2015, Mol Cell 59:149-161).
  • the constructs described herein comprise the following components: (1) AAV8 inverted terminal repeats that flank the expression cassette; (2) control elements, which include a) the CB7 promoter, comprising the CMV enhancer/chicken P-actin promoter, b) a chicken p-actin intron and c) a rabbit P-globin poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid or protein product of interest.
  • control elements which include a) the CB7 promoter, comprising the CMV enhancer/chicken P-actin promoter, b) a chicken p-actin intron and c) a rabbit P-globin poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid or protein product of interest.
  • the constructs described herein comprise the following components: (1) AAV 8 inverted terminal repeats that flank the expression cassette; (2) control elements, which include a) a hypoxia-inducible promoter, b) a chicken p-actin intron and c) a rabbit P-globin poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid or protein product of interest.
  • the viral vectors provided herein may be manufactured using host cells, e.g., mammalian host cells, including host cells from humans, monkeys, mice, rats, rabbits, or hamsters.
  • host cells e.g., mammalian host cells, including host cells from humans, monkeys, mice, rats, rabbits, or hamsters.
  • Nonlimiting examples include: A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC I, BSC 40, BMT 10, VERO. W138. HeLa. 293, Saos. C2C12, L. HT1080, HepG2. primary fibroblast, hepatocyte, and myoblast cells.
  • the host cells are stably transformed with the sequences encoding the transgene and associated elements (i.e., the vector genome), and genetic components for producing viruses in the host cells, such as the replication and capsid genes (e.g. , the rep and cap genes of AAV).
  • viruses e.g. , the rep and cap genes of AAV.
  • the replication and capsid genes e.g. , the rep and cap genes of AAV.
  • Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis.
  • Virions may be recovered, for example, by CsCh sedimentation.
  • baculovirus expression systems in insect cells may be used to produce AAV vectors.
  • Aponte-Ubillus et al. 2018, Appl. Microbiol. Biotechnol. 102:1045-1054, which is incorporated by reference herein in its entirety for manufacturing techniques.
  • in vitro assays can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector.
  • a vector described herein e.g., the PER.C6® Cell Line (Lonza), a cell line derived from human embryonic retinal cells, or retinal pigment epithelial cells, e.g., the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®), can be used to assess transgene expression.
  • cell lines derived from liver or other cell types may be used, for example, but not limited, to HuH-7, HEK293, fibrosarcoma FIT-1080, HKB-11, and CAP cells.
  • characteristics of the expressed product i.e., transgene product
  • characteristics of the expressed product can be determined, including determination of the glycosylation and tyrosine sulfation patterns, using assays known in the art.
  • Another aspect relates to therapies which involve administering a transgene via a rAAV vector according to the invention to a subject in need thereof, for delaying, preventing, treating, and/or managing a disease or disorder, and/or ameliorating one or more symptoms associated therewith.
  • a subject in need thereof includes a subject suffering from the disease or disorder, or a subject pre-disposed thereto, e.g, a subject at risk of developing or having a recurrence of the disease or disorder.
  • a rAAV carrying a particular transgene will find use with respect to a given disease or disorder in a subject where the subject’s native gene, corresponding to the transgene, is defective in providing the correct gene product, or correct amounts of the gene product.
  • the transgene then can provide a copy of a gene that is defective in the subject.
  • the transgene comprises cDNA that restores protein function to a subject having a genetic mutation(s) in the corresponding native gene.
  • the cDNA comprises associated RNA for performing genomic engineering, such as genome editing via homologous recombination.
  • the transgene encodes a therapeutic RNA, such as a shRNA, artificial miRNA, or element that influences splicing.
  • the rAAVs of the present invention find use in delivery to target tissues, or target cell types, including cell matrix associated with the target cell ty pes, associated with the disorder or disease to be treated/pre vented.
  • a disease or disorder associated with a particular tissue or cell type is one that largely affects the particular tissue or cell type, in comparison to other tissue of cell types of the body, or one where the effects or symptoms of the disorder appear in the particular tissue or cell type.
  • Methods of delivering a transgene to a target tissue of a subject in need thereof involve administering to the subject tan rAAV where the peptide insertion is a homing peptide.
  • a rAAV vector comprising a peptide insertion that directs the rAAV to target the eye or ocular tissues of the subject, in particular, where the peptide insertion facilitates the rAAV in transducing ocular tissue with high efficiency, including satellite cells, yet results in lower transduction of liver cells.
  • rAAV vectors can be selected from the libraries herein that comprise a peptide insertion that directs ocular tissue transduction, relative to the parental rAAV vector without a peptide insertion.
  • ocular diseases/ Nonlimiting examples of ocular diseases include anterior ischemic optic neuropathy; acute macular neuroretinopathy; Bardet-Biedl syndrome; Behcet's disease; branch retinal vein occlusion; central retinal vein occlusion; choroideremia; choroidal neovascularization; chorioretinal degeneration; cone-rod dystrophy; color vision disorders (e.g., achromatopsia, protanopia.
  • anterior ischemic optic neuropathy anterior macular neuroretinopathy
  • Bardet-Biedl syndrome Behcet's disease
  • branch retinal vein occlusion central retinal vein occlusion
  • choroideremia choroidal neovascularization
  • chorioretinal degeneration chorioretinal degeneration
  • cone-rod dystrophy color vision disorders (e.g., achromatopsia, protanopia.
  • deuteranopia, and tritanopia congenital stationary night blindness; diabetic uveitis; epiretinal membrane disorders; inherited macular degeneration; histoplasmosis; macular degeneration (e.g., acute macular degeneration, non-exudative age related macular degeneration, exudative age related macular degeneration, dry age related macular degeneration (dry' AMD) or dry' age related macular degeneration with geographic atrophy (GA)); diabetic retinopathy; edema (e.g., macular edema, cystoid macular edema, diabetic macular edema); glaucoma; Leber congenital amaurosis; Leber's hereditary optic neuropathy; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal dysfunction; ocular trauma; ocular tumors; proliferative vitreoretinopathy (PVR); retinopathy of prematurity;
  • the rAAV vector has a capsid with ocular tropism, directing the rAAV to target the eye or ocular tissues of the subject.
  • the term “retinal celf’ refers to one or more of the cell types found in or near the retina, including amacrine cells, bipolar cells, horizontal cells, Muller glial cells, photoreceptor cells (e.g., rods and cones), retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retina ganglion cells, and photosensitive ganglion cells), retinal pigmented epithelium, endothelial cells of the inner limiting membrane, and the like.
  • Ocular tissues include anterior segment tissues, including the iris, cornea, lens, ciliary body, Schlemnf s canal, and trabecular meshwork, and posterior segment tissues, such as the retina or RPE-choroid, and optic nerve.
  • the rAAV are administered so as to target ocular tissues and may be administered by suprachoroidal administration (i.e., into the suprachoroidal space). In other embodiments, the rAAV is administered by intravitreal, intraocular, or intracameral administration.
  • the rAAV vectors of the invention also can facilitate delivery', in particular, targeted delivery, of oligonucleotides, drugs, imaging agents, inorganic nanoparticles, liposomes, antibodies to target cells or tissues.
  • the rAAV vectors also can facilitate delivery, in particular, targeted delivery, of non-coding DNA, RNA, or oligonucleotides to target tissues.
  • the agents may be provided as pharmaceutically acceptable compositions as known in the art and/or as described herein. Also, the rAAV molecule of the invention may be administered alone or in combination with other prophylactic and/or therapeutic agents.
  • the dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophy tactically effective.
  • the dosage and frequency will ty pically vary' according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease, the route of administration, as well as age. body weight, response, and the past medical history of the patient, and should be decided according to the judgment of the practitioner and each patient's circumstances. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician 's Desk Reference (56 th ed., 2002).
  • Prophylactic and/or therapeutic agents can be administered repeatedly. Several aspects of the procedure may vary such as the temporal regimen of administering the prophylactic or therapeutic agents, and whether such agents are administered separately or as an admixture.
  • the amount of an agent of the invention that will be effective can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • Prophylactic and/or therapeutic agents can be tested in suitable animal model systems prior to use in humans.
  • animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in the art may be used. Such model systems are widely used and well known to the skilled artisan. In some embodiments, animal model systems for an ocular condition are used that are based on rats, mice, or other small mammal other than a primate.
  • a clinical trial can be designed to test a rAAV molecule of the invention for efficacy and toxicity in human patients.
  • Toxicity and efficacy of the prophylactic and/or therapeutic agents of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • a rAAV molecule of the invention generally will be administered for a time and in an amount effective for obtain a desired therapeutic and/or prophylactic benefit.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range and/or schedule for dosage of the prophylactic and/or therapeutic agents for use in humans.
  • the dosage of such agents lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • a therapeutically effective dosage of an rAAV vector for patients is generally from about 0.1 ml to about 100 ml of solution containing concentrations of from about IxlO 9 to about IxlO 16 genomes rAAV vector, or about IxlO 10 to about IxlO 13 , about IxlO 12 to about IxlO 16 , or about IxlO 14 to about IxlO 16 rAAV genomes.
  • concentrations of from about IxlO 9 to about IxlO 16 genomes rAAV vector or about IxlO 10 to about IxlO 13 , about IxlO 12 to about IxlO 16 , or about IxlO 14 to about IxlO 16 rAAV genomes.
  • Levels of expression of the transgene can be monitored to determine/adjust dosage amounts, frequency, scheduling, and the like.
  • Treatment of a subject with a therapeutically or prophy lactically effective amount of the agents of the invention can include a single treatment or can include a series of treatments.
  • pharmaceutical compositions comprising an agent of the invention may be administered once a day, twice a day, or three times a day.
  • the agent may be administered once a day, every other day, once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, or once per year.
  • the effective dosage of certain agents e.g., the effective dosage of agents comprising a dual antigen-binding molecule of the invention, may increase or decrease over the course of treatment.
  • ongoing treatment is indicated, e.g. , on a long-term basis, such as in the ongoing treatment and/or management of chronic diseases or disorders.
  • an agent of the invention is administered over a period of time, e.g., for at least 6 months, at least one year, at least tw o years, at least five years, at least ten years, at least fifteen years, at least twenty years, or for the rest of the lifetime of a subject in need thereof.
  • the rAAV molecules of the invention may be administered alone or in combination with other prophylactic and/or therapeutic agents.
  • Each prophylactic or therapeutic agent may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect.
  • Each therapeutic agent can be administered separately, in any appropriate form and by any suitable route.
  • the different prophylactic and/or therapeutic agents are administered less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours apart, or no more than 48 hours apart.
  • two or more agents are administered within the same patient visit.
  • Methods of administering agents of the invention include, but are not limited to, parenteral administration (e.g, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection), epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral mucosa, rectal, and intestinal mucosa, etc.).
  • parenteral administration e.g, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection
  • epidural e.g., epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral mucosa, rectal, and intestinal mucosa, etc.).
  • a method of suprachoroidal administration for treating a pathology of the eye comprising administering to the suprachoroidal space in the eye of a human subj ect in need of treatment a recombinant viral vector comprising a nucleotide sequence encoding a therapeutic product such that the therapeutic product is expressed and results in treatment of the pathology of the eye.
  • the administering step is by injecting the recombinant viral vector into the suprachoroidal space using a suprachoroidal drug delivery' device.
  • the suprachoroidal drug delivery device comprises a microneedle.
  • the suprachoroidal drug delivery device is a microinjector.
  • SC suprachoroidal space
  • scleral flap technique catheters and standard hypodermic needles, as well as with microneedles.
  • a hollow-bore microneedle with a length matched to the thickness of the sclera, for example a 900 pm-long or 1100 pm-long microneedle (SCS Microinjector®, Clearside Biomedical. Inc.) inserted in the eye has been shown to deliver therapeutic agents to the posterior segment of the eye via the suprachoroidal space (Rai UDJ, et al. The suprachoroidal pathway: a new drug delivery' route to the back of the eye. Drug Discov Today.
  • Recombinant AAV vector compositions such as the rAAV compositions described herein, achieve delivery of the transgene to the posterior segment of the eye, including retinal cells. RPE and photoreceptor cells, via suprachoroidal administration (Ewing, TM. et al. 2022 Retinal Physician 19:24-26).
  • a method of administering an rAAV vector composition comprises inserting a needle, e.g. a microneedle, into the suprachoroidal space of the eye without the needle penetrating through the choroid (“suprachoroidal administration or injection’').
  • rAAV vector compositions can be injected into the eye using a microneedle, ty pically locating the needle approximately 4-4.5 mm from the limbus, into the pars plana.
  • the microneedle is inserted into the sclera at a perpendicular angle, then the needle hub is depressed to create a sealing gasket effect, then the injection of a pharmaceutical composition comprising the rAAV vector is performed over 5-10 seconds.
  • the administration step is performed under local anesthesia (e.g. anesthesia administered before the suprachoroidal injection).
  • topical steroid or other anti-inflammatory may be applied to the eye before or after insertion of the needle.
  • Oxular Limited is developing a SCS device and delivery system (Oxulumis) that advances an illuminated cannula in the suprachoroidal space.
  • the Orbit device (Gyroscope) is a specially-designed system enabling cannulation of the suprachoroidal space with a flexible cannula (for example, the flexible cannula is inserted between the sclera and the choroid).
  • a microneedle inside the cannula is advanced into the subretinal space to enable targeted dose delivery.
  • Ab intemo access to the SCS can also be achieved using micro-stents, which serve as minimally-invasive glaucoma surgery (MIGS) devices.
  • MIGS minimally-invasive glaucoma surgery
  • Examples include the CyPass® MicroStent (Alcon, Fort Worth, Texas, US) and iStent® (Glaukos), which are surgically implanted to provide a conduit from the anterior chamber to the SCS to drain the aqueous humor without forming a filtering bleb.
  • the suprachoroidal drug delivery device is a syringe with a 1 millimeter 30 gauge needle.
  • the syringe has a larger circumference (e.g., 29 gauge needle).
  • a microneedle or syringe is used to administer the rAAV compositions described herein.
  • a microneedle or syringe comprises a needle having an effective length of about 2000 microns or less.
  • a microneedle or syringe comprises a needle having an effective length between about 500 microns to about 2000 microns, or between about 800 microns to about 1200 microns.
  • a microneedle or syringe comprises a needle having an effective length of about 800 pm, about 850 pm, about 900 pm. about 950 pm, about 1000 pm. about 1100 pm, or about 1200 pm.
  • a microneedle or syringe is selected based on the viscosity of a pharmaceutical composition (e.g., liquid formulation).
  • a microneedle is selected based on the pressure resulted in the eye (e.g., in the SCS) when a pharmaceutical composition (e.g.. liquid formulation) is administered.
  • a pharmaceutical composition e.g., liquid formulation
  • the pressure in the SCS is lower when a wider microneedle is used as compared to the pressure obtained when a narrower microneedle is used.
  • 10 gauge needle, 11 gauge needle, 12 gauge needle are examples of 10 gauge needle, 11 gauge needle, 12 gauge needle.
  • gauge needle 13 gauge needle. 14 gauge needle. 15 gauge needle. 16 gauge needle, 17 gauge needle, 18 gauge needle, 19 gauge needle, 20 gauge needle, 21 gauge needle, 22 gauge needle, 23 gauge needle, 24 gauge needle, 25 gauge needle, 26 gauge needle, 27 gauge needle, 28 gauge needle, 29 gauge needle, 30 gauge needle, 31 gauge needle, 32 gauge needle, 33 gauge needle, or 34 gauge needle is used. In some embodiments, a 27 gauge needle is used. In some embodiments, a 28 gauge needle is used. In some embodiments, a 29 gauge needle is used. In some embodiments, a 30 gauge needle is used. In some embodiments, a 31 gauge needle is used. In some embodiments, a gauge that is smaller than a 27 gauge needle is used.
  • a gauge that is larger than a 27 gauge needle is used. In some embodiments, a gauge that is smaller than a 28 gauge needle is used. In some embodiments, a gauge that is smaller than a 30 gauge needle is used. In some embodiments, a gauge that is higher than a 30 gauge needle is used.
  • Devices used to carry out the methods described herein comprise any one of the devices disclosed in International Publication No. WO2011139713, International Publication No. W02014036009, International Publication No. WO2014074823, International Publication No. WO2014179698, International Publication No. WO2015126694, International Publication No. W02016044404, International Publication No. W02016040635, International Publication No. WO2017156227, International Publication No. WO2017218613, International Publication No. WO2019053466, International Publication No. W02019202603, International Publication No. WO2021188803, and US Patent No. 11.273,072 each of which are hereby incorporated by reference in their entireties.
  • a pharmaceutical composition or a reference pharmaceutical composition provided herein is suitable for administration by one, two or more routes of administration (e.g., suitable for suprachoroidal and subretinal administration).
  • the agents of the invention are administered intravenously and may be administered together with other biologically active agents.
  • agents of the invention may be delivered in a sustained release formulation, e.g., where the formulations provide extended release and thus extended half-life of the administered agent.
  • Controlled release systems suitable for use include, without limitation, diffusion-controlled, solvent-controlled, and chemically-controlled systems.
  • Diffusion controlled systems include, for example reservoir devices, in which the molecules of the invention are enclosed within a device such that release of the molecules is controlled by permeation through a diffusion barrier.
  • Common reservoir devices include, for example, membranes, capsules, microcapsules, liposomes, and hollow fibers.
  • Monolithic (matrix) device are a second type of diffusion controlled system, wherein the dual antigenbinding molecules are dispersed or dissolved in a rate-controlling matrix (e.g., a polymer matrix).
  • a rate-controlling matrix e.g., a polymer matrix
  • Agents of the invention can be homogeneously dispersed throughout a rate-controlling matrix and the rate of release is controlled by diffusion through the matrix.
  • Polymers suitable for use in the monolithic matrix device include naturally occurring polymers, synthetic polymers and synthetically modified natural polymers, as well as polymer derivatives.
  • a pump may be used in a controlled release system (see Langer, supra, Sefton, CRC Crit. Ref. Biomed. Eng., 14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J. Med., 321 :574, 1989).
  • polymeric materials can be used to achieve controlled release of agents comprising dual antigen-binding molecule, or antigen-binding fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance. Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem., 23:61, 1983; see also Levy et al., Science, 228: 190, 1985; During et al., Aww.
  • a controlled release system can be placed in proximity of the therapeutic target (e.g., an affected joint), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)).
  • Other controlled release systems are discussed in the review by Langer, Science, 249: 1527 1533, 1990.
  • rAAVs can be used for in vivo delivery of transgenes for scientific studies such as optogenetics, gene knock-down with miRNAs, recombinase delivery for conditional gene deletion, gene editing with CRISPRs, and the like.
  • the invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an agent of the invention, said agent comprising a rAAV molecule of the invention.
  • the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as saline solutions and aqueous dextrose and glycerol solutions that can also be employed as liquid carriers, particularly for injectable solutions.
  • compositions are provided for use in accordance with the methods of the invention, said pharmaceutical compositions comprising a therapeutically and/or prophylactically effective amount of an agent of the invention along with a pharmaceutically acceptable carrier.
  • the agent of the invention is substantially purified (/. ⁇ ?., substantially free from substances that limit its effect or produce undesired side-effects).
  • the host or subject is an animal, e.g., a mammal such as non-primate (e.g, cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey such as, a cynomolgus monkey and a human).
  • the host is a human.
  • kits that can be used in the above methods.
  • a kit comprises one or more agents of the invention, e.g., in one or more containers.
  • a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of a condition, in one or more containers.
  • the invention also provides agents of the invention packaged in a hermetically sealed container, such as a clear glass vial or polymer vial, such as a cyclo olefin polymer (COP) vial or Crystal Zenith® (Daikyo) vial.
  • a hermetically sealed container such as a clear glass vial or polymer vial, such as a cyclo olefin polymer (COP) vial or Crystal Zenith® (Daikyo) vial.
  • COP cyclo olefin polymer
  • Crystal Zenith® Crystal Zenith®
  • compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) as well as pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient).
  • Bulk drug compositions can be used in the preparation of unit dosage forms, e.g., comprising a prophylactically or therapeutically effective amount of an agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier.
  • the invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the agents of the invention. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of the target disease or disorder can also be included in the pharmaceutical pack or kit.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.
  • compositions of the invention are supplied in unit dosage form, for example, as a sterile liquid or dry lyophilized powder in a hermetically sealed vial indicating the quantity of agent or active agent.
  • the following examples report a method of making rAAV libraries containing numerous rAAV capsids having surface-exposed peptides inserted at designated insertion sites of the capsid.
  • the recombinantly engineered capsids are screened to identify candidates for particularly properties, such as tissue tropism.
  • the invention is illustrated by way of examples, describing the construction of rAAV capsids engineered to contain 7-mer or 9-mer peptides, wherein the method is designed to increase library diversity by reducing the parental capsid formation during manufacture, thereby reducing its abundance and therefore its representation in the library, thereby enhancing screening and identifying capsids with peptide inserts relative to the parental capsid.
  • a custom Rep-only trans-plasmid was created in which the Cap sequence was removed.
  • a custom cis-plasmid was created in which the CMV enhancer-promoter and Rep intron precedes the AAV Cap gene of interest, followed by the RBG poly A.
  • Nucleotide sequence encoding the AAV8 parental capsid (SEQ ID NO: 33) was used as a starting template for i) producing an AAV 8 vector library having random peptide insertions after amino acid residue G455, to generate a AAV8.VR4 vector library, and ii) producing an AAV8 vector library having random peptide insertions after amino acid residue Q589 plus the amino acid glycine (G) 5’ of the insert and the amino acid alanine (A) 3’ of the 7-mer insert, to generate a AAV8.VR8 vector library, using the methods herein.
  • Libraries were produced by generating a randomized 7-mer peptide insertion library synthesized in a chip-based format (Genscript Biotech) in which each amino acid except cysteine was represented by a single codon to minimize redundancy and stop codons.
  • the synthesized insert was cloned into plasmid i) pRGX.CMV. rep-intron. AAV8.456. stopcodon or ii) pRGX.CMV. rep-intron.AAV8.590.stopcodon (FIG. 3), thereby replacing the stop codon at position 456 or 590 of the cap gene for AAV8, respectively.
  • Endura competent cells (Lucigen) colony counts on dilution plates were used to determine library size.
  • the library E. coli culture was scaled up to prepare 0.5 mg of library cA-plasmid. Sequence diversity of the library insert and amino acid evenness was confirmed by NGS. 7ro -plasmid pRGX. rep-only was generated by removing the cap gene from a standard repcap /ra s -plasmid. Next-generation sequencing of the libraries was used to characterize library diversity and the wildtype fraction. In AAV8 having the insertion library cloned into position 456 in VR-IV the diversity’ was 4.32 x!07. See also Table 1. This site occurs at the top of the VR-IV flexible loop and has previously been shown to tolerate peptide insertions (PCT International Publication No.
  • FIGs. 1 and 2 depict analysis of variable region four or eight of the adeno-associated virus type 8 (AAV8 VR-IV or AAV8 VR-VIII) by amino acid sequence comparison to that of other AAVs VR-IV (FIG. 1), and an AAV9 protein model (FIG. 2) depicting the potential interactions between VR-IV, VR-V (three asparagines) and VR-VIII.
  • AAV8 has similar regions and interactions. Nam, H.-J. et al.
  • Each library’ was produced at 20L scale, as described above, by the Vector Core group at REGENXBIO Inc.
  • Library diversity and titer measurements determined a high level of diversity and the production lots had final BDS titer ranging from 3.5E13-8.2E13 (Table 1 and FIG. 4).
  • NGS testing obtained approximately 300 million reads per library.
  • NGS analysis of initial vector libraries had high parental vector levels, as indicated for example in an similarly constructed AAV5.VR8 library’ without a stop codon, by the larger representation of parental vector following production compared to the percent parental plasmid in the initial preparation of the library (FIG. 5A).
  • the drop-in vector stop codon abundance levels indicate selection against cross-packaging (FIG. 5B). Capsid crosspackaging and chimerism can be an issue in AAV library production.
  • Library cross-packaging refers to the packaging of vector genomes within capsids whereby the sequence of capsid and genome are mismatched. Chimerism occurs when capsids are assembled from VP proteins of multiple different variants. These phenomena occur during AAV library' production due to the entrance of multiple cap gene variants in each cell during triple transfection.
  • we optimized the amount of cA-plasmid during triple transfection and found that at a ratio of 100 cis-plasmids per cell, high production titers could be retained while minimizing cross-packaging. It has been previously described that ⁇ 1% of transfected DNA reaches the cell nucleus, therefore it is estimated that we achieve approximately one library variant per cell on average (Nguyen. Tam N.T. et al. 2021 Molecular Therapy: Methods & Clinical Development 21 : 642-655).
  • the parental fraction of AAV5 in an AAV5 library following AAV packaging was characterized by LC-MS for detection of the VP3 protein.
  • the parental VP3 theoretical wildtype mass is 59,551.64 Da and AAV5 VP3 with a peptide insertion is 59,951 to 60,855 Da.
  • FIG. 5A and FIG. 5B illustrate (by LC-MS analysis of VP3 proteins in each version of the library) that the addition of stop codon (FIG. 5B) significantly reduces the packaging of parental vector thereby reducing its overrepresentation in the library.
  • NGS analysis of non-mutated sequence in the plasmid and vector library showed a 5-fold increase in non-stop codon sequence between the plasmid and vector, and a 22 -fold reduction (1.4% to 0.066%) in the abundance of the unmutated stop codon sequence between the library plasmid and library vector genomes was achieved (FIG. 5D). If cross-packaging was highly abundant, one would expect a similar level of the stop codon sequence in both library plasmid and vector genomes.
  • mass range for the AAV8.456 library VP3 by LC-MS was -60509 Da to -60711 Da and there was no detected signal for wildtype AAV8 VP3 mass species (at around 59800 Da) (FIG. 5E).
  • Mass range for AAV 8.590 library VP3 was -60607 Da to -60868 Da with no detected signal for wildtype AAV8 VP3 mass species (at around 59800 Da) (FIG. 5F).
  • LC/MS analysis of vector libraries The LC/MS analysis of AAV capsid proteins was performed on a VanquishTM UHPLC (Thermo Fisher Scientific Inc.) coupled with a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.). The separation was performed on a Waters UPLC C4 column using reversed phase chromatography over a 35 minute reversed-phase gradient. Proteins eluting from the column were detected by high resolution mass spectrometer. Biopharma Finder 4.0 software is used for mass deconvolution.
  • Bioinformatic library sequence analysis A Linux-based bioinformatics pipeline was developed to analyze the sequence composition of AAV libraries before and after in vivo administration (from collected tissues, see below) from raw fastq files generated by the Illumina MiSeq or HiSeq.
  • the pipeline used the cutadapt tool to trim sequences flanking the peptide insertion and discard reads that are too long or missing the flanking sequences.
  • the starcode tool was then used to cluster sequences within a Levenstein distance of 2 to reduce sequencing errors. DNA sequences were then converted to amino acid sequences, identical sequences aggregated and sequences with stop codons discarded. For each peptide sequence the total counts, relative abundance in the sample, and enrichment score was reported (relative abundance in sample divided by relative abundance in the input library).
  • Cynomolgus monkeys (Macaca fascicularis) between 2 and 4 years of age were quarantined for four weeks at the study facility with daily observations. Serum was collected and saved for possible herpes B virus evaluation, two negative intradermal tuberculosis tests were conducted, and fecal flotation for parasites was performed. The animals were housed individually in stainless steel cages. The housing was in compliance with the Guide for the Care and Use of Laboratory Animals, DHHS, (NIH) No. 86-23, and the Animal Welfare Act (9 CFR 3). [00156] Blood was collected immediately prior to dosing, and serum collected and processed.
  • Tissues were flash frozen in liquid nitrogen and then maintained on dry ice prior to storage at -70 to -90 °C.
  • DNA and RNA was extracted from all tissues by standard techniques and next generation sequencing (NGS) and quantitative PCR (mRNA transcript expression) was performed, respectively.
  • NGS next generation sequencing
  • mRNA transcript expression quantitative PCR
  • a custom bioinformatics platform is described above for analyzing cDNA counts and RNA-seq data to determine relative abundance in the samples, and thus enrichment scores.
  • Analysis of the vector repertoire in multiple ocular tissues revealed that many peptides in the AAV8.VR4 library or AAV8.VR8 library 7 were detected in one or more of these ocular tissues such as retina and RPE-choroid, e.g. tissues that would provide a desirable depot of vector thus contributing to transgene product expression.
  • the pool of top hits from these libraries was further evaluated in a similar NHP study. For example, in round 2 studies, eyes were collected and tissue punches of 2-15 mg of tissue from retina, RPE-choroid, sclera, cornea, iris-ciliary 7 body, trabecular meshwork, and remaining tissues were frozen and later lysed and aliquoted for analysis.
  • AAV8.VR4 Libraries Following the analy sis of a vector repertoire containing a diverse set of peptides having tropism for one or more ocular tissue samples in the first round of animal testing (Round 1), a large pool from AAV8.VR4 was further analyzed by dosing NHPs at 1.52el3 GC/kg (pooled capsids). The bioinformatics tools were again used to determine vector performance ranking based on readouts such as relative abundance (RA), ES counts, etc. (e.g. using Dunnetfs multiple comparison test p-values) to determine an enrichment score.
  • RA relative abundance
  • ES counts etc.
  • AAV8.VR8 Libraries A large pool from AAV8.VR8 (Round 1 NHP study) was then further analyzed by dosing NHPs at 1.52el3 GC/kg (pooled capsids). The bioinformatics tools were then used analogously to determine vector performance ranking based on readouts such as relative abundance (RA to determine an enrichment score from the Round 2 study, as described hereinabove. A representative list of 10 hits (peptides) emerged, as in Table 3B for example, based on selection in retina compared to the parental spiked-in vector control (AAV8).
  • AAV8.VR8 Libraries A large pool from AAV8.VR8 (Round 1 NHP study) was then further analyzed by dosing NHPs at 1.52el3 GC/kg (pooled capsids). The bioinformatics tools were then used analogously to determine vector performance ranking based on readouts such as relative abundance (RA to determine an enrichment score from the Round 2 study, as described hereinabove. A
  • Table 3B AAV8.VR8 Peptide Inserts [00159] Following assessment using the bioinformatics tools described, representative AAV.590 capsids from the Round 2 library' reveal that the relative abundance of variant AAV8 capsids in retina is greatly enriched. >40-fold higher, compared to the parental AAV8 capsid (shown as fold-change compared to control capsid spiked-in to the pool) (FIG. 6B).
  • AAV3B libraries (not described herein) comprised the AB and CE batches (SCS5 and SCS6, respectively) and the AAV8 libraries containing capsids of Tables 3A and 3B comprised the DF batch (represented in SCS6 and SCS7).
  • test article (SCS5, 6 or 7) was dosed in three eyes by SCS administration at a dose of 3E12 GC per eye as described in Table 4.
  • tissue punches of 2-15 mg of tissue from retina, RPE-choroid, sclera, cornea, iris-ciliary body, trabecular meshwork, and remaining tissue samples were collected using aseptic technique and RNAse-free instruments and workspaces. Tissues were flash frozen in liquid nitrogen and then maintained on dry ice prior to storage at -70 to -90 °C until analyzed.
  • DNA Kerfisher DNA
  • mRNA Devereds mRNA direct
  • each vector in the pool measured by NGS in retina and RPE-choroid tissue is shown in FIG. 7A-B, represented as fold change RA relative to the control parental vector, and allowed assessment of the pool input composition.
  • each capsid was represented similarly whether in the 80% DF pool and the 20% DF pool.
  • FIGs. 8A-B show that several AAV8.456 and AAV8.590 peptide insertion capsids mediated about a 100-fold increase in mRNA expression in RPE-choroid (CHR) and retina (RER) in the 80% DF pool. Some of these capsids also mediated up to a 10- fold increase in mRNA expression of transgene in sclera (SCR)(FIG. 8C). These patterns were highly similar for the low dose pool (20% DF)(FIGs. 8D-F). Interestingly, the same evolved AAV8 peptide insertion variants mediated about 10-fold increased vector genome (vg) copies in retina and RPE compared to wild-type AAV8 by SCS administration (data not shown).
  • vg vector genome
  • FIGs. 9A-9B also illustrate the enrichment of SCS-delivered vectors having engineered capsids packaging a fluorescent protein, showing the majority of the tested capsids resulted in greater than 1 OX abundance over parental AAV 8 capsid.
  • the data from the present NHP study for AAV8.456 and AAV8.590 mRNA distribution in ocular tissues shows that RNA expression of transgene from these variant capsids is greatly enriched and transduction of retina and RPE-choroid tissue over scleral tissue is better than wildtype AAV8 upon SCS administration which may be desirable for certain transgenes.
  • AAV capsid sequences modified by peptide insertions with (AAV8.590 library' hits) or without additional substitutions (AAV8.456 library' hits) were further evaluated in in vivo for bio-distribution in test animals using next generation sequencing (NGS) and quantitative PCR.
  • NGS next generation sequencing
  • formalin fixed eyes may' be, e.g. sectioned at 40pm thickness on a vibrating blade microtome (VT1000S, Leica) and the floating sections probed with antibodies against transgene (or viewed for fluorescence if fluorescent transgene) to look at the cellular distribution of the delivered vectors.
  • VT1000S vibrating blade microtome
  • NHP Library Down-selection Summary As described hereinabove, several high diversity peptide insertion libraries (up to 10 8 variants per library) were designed and produced, each encoding AAV cap under the control of the CMV promoter with randomized 7-mer insertions at VR-IV or VR-VIII of AAV8.
  • the libraries were delivered intraocularly to nonhuman primates (NHP, cynomolgus macaques). Leveraging a directed evolution platform NAVIGATE (Novel AAV Vector Intelligent Guided Adaptation Through Evolution), 2-4k novel variants overall were identified and enriched in retina and RPE-choroid from each high diversity library, which were then re-packaged and administered to NHP via SCS delivery.
  • capsid variants (12 AAV8. VR-IV and 10 AAV 8. VR-VIII) were chosen for final library' down-selection.
  • Each variant (as well as parental control AAV8) was produced individually with genomes encoding a barcoded CAG.tdTomato cassette, pooled, and delivered to NHP, again by SCS delivery 7 , at a dose of 3el2 GC/eye.
  • FIGs. 10A- B Four capsids/vector with high abundance are shown in Table 5C (with further results of the individual capsids in Tables 5D-5G).
  • AAV8.VR-IV/AAV8.VR-VIII insertion variants were up to >30-fold and up to >50-fold improved, respectively, when compared to the parental serotypes.
  • Most AAV8-based insertion variants also expressed >20-fold more transgene mRNA in RPE-choroid compared to AAV8, with the top variant producing in excess of 200-fold more mRNA than AAV8.
  • Numerous variants with advantageous off-target transduction profiles w ere also identified. For example, the majority of AAV8.VR-IV and AAV8.VR-VIII variants that showed a greater than 10-fold increase in retinal transduction yet did not transduce liver, heart, or kidney to a greater degree than AAV 8.
  • a variant capsid library and a subset of single vectors was assessed via SCS delivery in Yucatan mini -pigs.
  • pooled capsids (vectors) were administered to one cohort (2 animals, 2 eyes collected each). Following 4 weeks, animals were sacrificed and eyes and several peripheral tissues were collected (Day 15 and Day 29).
  • scFv genome cDNA
  • transcripts mRNA
  • TP transgene product
  • Novel AAV8 variants from both libraries outperform AAV8 following single vector SCS delivery’ of scFvOl transgene in mini-pigs in a dose-dependent manner.
  • AAV8 variants achieve TP concentrations up to 344ng/mg in retina, consistent with biodistribution.
  • AAV8.3 achieves >153X AAV8 expression levels if transgene product in AAV8 in AH.
  • results show that transgene product (TP) in aqueous humor for the AAV8.456.RIQMGTK (8.1), AAV8.456.RQKNAMV (8.2), AAV8.590.GDNTTFRRA (8.3), and AAV8.590.GRTIRGDLA (8.4) vectors were increased relative to wildtype AAV8 vector.
  • AAV8.456.RIQMGTK (8.1) and AAV8.456.RQKNAMV (8.2) vectors exhibited about 50- fold and 150-fold change, respectively, in TP concentration relative to wtAAV8 vector at the high dose at Day 15.
  • Findings from the AAV8 insertion variants were largely translatable from NHP to mini-pigs, with top hits achieving improvement relative to AAV8 of >60-fold and >100-fold transduction in RPE-choroid and retina, respectively.
  • Variants evaluated as single vectors achieved up to 150-fold higher secreted transgene protein expression in the aqueous humor as compared to the AAV8 control.
  • Transduction of iCell RPE cells and an ARPE-AAVR cell line with either the 63-member library (SCS5, 6, 7 pools) or a subset of top hits as single vectors established that most variants exhibit superior transduction efficiency compared to AAV8 with top variants achieving >20-fold improvement in both the library and single vector settings.
  • a Uniform Manifold Approximation and Projection (UMAP) method was utilized to display and analyze the data for peptide insertion capsids in a ty pe of dimensionality reduction tool.
  • the peptides of Table 3A and 3B and all the input peptides in the libraries that were detected in at least one tissue sample were clustered. For example, combined peptides (> 20000 peptides in total for AAV8.456 and >10000 in total for AAV8.590 detected in at least one sample) from round 2 RPE and retina samples were analyzed in several clusters where no filter was applied to the input counts.
  • the enrichment scores of a peptide detected in both RPE and Retina libraries were averaged, and then the density plot of the resulting merged enrichment scores of all peptides detected in at least 1 sample and in the input library were plotted.
  • the peptide insertion sequence a) has an amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: xviii)Xi and X2 are each any amino acid; X?
  • Xi and X2 are each any amino acid; X3 is R or K or Q; X4 is V or K; Xs is K or Q or S or T; Xs is S or T or Q or A or I or V; and X71S V or Q or T or P or S (SEQ ID NO: 116); xx) Xi is any amino acid; X2 is R or K or F or P or N; X3 and X4 are each any amino acid; Xs is R or P or Q; Xs and X7 are each any amino acid (SEQ ID NO: 117); xxi) Xi is any amino acid; X2 is R or K or F or P or N; X3 is and
  • Xzand X3 are each any amino acid; X4 is S or Q or T or G; X5 and Xe are each any amino acid; X7 is K or R or Q or T or S (SEQ ID NO: 121); xxv) Xi is R; X2,is K; X3 is any amino acid; X4 is S or Q or T or G; X5 and Xe are each any amino acid; X7 is K or R or Q or T or S (SEQ ID NO: 122): xxvi) Xi is A or G or D; X2 is V or K or A; X3 is R or Q or V; X4 is K or R or S or H; X5 is K or A or S or P or R or P; Xe is any amino acid; X7 is K or T or Q or V (SEQ ID NO: 124); xxvii) Xi and X2 are each any amino acid; X3 is R or Q or T or D or V; X
  • X3, X4, and X5 are each any amino acid; Xe is R or K or Q; Xi is any amino acid (SEQ ID NO: 131); or b) the peptide insertion sequence has an amino acid sequence of X1-X2-X3-X4-X5-X6- X7-X8-X9, wherein: xxxiv) Xi is G; Xi. Xi X4, Xs, Xe are each any amino acid; Xs is R or K; Xs is any amino acid; X 9 is A (SEQ ID NO: 123).
  • Manufacturability assessment is crucial in helping to identify the suitability of a therapeutic molecule, to evaluate its fitness to a platform, and to reduce the process development effort due to unexpected results.
  • an in-house developed high throughput chromatography technology REGENXBIO Inc.
  • REGENXBIO Inc. high throughput chromatography technology
  • Process differences between these novel SCS capsids were observed. While the harvest titers, analyzed by digital droplet PCR (ddPCR). were all acceptable, there is a ⁇ 2.5 fold difference between the lowest and highest harvest titers for this panel of tested variants.
  • AAV8 variants particularly 8.1, 8.2, 8.3 and 8.4 resulted in harvest yields (titer) mostly comparable to ⁇ 1AAV8.
  • FIG. 15. The purification steps also demonstrated differences in the step yields and elution patterns for certain AAV8 variants.
  • the polishing steps showed that the AAV8 mutants all eluted earlier than, for example, AAV3B mutants, and when compared to AAV8 and AAV3B controls.
  • novel SCS capsids were selected to produce at 10L scale for a more complete manufacturability assessment, and the process profiles matched that of the smaller scale high throughput screening.
  • the final produced material all had similar product qualities based on capsid purity, genome integrity, and percentage of full capsids present in solution, but thermostability testing showed major differences, up to ⁇ 30°C, in melting temperatures. This information in combination with earlier biological activity data helped to determine the desirable capsids for further NHP studies.
  • iPSC-derived human RPE cells were transduced with variant AAV8.scFV01 vectors (carrying an antibody transgene formatted to express as a scFv protein).
  • iRPE cells were treated at an MOI of 3e5 vg/cell.
  • Transgene product (TP) level in apical and basal compartments was measured.
  • ARPE cells stable expressing AAV receptor (AAVR) were cultured in 96 well plates for 4 weeks prior to AAV transduction. Cells were also transduced with variant AAV8.scFv01 vectors at an MOI of 3e5 vg/cell, transgene product collected and quantitated.
  • AAV8 variants outperform AAV8 in vitro, achieving secreted transgene product levels approaching or exceeding those of AAV3B, which is traditionally highly potent in vitro.
  • AAV8 variants also performed slightly better than the AAV3B variants (AAV3B.3.1 or AAV3B3.2 variants) in iPSC cells (FIG. 14A).
  • Transduction efficiency of AAV8 variants in ARPE cells was comparable or better than AAV8 and AAV3B (FIG. 14B).
  • AAV capsid modifications performed by random peptide insertions in surface exposed loop of VR-IV were able to produce sufficient library titers in the production system described herein for analysis of transduction properties in NHP tissues.
  • the method provided for reducing carryover parental plasmid following cloning of the random insertions and was shown to reduce overrepresentation of the parental vector in the library’ biodistribution and transduction studies.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties. In particular, the invention provides engineered capsid proteins comprising peptide insertions from heterologous proteins inserted within or near variable region IV (VR-IV) or variable region VIII (VR-VIII) of the virus capsid, such that the insertion is surface exposed on the AAV particle. The invention also provides capsid proteins that direct rAAVs to target tissues, in particular, capsid proteins comprising peptides that are inserted into surface-exposed variable regions using a method for replacing a stop codon in the capsid gene with such peptide insert. The invention provides such methods for making the rAAV vectors into vector libraries and selecting the rAAV vectors having peptide inserts that target the rAAV to particular tissues of interest, and thus deliver therapeutics for treating disorders.

Description

RECOMBINANT ADENO-ASSOCIATED VIRUSES AND USES THEREOF
REFERENCE TO ELECTRONIC SEQUENCE LISTING
[0001] The contents of the electronic sequence listing submitted April 8, 2025 as an XML file named "‘38013 0042P1 SL,” created on April 8, 2025, and having a size of 163,840 bytes is hereby incorporated by reference in its entirety7.
1. FIELD OF THE INVENTION
[0002] The present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties when incorporated into a rAAV capsid. In particular, the invention provides engineered capsid proteins comprising peptide insertions inserted within or near variable region IV (VR-IV) of the virus capsid, such that the insertion is surface exposed on the AAV particle. The invention also provides capsid proteins that direct rAAVs to target tissues, in particular, capsid proteins derived from rAAV libraries, and provides such libraries constructed to reduce the parental vector production and thus overrepresentation of the parental capsid in the library and comprising random peptides inserted into surface-exposed variable regions to target rAAVs to and/or improve transduction of tissues of interest, including the ocular tissue, and deliver therapeutics for treating ocular disorders.
2. BACKGROUND
[0003] The use of recombinant adeno-associated viruses (AAV) as gene delivery vectors is a promising avenue for the treatment of many patients with unmet needs and/or rare disease. Dozens of naturally occurring AAV capsids have been reported, and mining the natural diversity of AAV sequences in primate tissues has identified over a hundred variants, distributed in clades. AAVs belong to the parvovirus family and are single-stranded DNA viruses with relatively small genomes and simple genetic components. Our current understanding of these capsids, their utility and function has allowed for efforts to further hone the efficiency and effectiveness of carrying therapeutic genome DNA, such as directing tissue tropism to deliver such DNA into target cells, while reducing the tropism to tissues where vector transduction and/or expression of transgene is undesirable, in order to safely ameliorate serious diseases. [0004] Due to low pathogenicity and the promise of long-term, targeted gene expression, recombinant AAVs (rAAVs) have been used as gene transfer vectors, in which therapeutic sequences are packaged into capsids. Such vectors have been used to deliver a variety of therapeutic genes, thus many gene therapy products are currently in clinical development. Recombinant AAVs, such as AAV9. have demonstrated desirable muscle and neurotropic properties and clinical trials using recombinant AAV8 for treatment of ocular disorders are underway. However, attempts to identify rAAV capsids with desirable properties in human subjects are limited by the methods that select for them.
[0005] When administered in the suprachoroidal space, for example, AAV vectors encounter barriers that prevent efficient transduction of ocular tissues. To reach retinal and RPE target tissues, rAAVs must pass around the choriocapillaris, cross the Bruch’s membrane and transit across the RPE. Several approaches can be taken to overcome these challenges, including the engineering of novel capsids with improved ocular transduction profiles that can provide improved transgene expression to the back of the eye.
[0006] There remains a need for rAAV vectors with enhanced tropism to particular tissues and properties for use, e.g., in high transduction to ocular tissues to delivery' therapies in treating disorders such as ocular disorders, including those amenable to suprachoroidal administration of rAAV. There also is a need for improved methods for identifying such rAAV vectors with enhanced tissue-specific targeting and/or enhanced tissue-specific transduction to deliver therapies using lower dosing than is currently available.
3. SUMMARY OF THE INVENTION
[0007] Provided are recombinant adeno-associated viruses (rAAVs) having capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties such as tissue targeting, transduction or expression of the rAAV genome. In particular, provided are engineered capsid proteins comprising peptide insertions, derived from peptide libraries, inserted within or near variable region IV (VR-IV) of the virus capsid, or, in certain embodiments, within or near variable region VIII (VR-VIII), such that the peptide insertion is surface exposed on the AAV particle when the engineered capsid protein is incorporated into an rAAV particle. In embodiments, the peptide is 7 or 9 amino acids of one of the peptides having an amino acid sequence of SEQ ID NOs: 1-32 (Tables 3A and 3B). In embodiments, the peptide is a 7 amino acid peptide within consensus sequences of SEQ ID NOs: 115-131 (see Table 6). In embodiments, the insertion is immediately after an amino acid residue corresponding to one of the amino acids 451 to 461 or 585 to 593 of the AAV8 capsid protein (SEQ ID NO: 33 and as numbered, e.g., in FIG. 1), including after the amino acid 455 (i.e., between amino acid 454 and 455) or after the amino acid 589 of the AAV8 capsid or in the AAV8.AAA capsid protein; or in a capsid protein of a different AAV type after a residue that corresponds to the amino acid G455 or Q589 of AAV 8, see alignment in FIG. 1 or for AAV types not included in FIG. 1, a similar amino acid sequence alignment of the AAV8 capsid protein sequence (SEQ ID NO: 33); and the AAV capsid protein as would be well known in the art). The capsid protein may be an AAV8 capsid protein but may also be any AAV capsid protein, such as AAV type 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype hu.31 (AAVhu.31), serotype hu.32 (AAVhu.32). serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype hu.37 (AVVhu.37). serotype rh39 (AAVrh39), and serotype rh74 (AAVrh74), or a variant AAV8 capsid protein AAV8.AAA (496NNN/AAA498 amino acid substitutions), (see, e.g., alignments presented in FIG. 1) or may be a capsid protein which has 90%, 95% or 99% amino acid sequence identity to one of the foregoing capsid proteins. Thus, provided is an engineered capsid protein comprising a peptide insertion from a heterologous protein (i.e., not an AAV capsid protein) inserted immediately after or near an amino acid corresponding to the amino acid residue at position 455 or at position 589 of AAV8, as numbered in FIG. 1.
[0008] Also provided are engineered capsid proteins that direct rAAVs to target tissues, in particular, capsid proteins comprising peptides (derived from peptide libraries) or a peptide that promotes tissue targeting and/or cellular uptake and/or expression of the rAAV genome, that are inserted into surface-exposed variable regions and that target rAAVs to ocular tissue, including retina, RPE-choroid, and/or sclera tissue, and deliver therapeutics for treating ocular disorders. These peptides, including 7 or 9 contiguous amino acids of one of the peptides in Tables 3A and 3B (SEQ ID NOs: 1-32), are advantageously inserted into the amino acid sequence of the capsid protein (VP1, VP2 and/or VP3) such that, when the capsid protein is incorporated into the AAV particle, the inserted peptide is surface exposed. Also provided are consensus sequences for the 7 mer to 9 mer peptides based upon the peptides identified as described in Example 5. Consensus sequences are provided in Table 6 (SEQ ID NOs: 1 ISIS 1). These peptides are inserted immediately after one of the amino acid residues of, or after one of the amino acids corresponding to the amino acid, 585-593 of VR-VIII of the AAV8 capsid (SEQ ID NO: 33) or an AAV8 capsid with substitutions e.g. AAV8.AAA capsid (SEQ ID NO: 114, e.g. see FIG. 1 for alignment), or immediately after one of the amino acid residues of, or after one of the amino acids corresponding to, 451-461 of VR-IV of the AAV8 capsid or AAV8.AAA capsid and amino acids corresponding to any one of positions 451-461 of the AAV8 capsid (SEQ ID NO: 33. see, e.g. FIG. 1 for alignment) or AAV8.AAA capsid. Exemplary modified capsid sequences are provided in Table 7. including capsid proteins having amino acid sequence of one of SEQ ID NOs: 50 to 113. In embodiments, provided are capsid proteins and AAV particles incorporating capsid proteins comprising an amino acid sequence of SEQ ID NO: 33 (AAV8 parental capsid). In other embodiments, provided are capsid proteins and AAV particles incorporating capsid proteins comprising an amino acid sequence of SEQ ID NO: 114 (AAV8.AAA parental capsid).
[0009] Provided are engineered capsid proteins comprising peptides that target specific tissues, including, when incorporated into an rAAV vector as a capsid to promote or increase cellular uptake and/or integration of an rAAV genome and/or expression of a transgene within the rAAV genome wherein the peptides are inserted into surface-exposed variable regions of the capsid protein. In certain embodiments, the peptides target and/or promote transduction or genome integration in cells of the eye, including the retina, RPE-choroid, the sclera or other ocular tissues, for example, at least 7 contiguous amino acids or at least 9 contiguous amino acids of any of the peptides in Tables 3A and 3B, and capsids containing one of these peptides, for example, immediately after one of the positions 451 -461, including after position 455 or 589, of AAV8 or AAV8.AAA, preferentially target the rAAV with the capsid to ocular tissue, including retina, RPE-choroid and/or sclera tissue, and, in embodiments detargeting peripheral tissues and/or the liver when administered to the eye. In embodiments, the capsid having the peptide insert targets and transduces ocular tissue. In other embodiments, the inserted peptide is at least 7 contiguous amino acids and may be 7 contiguous amino acids, 8 contiguous amino acids or 9 contiguous amino acids, and, in addition, the capsid is engineered to have one or more amino acid substitutions which may improve tropism, transduction or reduce immune neutralizing activity. Such amino acid modifications may include amino acid substitutions of the NNN (asparagines) at 496 to 498 with AAA (alanines) in the AAV8 capsid or corresponding substitutions in other AAV type capsids. Still other parental capsids having the peptide insertion may also have substitutions of S263F/S269T/A273T in AAV8. and corresponding substitutions in other AAV type capsids. W530R or Q474A in AAV8, and corresponding substitutions in other AAV type capsids, and/or A269S in AAV8, and corresponding substitutions in other AAV type capsids. [0010] Also provided are engineered capsid proteins that promote transduction of the rAAV in one or more ocular tissues, including one or more ocular cell types, upon ocular administration, including suprachoroidal administration (into the suprachoroidal space), wherein the capsid proteins comprise a peptide of Tables 3A and 3B (SEQ ID NOs: 1 to 32) or Table 6 (SEQ ID NOs: 115-131) that is inserted into a surface-exposed variable region (VR) of the capsid, e.g. VR-I, VR-IV or VR-VIII, or after the first amino acid of VP2, e.g, immediately after residue 138 of the AAV8 capsid (amino acid sequence of SEQ ID NO: 33) or immediately after the corresponding residue of another AAV capsid, or alternatively is engineered with one or more of the amino acid substitutions described herein, and transduction of the AAV having the engineered capsid in the ocular tissue is increased upon said administration compared (for example, 1 fold, 2 fold, 5 fold, 10 fold or 20 fold greater) to the transduction of the AAV having the corresponding unengineered capsid (parental capsid) or a reference capsid such as AAV8 or AAV8.AAA. Such capsids may also exhibit reduced transduction of one or more tissues, including peripheral tissue and/or liver upon administration compared (for example, 1 fold, 2 fold, 5 fold, 10 fold or 20 fold greater) to the transduction of the AAV in ocular tissue or having the corresponding unengineered capsid (parental capsid) or a reference capsid such as AAV8 or AAV8.AAA when administered to the eye. In certain embodiments, transduction is measured by detection of transgene, such as DNA, RNA transcript or expressed protein in the cell, e.g. reporter transgenes may be utilized and measured such as GFP fluorescence.
[0011] In certain embodiments, provided are rAAVs incorporating the engineered capsids described herein, including rAAVs with genomes comprising a transgene of therapeutic interest, including for an ocular disease or disorder. Plasmids and cells for production of a pool (stock) of plasmids for the production of the rAAVs are described herein. Packaging cells and methods for the production of the rAAVs comprising the engineered capsids are also provided herein. Method of treatment by delivery of, and pharmaceutical compositions comprising, the engineered rAAVs described herein are provided. Also provided are methods of manufacturing the rAAVs with the engineered capsids described herein.
[0012] Provided are methods of treating ocular indications, including Age-Related Macular Degeneration (AMD) and associated geographic atrophy (GA), by ocular, including suprachoroidal, administration to a patient suffering therefrom, a pharmaceutical composition comprising the rAAV capsid protein comprising a peptide insertion of at least 4 and up to 9 contiguous amino acids and a transgene. Also provided are pharmaceutical compositions formulated for administration to the suprachoroidal space with a microneedle or microinjector for treatment of dry AMD, including dry AMD with geographic atrophy.
[0013] Also provided are methods of making the capsid libraries having the peptide inserts. In embodiments, the method comprises providing a starting plasmid that contains a gene expression cassette encoding a capsid gene, wherein a stop codon is placed at a target insertion site within the capsid gene, randomizing a repertoire of nucleic acids encoding randomized peptides to produce a peptide library; creating individual plasmids based on the starting plasmid 1) each having a nucleic acid encoding a random peptide from the peptide library inserted at the target insertion site of the capsid gene thus replacing the stop codon, and 2) each encoding a barcode for identification of the capsid gene having the insert placed before the 5'- or after the 3'-end of the capsid gene; collecting the individual plasmids to form a population or collection of plasmids encoding the capsids with peptide inserts and transfecting with the collection of plasmids with plasmids encoding a recombinant rAAV genome containing a transgene, including one that is detectable, and necessary genes to produce a collection of rAAV vectors encapsidating the rAAV genome containing the transgene, wherein the rAAV vectors have capsids with the encoded capsid protein containing a library' peptide insert. In embodiments, the parental AAV is AAV8, AAV8.AAA, or any other suitable AAV serotype, for example, as in Table 7. The insertion site may be in VR-IV. including immediately after one of amino acids 451 -461 of AAV8 or corresponding to one of those residues or in VR-IV or VR-VIII, including immediately after amino acid 455 or 589 of AAV8 or corresponding to that position in a different AAV capsid type (see FIG. 1 for alignment).
[0014] The library of modified capsids is harvested from these cells. In embodiments, the rAAV library population produced has high levels of capsids having the peptide inserts, including 85%, 90%, 95% or 98%, 99% or even 100%.
[0015] The invention is illustrated by way of examples infra describing the construction of engineered rAAV 8 capsids having peptide inserts designed from rAAV libraries enabling the detection of desirable properties such as tissue targeting.
3.1. Embodiments
1. A recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insertion of at least 4 and up to 9 contiguous amino acids, wherein the peptide insertion is immediately after an amino acid residue corresponding to one of amino acids 451 to 461 or amino acids 585-593 of a parental capsid protein, wherein the parental capsid protein is an AAV8 capsid protein having an amino acid sequence of SEQ ID NO: 33 or a capsid protein that has 90%, 95%, or 99% sequence identity thereto, wherein said peptide insertion has an amino acid sequence of one of SEQ ID NOs: 1-32, and wherein an rAAV vector comprising the capsid protein comprising the peptide insertion has enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein. recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insertion of at least 4 and up to 9 contiguous amino acids, wherein the peptide insertion is immediately after an amino acid residue corresponding to one of amino acids 451 to 461 or amino acids 585-593 of a parental capsid protein, wherein the parental capsid protein is an AAV8 capsid protein having an amino acid sequence of SEQ ID NO: 33 or a capsid protein that has 90%, 95%, or 99% sequence identity thereto, wherein said peptide insertion has an amino acid sequence of one of SEQ ID NOs: 115-131, and wherein an rAAV vector comprising the capsid protein comprising the peptide insertion has enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein. he rAAV capsid protein of embodiment 1 or 2, wherein said parental capsid protein is serotype 8 having 496NNN/AAA498 substitutions (AAV8.AAA) and has an amino acid sequence of SEQ ID NO: 114. he rAAV capsid protein of embodiment 3, wherein said peptide insertion occurs immediately after one of amino acids Q451, T452, T453, G454, G455, T456, A457, N458, T459, Q460, or T461 of the parental capsid. he rAAV capsid protein of embodiment 4, wherein said peptide insertion occurs immediately after amino acid G455 of the parental capsid. he rAAV capsid protein of any one of embodiments 1-5 wherein the peptide insertion is 7 to 9 amino acids of one of the amino acid sequences of SEQ ID NOs: 1-12 or 115- 122. he rAAV capsid protein of embodiment 6 which has an amino acid sequence of one of
SEQ ID NOs: 50 to 61. he rAAV capsid protein of embodiment 1 or 2, wherein the peptide insertion occurs immediately after one of amino acids 585 to 593 of the parental capsid. he rAAV capsid protein of embodiment 8. wherein the peptide insertion occurs immediately after amino acid Q589 of the parental capsid. he rAAV capsid protein of embodiments 8 or 9 wherein the peptide insertion is 7 to 9 amino acids of one of the amino acid sequences of SEQ ID NO: 13-32 or 123-131.he rAAV capsid protein of embodiment 10 which has an amino acid sequence of one of SEQ ID NOs: 62 to 113. he rAAV capsid protein of any one of the preceding embodiments which has enhanced tropism for retina or RPE choroid tissue relative to the parental capsid protein.he rAAV capsid protein of embodiment 12, wherein an rAAV vector comprising the capsid protein exhibits at least about 2 fold, 5 fold, 10 fold, 15 fold. 20 fold, 25 fold or 100 fold greater transduction of retina and/or RPE choroid tissue than an rAAV vector comprising the parental capsid protein. he rAAV capsid protein of any one of the preceding embodiments wherein, upon administration to an eye, a rAAV vector comprising the capsid protein has reduced transduction of peripheral tissues than a rAAV vector comprising the parental capsid protein. he rAAV capsid protein of embodiment 14 wherein, upon administration to an eye. the rAAV vector comprising the capsid protein has an at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 25 fold or 40 fold lower transduction of liver than an rAAV vector comprising the parental capsid protein. nucleic acid comprising a nucleotide sequence encoding the rAAV capsid protein of any one of the preceding embodiments, or encoding an amino acid sequence having at least 80% identity therewith, wherein an rAAV vector comprising the rAAV capsid protein of any of the preceding claims retains enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein. he nucleic acid of embodiment 16 which encodes the rAAV capsid protein of any one of embodiments 1-15. packaging cell capable of expressing the nucleic acid of embodiment 16 or embodiment 17 to produce AAV vectors comprising the capsid protein encoded by said nucleotide sequence. rAAV vector comprising the rAAV capsid protein of any one of embodiments 1-15.he rAAV vector of embodiment 18, further comprising a rAAV genome comprising a transgene flanked by AAV ITR sequences, wherein the transgene encodes a therapeutic for an ocular disease. pharmaceutical composition comprising the rAAV vector of embodiment 19 or 20 and a pharmaceutically acceptable carrier. he pharmaceutical composition of embodiment 21 formulated for administration to the suprachoroidal space. he pharmaceutical composition of embodiment 21, wherein said composition is formulated for administration to the suprachoroidal space with a microneedle or microinjector device. method of delivering a transgene to a cell, said method comprising contacting said cell with the rAAV vector of embodiment 19 or 20. method of delivering a transgene to ocular tissue of a subject in need thereof, said method comprising administering to said subject the rAAV vector of embodiment 19 or 20. he method according to embodiment 25, wherein said rAAV vector is administered to the suprachoroidal space in the eye. he method according to any of claims 24 to 26. wherein said target tissue is retinal or
RPE choroidal tissue. pharmaceutical composition for use in delivering a transgene to a cell in a subject in need thereof, wherein the pharmaceutical composition comprises the rAAV vector of embodiment 19 or 20. he pharmaceutical composition for use in delivering a transgene to ocular tissue of a subject in need thereof, wherein the pharmaceutical composition comprises the rAAV vector of embodiment 19 or 20. he pharmaceutical composition for use according to embodiment 28 or 29, wherein said rAAV vector is administered to the suprachoroidal space in the eye. he pharmaceutical composition for use according to embodiment 30, wherein said rAAV vector is administered by a microneedle device or microinjector devicehe pharmaceutical composition for use according to any one of embodiments 28-31, wherein said target tissue is retinal or RPE choroidal tissue. pharmaceutical composition for use in treating Age-Related Macular Degeneration
(AMD) in a subject or a method of treating AMD in a human subject in need thereof comprising administering a therapeutically effective amount of recombinant adeno- associated virus (rAAV) vector comprising the recombinant adeno-associated virus (rAAV) capsid protein of any one of embodiments 1 to 15. he pharmaceutical composition or method or treatment according to embodiment 33, wherein the AMD is dry AMD with geographic atrophy. he pharmaceutical composition or method for treating according to embodiment 33 or
34, wherein the rAAV is administered suprachoroidally. he pharmaceutical composition or method for treatment according to any one of embodiments 33 to -35 wherein the rAAV is delivered by suprachoroidal administration optionally by a microneedle or microinjector device. 4. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts alignment of AAVs 1-8, hu31. hu32, AAV9, and rhlO capsid sequences (VP1) highlighting the VR-IV insertion site for these capsids (see VR4 corresponding to amino acids 451 to 461 of the AAV9 capsid protein and VR8 corresponding to amino acids 585 to 593 of the AAV9 capsid protein). A Clustal Multiple Sequence Alignment of AAV capsids was performed and illustrates that amino acid substitutions (shown in bold in the bottom rows) can be made to AAV9, AAVhu32 or other capsids by “recruiting” amino acid residues from the corresponding position of other aligned AAV capsids. Sequences shown in gray are hypervariable regions (HVR). The amino acid sequences of the AAV capsids are assigned SEQ ID NOs as follows: AAV1 is SEQ ID NO: 34; AAV2 is SEQ ID NO: 35; AAV3 is SEQ ID NO: 36; AAV4 is SEQ ID NO: 37; AAV5 is SEQ ID NO: 38; AAV6 is SEQ ID NO: 39; AAV7 is SEQ ID NO: 40; AAV8 is SEQ ID NO: 33; AAV9 is SEQ ID NO: 43; AAVrhlO is SEQ ID NO: 44; hu31 is SEQ ID NO: 41; and hu32 is SEQ ID NO: 42.
[0016] FIG. 2 illustrates a protein model of variable region four and eight of the adeno- associated virus type 9 (AAV9 VR-IV and AAV9 VR-VIII, respectively) indicating the NNN sequence at amino acids 498-500 of the AAV9 capsid sequence and the point of peptide insertion in VR-IV.
[0017] FIG. 3 depicts a representative genome construct of the capsid gene for use in construction of rAAV libraries having from 5’ to 3’: 5'-inverted terminal repeat (ITR), CMV enhancer-promoter, Rep intron, the AAV Cap gene of interest. polyA sequence, and 3 ’-ITR. The illustration depicts insertion of a random peptide library in the place of a stop codon (see arrow) that was inserted into the Cap gene variable region before construction of the library (to reduce expression of wildtype sequence in the library).
[0018] FIG. 4 demonstrates the PCR amplicon obtained from representative libraries Al to G1 used for NGS analysis of library diversity.
[0019] FIGs. 5A-5F show-s (A) a mass spectrometry VP3 profile for a representative AAV5 library produced without a stop in the capsid sequence template and (B) with a stop codon in the template. (C) A representative AAV9 round 1 library adequately produced with the stop codon template. (D) NGS analysis of non-mutated sequence in the plasmid and vector library shows a 5-fold increase in non-stop codon sequence between the plasmid and vector and a 22- fold decrease in stop codon sequence, indicating selection against cross-packaging. (E) Liquid chromatograph-mass spectrometry (LC-MS) of VP3 proteins of the libraries shows that mass range for the AAV8.456 library VP3 was -60509 Da to -60711 Da and there was no detected signal for wildtype AAV 8 VP3 mass species (at around 59800 Da). (F) LC-MS shows that mass range for AAV8.590 library VP3 was -60607 Da to -60868 Da with no detected signal for wildtype AAV8 VP3 mass species (at around 59800 Da).
[0020] FIGs. 6A-6B. Presents results of biodistribution after dosing NHPs in retina. (A) Relative abundance (RA) and coefficient of variance (CV) of AAV8.VR4 capsids library capsids as indicated in retina relative to parental (AAV8) spiked-in vector control and (B) Relative abundance (RA) and coefficient of variance (CV) of AAV8.VR8 capsids library capsids as indicated in retina relative to parental (AAV8) spiked-in vector control.
[0021] FIGs. 7A-7B. Presents abundance of each vector in the pool measured by NGS in retina and RPE-choroid tissue for each insertion capsid compared to parental capsid, represented as fold change RA relative to the control parental vector for both the 80% (A) and 20% (B) input pools.
[0022] FIGs. 8A-8F are bar graphs depicting the fold increase in mRNA transgene expression relative to the transgene expression from parental AAV8 when pooled capsids are administered into the suprachoroidal space in the 80% DF pool in (A) RPE-Choroid (CHR), (B) retina (RER), and (C) sclera (SCR) and in the 20% DF pool in (D) RPE-Choroid (CHR), (E) retina (RER), and (F) sclera (SCR).
[0023] FIGs. 9A-9B are plots illustrating engineered capsids from the AAV8.VR-IV library all having a higher abundance than parental AAV8 in both retina and RPE-C, with all performing more than lOx better than AAV8 in retina and nearly all in RPE-C. AAV8 capsids 8.1 and 8.2 were selected for further evaluation.
[0024] FIGs. 10A-10B illustrate the fold-change difference of round 3 engineered capsids with increased transduction of (A) retina and (B) RPE-choroid compared to parental AAV8 following an SCS dose of 3x1012 GC/eye to NHPs. One-way ANOVA Dunnett’s test vs AAV8: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0025] FIGs 11A-11D represent (A) aqueous humor (AH) transgene product (TP) levels measured by ELISA following single vector SCS administration in mini-pigs at a dose of 3xl0n and 3xl012 vg/eye (AAV vectors carry a CAG.scFvOl transgene). (B) TP expression level following transduction of iRPE cells at an MOI of 3xl05 vg/cell. TP levels following single vector SCS administration in NHPs at a dose of 3xl012 vg/eye in (C) aqueous humor and in D) retina tissue punches. [0026] FIGs 12A-12B are bar graphs showing transgene product expression (A) and biodistribution (B) of PEPIN3.1, PEPIN8.1, PEPIN8.2 and PEPIN8.4 in the retina at 3el2 GC/eye (solid) and 3el 1 GC/eye (brick).
[0027] FIGs 13A-13C are bar graphs showing that AAV 8 variants PEPIN8. 1 , PEPIN8.2 and PEPIN8.3 produce greater than 10 times more protein in the retina (A), RPE-C (B) and vitreous humor (C) than AAV8 following SCS delivery of scFvOl transgene in NHPs (n=6 eyes at 3el2 GC/eye).
[0028] FIGs 14A-14B are bar graphs showing in vitro transduction efficiency of rAAV comprising various capsids (AAV8. AAV3B. 3.1, 3.2, 8.1. 8.2, 8.3, and 8.4) in iRPE cells at day 21 (A) and ARPE cells at 48 hours (B).
[0029] FIG. 15 illustrates productivity in a NAVXpress® manufacturing platform, showing harvest yields (titer GC/mL) for AAV8 variants 8.1, 8.2, 8.3, and 8.4 were mostly comparable to AAV8.
5. DETAILED DESCRIPTION
[0030] Provided are recombinant adeno-associated viruses (rAAVs) having capsid proteins engineered to include amino acid sequences that confer and/or enhance desired properties, such as tissue targeting, transduction and integration of the rAAV genome. In particular, provided are engineered capsid proteins comprising peptide insertions of 7 or 9 contiguous amino acids, from random peptide libraries, inserted within or near variable region IV (VR-IV) or VR-V1I1 of the virus capsid, such that the peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle. Also provided are recombinant capsid proteins, and libraries of the individual rAAVs comprising them, that have inserted peptides that target specific tissues and/or promote rAAV cellular uptake, transduction and/or transgene expression, for example, see Table 7. Provided are capsids having 7 to 9 amino acid peptides which enhance targeting, transduction and/or integration of the rAAV genome in ocular tissue with peptides inserted in VR-IV of AAV 8 capsid protein with peptides having or comprising the amino acid sequences of SEQ ID NOs: 1-32 or 115-131. Provided are novel AAV capsids which harbor peptide insertions that have undergone selection in non-human primates to identify variants with improved biodistribution to retina and RPE-choroid following suprachoroidal administration. 5.1. Definitions
[0031] The term “AAV” or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses. The AAV can be an AAV derived from a naturally occurring “wild-type” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and/or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene. An example of the latter includes a rAAV having a capsid protein comprising a peptide insertion into the amino acid sequence of the naturally-occurring capsid.
[0032] The term “rAAV” refers to a “recombinant AAV.” In some embodiments, a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.
[0033] The term “rep-cap helper plasmid” refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from rAAV genomes lacking functional rep and/or the cap gene sequences.
[0034] The term “cap gene” refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus. For AAV, the capsid protein may be VP1, VP2, or VP3.
[0035] The term “rep gene” refers to the nucleic acid sequences that encode the non- structural protein needed for replication and production of virus.
[0036] The terms “nucleic acids” and “nucleotide sequences” include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tri tylated bases. Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes. The nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.
[0037] As used herein, the terms “subject”, “host”, and “patient” are used interchangeably. As used herein, a subject is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), or, in certain embodiments, a human. [0038] '‘Library” or '‘libraries” generally refer to a repertoire of capsid genes (each unique and usually placed recombinantly into a vehicle, such as in a plasmid) or rAAV vectors produced from the unique capsids.
[0039] As used herein, the term “conservative amino acid substitution” means substitutions made in accordance with Tables A and B.
Table A: Amino Acid Substitutions
Table B: Amino Acid Abbreviations
[0040] It is understood that one way to define the variants and derivatives of the disclosed capsid proteins herein is to define them in terms of homology /identity to specific known sequences. Specifically disclosed are variants of capsids herein disclosed which have at least, 70% or at least 75% or at least 80% or at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% identity to the capsid sequences specifically recited herein. Those of skill in the art readily understand how to determine the identity of two proteins. Variants of capsids described herein may have 1. 2, 3. 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acid substitutions, including conservative amino acid substitutions. Variants of the 7 or 9 amino acid peptides described herein include peptides having 1, 2, or 3 amino acid substitutions, including conservative amino acid substitutions. [0041] The term “therapeutic agent” refers to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. As used herein, a “therapeutically effective amount” refers to the amount of agent, (e.g.. an amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder, when administered to a subject suffering therefrom. Further, a therapeutically effective amount w ith respect to an agent of the invention means that amount of agent alone, or when in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of the disease or disorder.
[0042] As used herein, the term “prophylactic agent” refers to any agent which can be used in the prevention, delay, or slowing down of the progression of a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene. As used herein, a “prophylactically effective amount” refers to the amount of the prophylactic agent (e.g., an amount of product expressed by the transgene) that provides at least one prophylactic benefit in the prevention or delay of the target disease or disorder, when administered to a subject predisposed thereto. A prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the occurrence of the target disease or disorder; or slow the progression of the target disease or disorder; the amount sufficient to delay or minimize the onset of the target disease or disorder; or the amount sufficient to prevent or delay the recurrence or spread thereof. A prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the exacerbation of symptoms of a target disease or disorder. Further, a prophylactically effective amount with respect to a prophylactic agent of the invention means that amount of prophylactic agent alone, or when in combination with other agents, that provides at least one prophylactic benefit in the prevention or delay of the disease or disorder.
[0043] A prophylactic agent of the invention can be administered to a subj ect “pre-disposed” to a target disease or disorder. A subject that is “pre-disposed” to a disease or disorder is one that show's symptoms associated with the development of the disease or disorder, or that has a genetic makeup, environmental exposure, or other risk factor for such a disease or disorder, but where the symptoms are not yet at the level to be diagnosed as the disease or disorder. For example, a patient with a family history of a disease associated with a missing gene (to be provided by a transgene) may qualify as one predisposed thereto. 5.2. Recombinant AAV Capsids and Vectors
[0044] One aspect relates to capsid protein libraries and the recombinant adeno-associated virus (rAAV) vectors thereof, the capsid proteins within the library engineered to comprise a peptide insertion from a random peptide library, wherein the peptide is not an AAV protein or peptide fragment thereof, where the peptide insertion is surface exposed when packaged as an AAV particle. In some embodiments, the peptide insertion occurs within (i.e., between two amino acids without deleting any capsid amino acids) variable region IV (VR-1V) of an AAV8 capsid or AAV8.AAA capsid, or a corresponding region for another type AAV capsid (see alignment in FIG. 1). In some embodiments, the peptide insertion occurs within (/.<?., between two amino acids without deleting any capsid amino acids) variable region VIII (VR-VIII) of an AAV8 capsid or AAV8.AAA, or a corresponding region of a capsid for another AAV type (see alignment in FIG. 1). In some embodiments, the peptide insertion is from a heterologous protein or domain (that is not an AAV capsid protein or domain), which directs the rAAV particles to target tissues and/or promote rAAV uptake, transduction and/or transgene expression. Also provided are nucleic acids encoding the engineered capsid proteins and variants thereof, packaging cells for expressing the nucleic acids to produce rAAV vectors, rAAV vectors further comprising a transgene, and pharmaceutical compositions of the rAAV vectors, as well as methods of using the rAAV vectors to deliver the transgene to a target cell type or target tissue of a subject in need thereof.
[0045] In the various embodiments, the target tissue may be ey e/retina tissue (including RPE- choroid or sclera tissue), and the capsid with the peptide insertion specifically recognizes and/or binds to and/or homes to that tissue, or for example, one or more specific cell types, such as within the target tissue, or cellular matrix thereof. In particular, peptides that can target rAAVs to ocular tissue can be useful for delivering therapeutics for treating ocular disorders and may be delivered by ocular, including, suprachoroidal administration. In embodiments, rAAV capsids containing the engineered capsid protein with the peptide insert have increased uptake, transduction, and/or transgene expression than the parental capsid or a reference capsid, including AAV8 or AAV8.AAA. The engineered capsids may further have reduced transduction in tissues such as peripheral tissues and/or liver relative to the targeted tissue or relative to transduction with the parental capsid or a reference capsid, including AAV8 or AAV8.AAA when administered to the eye.
[0046] In various embodiments, the target tissue may be ocular tissue, and particularly retina, RPE-choroid and/or sclera tissue. [0047] In embodiments either the parental capsid and/or the peptide insert detarget the rAAV vector comprising the parental capsid and/or the engineered capsid protein (having the peptide insert) from one or more tissue types, including liver and/or peripheral tissues.
5.2.1 rAAV Vectors with Peptide Insertions
[0048] A peptide insertion described as inserted "at" a given site refers to insertion immediately after that is having a peptide bond to the carboxy group of, the residue normally found at that site in the wild type virus. For example, insertion at G455 in AAV8 means that the peptide insertion appears between G455 and the consecutive amino acid (G456) in the AAV8 wildtype capsid protein sequence (SEQ ID NO: 33). Additionally, insertion at Q589 in AAV8 means that the peptide insertion appears between Q589 and the consecutive amino acid (N560) in the AAV8 wildtype capsid protein sequence (SEQ ID NO: 33). In embodiments, there is no deletion of amino acid residues at or near (within 5, 10, 15 residues or within the structural loop that is the site of the insertion) the point of insertion.
[0049] In embodiments, the capsid protein is an AAV8 capsid protein (SEQ ID NO: 33, or a capsid protein having an amino acid sequence that is 90%, 95%, or 99% identical to SEQ ID NO: 33) or an AAV8.AAA capsid protein and the insertion occurs immediately after at least one of the amino acid residues 451 to 461. In embodiments, the peptide insertion occurs immediately after amino acid T451, T452, G453, G454, T455, A456, N457, T458. Q459, T460, or L461 of the AAV8 capsid (ammo acid sequence SEQ ID NO: 33). In certain embodiments, the peptide is inserted between residues G455 and G456 of AAV8 capsid protein or between the residues corresponding to G455 and G456 of an AAV capsid protein other than an AAV8 capsid protein (amino acid sequence SEQ ID NO: 33). In other embodiments, the peptide is inserted immediately after one of the amino acid residues of 585 to 593 in VR-VIII of the AAV8 capsid protein (SEQ ID NO: 33), including between residues Q589 and N560 of AAV8 capsid protein or immediately after one of the amino acid residues corresponding to 585 to 593 of the AAV8 capsid protein or between the residues corresponding to Q589 and N560 of an AAV capsid protein other than an AAV8 capsid protein (amino acid sequence SEQ ID NO: 33) (see Fig. 1 for alignment). In embodiments, the capsid protein is an AAV8.AAA capsid protein and the insertion occurs immediately after at least one of the amino acid residues 451 to 461. In embodiments, the peptide insertion occurs immediately after amino acid T451, T452, G453, G454, T455, A456, N457. T458, Q459, T460. or L461 of the AAV8.AAA capsid (amino acid sequence SEQ ID NO: 114). In certain embodiments, the peptide is inserted between residues G455 and G456 of AAV8.AAA capsid protein or between the residues corresponding to G455 and G456 of an AAV8.AAA capsid protein other than an AAV8 capsid protein (amino acid sequence SEQ ID NO: 114). In other embodiments, the peptide is inserted immediately after one of the amino acid residues of 585 to 593 in VR-VIII of the AAV8.AAA capsid protein (SEQ ID NO: 114), including between residues Q589 and N560 of AAV8.AAA capsid protein or immediately after one of the amino acid residues corresponding to 585 to 593 of the AAV8.AAA capsid protein or between the residues corresponding to Q589 and N560 of an AAV capsid protein other than an AAV8.AAA capsid protein (amino acid sequence SEQ ID NO: 33) (see Fig. 1 for alignment). In embodiments, the capsid protein has an amino acid sequence of one of SEQ ID NOs: 50-113).
[0050] In other embodiments, the capsid protein is from at least one AAV type selected from AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6). serotype 7 (AAV7), serotype rh8 (AAVrh8). serotype 9 (AAV9), serotype 9e (AAV9e), serotype rhlO (AAVrhlO), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), seroty pe hu.37 (AAVhu.37), and seroty pe rh74 (AAVrh74, versions 1 and 2) (see FIG. 1 for alignment with AAV l-AAV9, hu31. Hu32, and rhlO capsid sequences) or a capsid that is at least 90%, 95% or 99% identical in amino acid sequence to the amino acid sequence of the VP1, VP2, or VP3 of the foregoing capsid proteins, and the insertion occurs immediately after an amino acid residue corresponding to at least one of the amino acid residues 451 to 461 of AAV8. The alignments of these different AAV serotypes, as show n in FIG. 1, indicates “corresponding” amino acid residues in the different capsid amino acid sequences such that a “corresponding” amino acid residue is lined up at the same position in the alignment as the residue in the reference sequence. In some embodiments, the peptide insertion occurs immediately after one of the amino acid residues within: 450-459 of AAV1 capsid (SEQ ID NO: 34); 449-458 of AAV2 capsid (SEQ ID NO: 35); 449-459 of AAV3 capsid (SEQ ID NO: 36); 443-453 of AAV4 capsid (SEQ ID NO: 37); 442-445 of AAV5 capsid (SEQ ID NO: 38); 450-459 of AAV6 capsid (SEQ ID NO: 39); 451-461 of AAV7 capsid (SEQ ID NO: 40); 451-461 of AAV8 capsid (SEQ ID NO: 33); 451-461 of AAV9 capsid (SEQ ID NO: 43); 451-461 of AAVhu.32 capsid (SEQ ID NO: 42); 452-461 of AAVrhlO capsid (SEQ ID NO: 44); 452-461 of AAVrh20 capsid (SEQ ID NO: 47); 452-461 of AAVhu.37 (SEQ ID NO: 45); 452-461 of AAVrh74 (SEQ ID NO: 48 or SEQ ID NO: 49); or 452-461 of AAVrh31 (SEQ ID NO: 41), in the sequences depicted in FIG. 1. In certain embodiments, the rAAV capsid protein comprises a peptide insertion immediately after (i.e., C-terminal to) amino acid 589 of AAV8 capsid protein (having the amino acid sequence of SEQ ID NO: 33 and see FIG. 1) or AAV8.AAA (having SEQ ID NO: 114), where said peptide insertion is surface exposed when the capsid protein is packaged as an AAV particle. In other embodiments, the rAAV capsid protein has a peptide insertion that is not immediately after amino acid 589 of AAV8 or AAV8.AAA. or corresponding to amino acid 589 of AAV8.
[0051] Also provided are AAV vectors comprising the engineered capsids. In some embodiments, the AAV vectors are non-replicating and do not include the nucleotide sequences encoding the rep or cap proteins (these are supplied by the packaging cells in the manufacture of the rAAV vectors). In some embodiments, AAV-based vectors comprise components from one or more serotypes of AAV. In some embodiments, AAV based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15. AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more thereof. In some embodiments, AAV based vectors provided herein comprise components from one or more of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B. AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more serotypes. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g. 85%. 86%. 87%. 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4. AAV5, AAV6, AAV7. AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8. AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV21YF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof. These engineered AAV vectors may comprise a genome comprising a transgene encoding a therapeutic protein.
[0052] In embodiments, the recombinant AAV for use in compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety ). In embodiments, the recombinant AAV for use in compositions and methods herein is AAV.7m8 (including variants thereof) (see, e.g., US 9,193,956; US 9,458,517; US 9,587,282; US 2016/0376323, and WO 2018/075798, each of which is incorporated herein by reference in its entirety)- In embodiments, the AAV for use in compositions and methods herein is any AAV disclosed inUS 9,585,971, such as AAV-PHP.B. In embodiments, the AAV for use in compositions and methods herein is an AAV2/Rec2 or AAV2/Rec3 vector, which has hybrid capsid sequences derived from AAV8 and serotypes cy5, rh20 or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4): e60361, which is incorporated by reference herein for these vectors). In embodiments, the AAV for use in compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7.282,199; US 7,906.111; US 8,524,446; US 8,999,678; US 8,628,966; US 8,927,514; US 8,734,809; US 9,284,357; US 9,409,953; US 9,169,299; US 9,193,956; US 9,458,517; US 9,587,282; US 2015/0374803; US 2015/0126588; US 2017/0067908; US 2013/0224836; US 2016/0215024; US 2017/0051257; PCT/US2015/034799; and PCT/EP2015/053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7.906.111; 8.524,446; 8,999.678; 8.628.966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335.
[0053] In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al.. 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments. rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo etal.. 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1. HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16. each of which is incorporated by reference in its entirety. [0054] In some embodiments, rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of '051 publication), WO 2005/033321 (see, e.g, SEQ ID NOs: 123 and 88 of '321 publication), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of '397 publication). WO 2006/068888 (see, e.g.. SEQ ID NOs: 1 and 3-6 of '888 publication). WO 2006/110689, (see, e.g.. SEQ ID NOs: 5-38 of '689 publication) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of '964 publication), WO 2010/127097 (see, e.g., SEQ ID NOs: 5-38 of '097 publication), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294 of '508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of '924 publication), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g, SEQ ID NO: 2 of '051 publication). WO 2005/033321 (see, e.g. SEQ ID NOs: 123 and 88 of '321 publication), WO 03/042397 (see, e.g, SEQ ID NOs: 2, 81, 85, and 97 of '397 publication), WO 2006/068888 (see, e.g, SEQ ID NOs: 1 and 3-6 of '888 publication), WO 2006/110689 (see, e.g., SEQ ID NOs: 5-38 of '689 publication) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7. 9, 20. 22. 24 and 31 of 964 publication), W0 2010/127097 (see, e.g.. SEQ ID NOs: 5-38 of '097 publication), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294 of '508 publication), and U.S. Appl. Publ. No. 20150023924 (see, e.g, SEQ ID NOs: 1, 5-10 of '924 publication).
[0055] In additional embodiments, rAAV particles comprise a pseudo typed AAV capsid. In some embodiments, the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan etal., J. Virol., 75:7662-7671 (2001); Halbert etal., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).
[0056] In certain embodiments, a single-stranded AAV (ssAAV) may be used. In certain embodiments, a self-complementary vector, e.g., scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171-82; McCarty et al, 2001, Gene Therapy, 8(16): 1248-1254; US 6,596,535; US 7,125,717; and US 7,456,683, each of which is incorporated herein by reference in its entirety).
[0057] Generally, the peptide insertion is sequence of contiguous amino acids from a heterologous protein or domain thereof. The peptide to be inserted typically is long enough to retain a particular biological function, characteristic, or feature of the protein or domain from which it is derived. The peptide to be inserted typically is short enough to allow the capsid protein to form a coat, similarly or substantially similarly to the native capsid protein without the insertion. In preferred embodiments, the peptide insertion is from about 4 to about 30 amino acid residues in length, about 4 to about 20, about 4 to about 15, about 5 to about 10, or about 7 amino acids in length. The peptide sequences for insertion are at least 4 amino acids in length and may be 5, 6, 7, 8, 9, 10, 11, 12, 13. 14. or 15 amino acids in length. In some embodiments, the peptide sequences are 16, 17, 18, 19, or 20 amino acids in length. In embodiments, the peptide is no more than 7 amino acids, 10 amino acids or 12 amino acids in length.
[0058] A “peptide insertion from a heterologous protein” in an AAV capsid protein refers to an amino acid sequence that has been introduced into the capsid protein and that is not native to the AAV serotype capsid into which it is inserted.
[0059] As used herein, the terms “homing” and “targeting” are used interchangeably. These peptides may also or alternatively promote rAAV cell uptake, transduction and/or genome integration in cells of the target tissue.
[0060] Examples of peptides for use as peptide insertions at any of the AAV capsid sites described herein are presented in Tables 3A and 3B and Table 6 below in the Examples (including the peptides having amino acid sequences of SEQ ID NOs: 1-32 and SEQ ID NOs: 115-131), and include at least 7 amino acid contiguous portions thereof, or 9 amino acid contiguous portions thereof (and includes variants having 1, 2 or 3 amino acid substitutions, including conservative amino acid substitutions) and have the functional attribute of the peptide being inserted alters the properties of the capsid, particularly its tropism. In certain embodiments, the recombinant AAV capsids and AAV vectors are engineered to include a peptide, or at least 7 or 9 amino acid contiguous portion thereof, from any of Tables 3A and 3B or Table 6 below (including peptides of amino acid sequences of SEQ ID NOs: 1-32 or SEQ ID NOs: 115-131), inserted into the AAV capsid sequence in such a way that the peptide insertion is displayed. In other embodiments, the peptides are inserted after an amino acid residue at positions 455 or 589 of the amino acid sequence of the AAV8 capsid (SEQ ID NO: 33) or of the AAV8.AAA capsid (SEQ ID NO: 114) a position corresponding thereto in any other AAV serotype (see FIG. 1 for capsid sequence alignments). Capsid protein (VP3) sequences of AAV8 capsid proteins having these peptide inserts are provided as amino acid sequences SEQ ID NOs: 50 to 1 13.
[0061] In another aspect, provided are heterologous peptide insertion libraries. A heterologous peptide insertion library' refers to a collection of rAAV vectors that carry' the same random peptide insertion at the same insertion site in the virus capsid to make the particular library, e.g, at a position within a given variable region of the capsid. Generally, the capsid proteins used comprise AAV genomes that contain modified rep and cap sequences to prevent the replication of the virus under conditions in which it could normally replicate (co-infection of a mammalian cell along with a helper virus such as adenovirus). The members of the peptide insertion libraries may then be assayed for functional display of the peptide on the rAAV surface, tissue targeting and/or gene transduction. Enhanced properties or desirable properties may be assessed upon comparison with the parental capsid from which the insertion library was made.
[0062] Provided are peptide insertion libraries and methods of making these libraries. An exemplary method of producing a capsid library with peptide inserts is descnbed herein in Example 1 and such libraries are screened herein as described in Examples 2 and 3. In such libraries, the nucleic acid encoding the parental capsid has a stop codon at the target insertion site for the population of nucleic acids encoding the peptides such that a capsid protein will only be expressed if the peptide insert is present, otherwise, translation will terminate prematurely. Production of the populations of rAAVs from the expression constructs results in an rAAV population with a high percentage of capsids with peptide inserts (including 80%, 85%, 90%, 95%, 98%, 99% or even 100%).
[0063] Also provided are methods of making the capsid libraries having the variable peptide inserts. In embodiments, the method comprises (1) providing a starting plasmid that contains a gene expression cassette comprising a nucleic acid sequence encoding an AAV capsid, wherein a stop codon is placed at the target insertion site within the capsid gene, (2) providing a repertoire of nucleic acids encoding randomized peptides to produce a peptide library; (3) creating individual plasmids based on the starting plasmid a) each having a nucleic acid encoding a random peptide from the peptide library inserted at the target insertion site of the capsid gene thus replacing the stop codon, and b) each encoding a barcode for identification of the capsid gene having the insert placed before the 5'- or after the 3'-end of the capsid gene; (4) collecting the individual plasmids to form a population or collection of plasmids encoding the capsids with peptide inserts and transfecting cells with the collection of plasmids and with plasmids encoding a recombinant rAAV genome containing a transgene, including one that is detectable, and constructs having the necessary genes to produce, under appropriate conditions, a collection of rAAV vectors encapsidating the rAAV genome containing the transgene, wherein the rAAV vectors have capsids with the encoded capsid protein containing a library peptide insert. In embodiments, the parental AAV is AAV8. AAV8.AAA, or any other suitable AAV serotype. The insertion site may be in VR-IV, including immediately after one of amino acids 455 of AAV8 or corresponding to one of those residues or in VR-VIII, including immediately after amino acid 589 of AAV8 or corresponding to that position in a different AAV capsid type (see FIG. 1 for alignment). The peptides may be 7 or 9 amino acids in length. [0064] In embodiments, the rep gene is on a separate expression plasmid from the plasmid encoding the cap gene with the inserts.
[0065] The library of modified capsids is harvested from these cells. In embodiments, the rAAV library population produced has high levels of capsids having the peptide inserts, including 85%. 90%. 95% or 98%, 99% or even 100%.
5.2.2 Capsids containing Ocular Tissue-Homing and other Targeting Peptides
[0066] The present inventors also have surprisingly discovered peptides that inserted into rAAV vectors “re-target” or enhance targeting properties of such AAV vectors to specific tissues, organs, or cells; in particular, providing peptides that cause rAAV vectors to target ocular tissue and/or other target tissues of interest, such as retinal tissue. RPE-choroid or scleral tissue. This can provide enhanced transport of rAAV particles encapsidating a transgene for optimizing distribution of the vector upon administration to the body, including by suprachoroidal administration. Such peptides, and modified vectors, are described below.
[0067] Another aspect of the present invention relates to capsid proteins selected from the random peptide insertion libraries comprising peptide insertions selected to confer or enhance ocular tissue-homing properties, or “ocular tropism”, including homing to ocular tissue. Also included are capsids and rAAV vectors having capsids comprising these peptide-containing capsid proteins. In other aspects, the peptides may target other tissues, such as, retinal, and other ocular tissues and may also detarget tissues such as liver and/or peripheral tissues when administered to the eye.
[0068] In certain embodiments, the peptide insertion consists of 7 or 9 contiguous amino acids of a peptide sequence of Tables 3A and 3B (SEQ ID NOs: 1-32) or may be a peptide within a consensus sequence of Table 6 (SEQ ID NOs: 115-131). The peptides disclosed herein were identified by screening libraries of peptides inserted in AAV capsids, which are screened for properties such as tropism for ocular tissue, retinal tissue, and other ocular tissues, such as RPE-choroid or scleral tissue, for example, through mouse and NHP biodistribution studies as described in the examples. In embodiments, the capsids having the peptide insertions have increased tropism for a target tissue, including human retinal cells, photoreceptor cells, retina, RPE-choroid, or other ocular tissue, relative to the parental capsid (that is having the capsid protein without the peptide insert) or a reference capsid, such as AAV8 or AAV8.AAA. The capsids with the peptide inserts may also distribute to liver and/or peripheral tissue at levels less than target tissues such as eye, retina, or other target tissue and/or less than a reference capsid, such as AAV8, including when administered to the eye.
[0069] In embodiments, the peptides that may be inserted into capsid proteins include those listed in Tables 3A and 3B and include peptides having amino acid sequences of 7 or 9 contiguous amino acids of one of the amino acid sequences of SEQ ID NOs: 1-32 or the consensus sequences of Table 6 (SEQ ID NOs: 115-131). In embodiments, the peptide is a 7 contiguous amino acid sequence of one of KPKPQQV (SEQ ID NO: 1), RTLKPQA (SEQ ID NO: 2), RKQVQSP (SEQ ID NO: 3), LQRASVM (SEQ ID NO: 4), RQKNAMV (SEQ ID NO: 5), RIMQTKT (SEQ ID NO: 6), RKTMAAV (SEQ ID NO: 7), RLIQGKP (SEQ ID NO: 8), TKLQAKP (SEQ ID NO: 9), RMKTVQT (SEQ ID NO: 10), RIQMGTK (SEQ ID NO: 11), or RPKSTMV (SEQ ID NO: 12). In embodiments, the peptide is inserted in VR-IV (including immediately after amino acid 455 of AAV8, AAV8.AAA). [0070] In embodiments, the peptide is a 7 or 9 contiguous amino acid sequence of one of SENRAQK (SEQ ID NO: 13), DNTTFRR (SEQ ID NO: 14), RTIRGDL (SEQ ID NO: 15), QNRVTAS (SEQ ID NO: 16), QNTIRTQ (SEQ ID NO: 17), ENVNRSK (SEQ ID NO: 18), MAVGGSK (SEQ ID NO: 19), EQAFKRM (SEQ ID NO: 20), HVNGRSS (SEQ ID NO: 21), EFTNKVR (SEQ ID NO: 22). GSENRAQKA (SEQ ID NO: 23), GDNTTFRRA (SEQ ID NO: 24), GRTIRGDLA (SEQ ID NO: 25), GQNRVTASA (SEQ ID NO: 26), GQNTIRTQA (SEQ ID NO: 27), GENVNRSKA (SEQ ID NO: 28), GMAVGGSKA (SEQ ID NO: 29), GEQAFKRMA (SEQ ID NO: 30), GHVNGRSSA (SEQ ID NO: 31). or GEFTNKVRA (SEQ ID NO: 32). In embodiments, the peptide is inserted in VR-VIII (including immediately after amino acid 589 of AAV8, AAV8.AAA).
[0071] In embodiments, the 7-mer to 9-mer peptide that is inserted into the AAV8 capsid is a peptide which has an amino acid sequence within a consensus sequence (see Example 5 and Table 6). and has or comprises an amino acid sequence of
X1-X2-X3-X4-X5-X6-X7, wherein: i) Xi and X2 are each any amino acid; Xs is R or K or Q or H; X4 is any amino acid; X5 is K or Q or S or T; Xe is S or T or Q or A or I or V; and X7 is V or Q or T or P or S (SEQ ID NO: 115); ii) Xi and X2 are each any amino acid; X3 is R or K or Q; X4 is V or K; X5 is K or Q or S or T; Xe is S or T or Q or A or I or V; and X7 is V or Q or T or P or S (SEQ ID NO: 116); iii) Xi is any amino acid; X2 is R or K or F or P or N; X3 and X4 are each any amino acid;
X5 is R or P or Q; Xe and X7 are each any amino acid (SEQ ID NO: 117); iv) Xi is any amino acid; X2 is R or K or F or P or N; X3 is and X4 are each any amino acid;
X5 is R or Q or P; X6 is S or Q or N; X7 is S or P or A (SEQ ID NO: 118); v) Xi, X2, and X3 are each any amino acid, and X4 is R or K or M or A; X5 is any amino acid; Xe is P or T or Q; and X7 is any amino acid (SEQ ID NO: 119); vi) Xi is K or Q; X2.and X3 are each any amino acid; X4 is R or K or M or A; X5 is any amino acid; Xe is P or T or Q; X7 is any amino acid; (SEQ ID NO: 120); vii)Xi, X2,and X3 are each any amino acid; X4 is S or Q or T or G; X5 and Xe are each any amino acid; X7 is K or R or Q or T or S (SEQ ID NO: 121); viii) Xi is R; X2,is K; X3 is any amino acid; X4 is S or Q or T or G; X5 and Xe are each any amino acid; X7 is K or R or Q or T or S (SEQ ID NO: 122); ix) Xi is A or G or D; X2 is V or K or A; X3 is R or Q or V; X4 is K or R or S or H; X5 is K or A or S or P or R or P; Xe is any amino acid; X7 is K or T or Q or V (SEQ ID NO: 124); x) Xi and X2 are each any amino acid; X3 is R or Q or T or D or V; X4, X5 and Xe are each any amino acid; X7 is R or Q or S or N or K (SEQ ID NO: 125); xi) Xi is K or S or V or Q or N; X2 is G or T or K or P or N; Xs is T or S or K or A; X4 is K or R or Q or G; X5 is R or S or K or G; Xe and X7 are each any amino acid (SEQ ID NO: 126); xii) Xi is any amino acid; X2 is R or V or Q or D or A; X3, X4, and X5 are each any amino acid; Xe is K or T or A or V; X7 is any amino acid (SEQ ID NO: 127); xiii) Xi is any amino acid; X2 is R or V or Q or D or A; X3 is R or G or A; X4, and X5 are each any amino acid; Xe is K or T or A or V; X7 is any amino acid (SEQ ID NO: 128); xiv) Xi is any amino acid; X2 is R or V or Q or D or A; X3 is R or G or A; X4 is T or D; X5 is any amino acid; Xe is K or T or A or V; X7 is any amino acid (SEQ ID NO: 129); xv) Xi is any amino acid; X2 is R or V or Q or D or A; X3 is R or G or A; X4 is T or D; Xs is any amino acid; Xe is K or T or A or V; X7 is S or V (SEQ ID NO: 130); xvi) XXXXX(R/K/Q)X Xi,X2, X3, X4, and Xs are each any amino acid; Xe is R or K or Q; X7 is any amino acid (SEQ ID NO: 131); or b) the peptide insertion sequence has an amino acid sequence of X1-X2-X3-X4-X5-X6- X7-X8-X9, wherein: xvii) Xi is G; X2, X3 X4, Xs, Xe are each any amino acid; Xs is R or K; Xs is any amino acid; X9 is A (SEQ ID NO: 123).
[0072] In embodiments, provided are capsids having an amino acid sequence of SEQ ID NO 50-113 (see Table 7).
[0073] In other embodiments, the peptide is a variant of one of the amino acid sequences of SEQ ID NOs: 1 to 32 which has 1, 2 or 3 amino acid substitutions, including conservative amino acid substitutions, while the peptide, when inserted into a capsid protein, retains its biological activity.
[0074] As detailed herein, the peptides may be inserted into wild type or variant capsid protein amino acid sequences at positions such that the peptide is surface displayed when the capsid protein is incorporated into an AAV capsid, for example, at sites that allow surface exposure of the peptide, such as within variable surface-exposed loops, and, in more examples, sites described herein corresponding to VR-I, VR-IV, or VR-VIII, or may be inserted after the first amino acid of VP2, e.g. after amino acid 137 (AAV4, AAV4-4, and AAV5) or at amino acid 138 (AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, rh.10. hu.31, AAVhu.32 and rh.10) (FIG. 1). In embodiments, the capsid protein is an AAV8 capsid protein or an AAV8.AAA capsid protein (or a capsid protein with 90%, 95% or 99% amino acid sequence identity to AAV8 or AAV8.AAA) and the peptide insertion occurs immediately after at least one of (or corresponding to) the amino acid residues 451 to 461 of the AAV8 capsid. In other embodiments, the capsid protein is from at least one AAV type selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrh20, AAVhu.31, AAVhu.32, AAVhu.37, AAVrh39, and AAVrh74 (versions 1 and 2) (see, for example, FIG. 1) or a capsid protein that has 90%, 95% or 99% amino acid identity to the capsid protein of AAV1. AAV2, AAV3, AAV4. AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVhu.31, AAVhu.32, AAVhu.37, AAVrh39, and AAVrh74 (versions 1 and 2). In embodiments, the peptide insertion occurs immediately after an amino acid residue 455 of AAV8 (SEQ ID NO: 33). In embodiments, the peptide insertion occurs immediately after an amino acid residue corresponding to 589 of AAV8 (SEQ ID NO: 33). The alignments of different AAV serotypes, as shown in FIG. 1, indicates corresponding amino acid residues in the different amino acid sequences.
[0075] In embodiments, the capsid protein having the peptide insert is one of the capsid proteins in Table 7, with amino acid sequence SEQ ID NOs: 50 to 113. In embodiments, the capsid protein is 90%, 95% or 99% identical to SEQ ID NOs: 50 to 113. except that it is identical with respect to the peptide insert and retains its biological activity.
[0076] In embodiments, the capsids with peptide inserts as described herein have increased tropism for target tissues, such as retina, and other target tissue relative to the parental capsid, i.e., containing the capsid protein that is identical except that it does not have the peptide insert, or a reference capsid, which may include AAV8, AAV8.AAA. or other capsid of interest that does not contain the peptide insert. In embodiments, the engineered capsid may have reduced tropism or is detargeted for tissues such as liver and/or peripheral tissues, including relative to target tissues, eye, retina, or others, and/or relative to the parental capsid or a reference capsid, which may include AAV8 or AAV8.AAA. In embodiments, the reference capsid does not contain the 496NNN/AAA498 amino acid substitution — for example if the parental capsid is AAV8.AAA. then the liver tropism/detargeting may be relative to AAV8 without the peptide insert, including in embodiments, when administered to the eye.
[0077] For example, as described in the Examples herein, the tissue tropism may be assessed by introducing an rAAV vector having the engineered capsid and a genome with a detectable transgene into a test animal, such as a mouse or NHP, for example by systemic, intravenous, intramuscular, intrathecal, subcutaneous, ocular, suprachoroidal administration or other administration, at an appropriate dosage (for example 1E12, 1E13 or 1E14 vg/kg) and then after an appropriate period of time harvesting the tissues of the animal and assessing the presence of the vector genome, mRNA transcribed from the genome, the ratio of the mRNA to the vector DNA, transgene protein product or activity, including relative to the parental or reference capsid.
[0078] Accordingly, provided are capsids as described herein which, when administered (for example, IV, IM, subcutaneous, suprachoroidal administration) to an animal, including a mouse or NHP, exhibit at least 2-fold, 5-fold, 10-fold, 15 fold, 20-fold, or 25 fold, or greater tropism for ocular tissue, including retinal, RPE-choroidal or scleral tissue, relative to a parental capsid or reference, such as AAV8 or AAV8.AAA, as measured by vector genome DNA, transgene mRNA, the ratio of mRNA to vector genome DNA, trans gene protein product, including protein product activity. In embodiments, the capsid preferentially transduces retinal cells or other ocular tissue in the eye of the animal. In embodiments, provided also are capsids as described herein which when administered (for example, IV, IM, subcutaneous, suprachoroidal administration) to an animal, including a mouse or NHP, exhibit at least 2-fold, 5-fold, 10-fold, 15 fold. 20-fold, 25 fold, 40-fold or 50-fold, less tropism for liver, either relative to a target ocular tissue relative to a parental capsid or reference, such as AAV8 or AAV8.AAA, as measured by amount of vector genome DNA, transgene mRNA, the ratio of mRNA to vector genome DNA, transgene protein product, including protein product activity.
5.2.3 Additional AAV Capsid Insertion Sites
[0079] The following summarizes insertion sites for the peptides described herein, including the peptides in Tables 3A and 3B set forth herein below in the VP1 protein of AAV types as numbered in the SEQ ID referenced and provided herein (see also, FIG. 1):
AAV1: 138; 262-272; 450-459; 595-593; and in an embodiment, between 453-454 (SEQ ID NO: 34). AAV2: 138; 262-272; 449-458; 584-592; and in an embodiment between 452-453 (SEQ ID NO: 35).
AAV3: 138; 262-272; 449-459; 585-593; and in an embodiment, between 452-453 (SEQ ID NO: 36).
AAV4: 137; 256-262; 443-453; 583-591; and in an embodiment, between 446-447 (SEQ ID NO: 37).
AAV5: 137; 252-262; 442-445; 574-582; and in an embodiment, between 445-446 (SEQ ID NO: 38).
AAV6: 138; 262-272; 450-459; 585-593; and in an embodiment, between 452-453 (SEQ ID NO: 39).
AAV7: 138; 263-273; 451-461; 586-594; and in an embodiment, between 453-454 (SEQ ID NO: 40).
AAV8: 138; 263-274; 451-461; 587-595; and in an embodiment, between 453-454 (SEQ ID NO: 33).
AAV9: 138; 262-273; 452-461; 585-593; and in an embodiment, between 454-455 (SEQ ID NO: 43).
AAV8.AAA: 138; 262-273; 452-461; 585-593; and in an embodiment, between 454- 455 (SEQ ID NO: 114).
AAVrhl O: 138; 263-274; 452-461 ; 587-595; and in an embodiment, between 454-455 (SEQ ID NO: 44).
AAVrh20: 138; 263-274; 452-461; 587-595; and in an embodiment, between 454-455 (SEQ ID NO: 47).
AAVrh74: 138; 263-274; 452-461; 587-595; and in an embodiment, between 454-455 (SEQ ID NO: 48 or SEQ ID NO: 49).
AAVhu.32: 138; 262-273; 452-461; 585-593; and in an embodiment, between 454-455 (SEQ ID NO: 42).
AAVhu.37: 138; 263-274; 452-461; 587-595; and in an embodiment, between 454-455 (SEQ ID NO: 45)
[0080] In embodiments, the peptide insertion occurs between amino acid residues 588-589 of the AAV8 capsid, or between corresponding residues of another AAV type capsid as determined by an amino acid sequence alignment (for example, as in FIG. 1). In embodiments, the peptide insertion occurs immediately after amino acid residue G455 or Q589 of the AAV8 capsid sequence, or immediately after corresponding residues of another AAV capsid sequence (FIG. 1)
[0081] In some embodiments, the capsid is chosen and/or further modified to reduce recognition of the AAV particles by the subject’s immune system, such as avoiding preexisting antibodies in the subject. In some embodiments. In some embodiments, the capsid is chosen and/or further modified to enhance desired tropism/targeting.
5.2.4 Generation of Modified capsids
[0082] In some embodiments, AAV capsids were modified by introducing selected single to multiple amino acid substitutions which increase effective gene delivery to ocular tissue, detarget the liver, and/or reduce immune responses of neutralizing antibodies, prior to making the rAAV library with random peptide insertions.
[0083] Exposure to the AAV capsid can generate an immune response of neutralizing antibodies. One approach to overcome this response is to map the AAV-specific neutralizing epitopes and rationally design an AAV capsid able to evade neutralization. A monoclonal antibody, specific for intact AAV9 capsids, with high neutralizing titer has recently been described (Giles et al, 2018, Mapping an Adeno-associated Virus 9-Specific Neutralizing Epitope To Develop Next-Generation Gene Delivery' Vectors). The epitope was mapped to the 3-fold axis of symmetry on the capsid, specifically to residues 496-NNN-498 and 588- QAQAQT-592 of, e.g., AAV9. Similar modifications were made to AAV8. Capsid mutagenesis demonstrated that single amino acid substitution within this epitope markedly reduced binding and neutralization. In addition, in vivo studies showed that mutations in the epitope conferred a "li ver-detargeting" phenotype to the mutant vectors, suggesting that the same residues are also responsible for AAV9 tropism. Liver detargeting has also been associated with substitution of amino acid 503 replacing tryptophan with arginine. Presence of the W503R mutation in the AAV 9 capsid was associated with low glycan binding avidity (Shen et al, 2012, Glycan Binding Avidity Determines the Systemic Fate of Adeno-Associated Virus Type 9).
[0084] In some embodiments, provided are capsids which were further modified by substituting asparagines at amino acid positions 498, 499, and 500 (such as AAV8.AAA) or 496, 497, and 498 (herein referred to as AAV8.AAA, SEQ ID NO: 114) with alanines.
[0085] In some embodiments, provided are capsids having three asparagines at amino acid positions 496. 497, and 498 of the AAV8 capsid replaced with alanines and optionally tryptophan at amino acid 503 of the AAV 8 capsid with arginine or capsids with substitutions corresponding to these positions in other AAV types. In some embodiments, provided are capsids having glutamine at amino acid position 474 of the AAV8 capsid substituted with alanine or capsids with substitutions corresponding to this position in other AAV types.
[0086] In some embodiments, the rAAVs described herein increase tissue-specific (such as. but not limited to, eye) cell transduction in a subject (a human, non-human-primate, or mouse subject) or in cell culture, compared to the rAAV not comprising the peptide insertion. In some embodiments, the increase in tissue specific cell transduction is at least 2, 10, 20, 30, 40, 50, 60. 70, 80, 90, or 100 fold more than that without the peptide insertion. For example, in some embodiments, there is a 50-80 fold increase in tissue specific cell transduction compared to transduction with the same AAV type without a peptide insert. The increase in transduction may be assessed using methods described in the Examples herein and known in the art.
5.3. Methods of Making rAAV Molecules
[0087] Another aspect of the present invention involves making molecules disclosed herein. In some embodiments, a molecule according to the invention is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid protein molecules herein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein. In some embodiments, the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99% or 99.9%, identity to the sequence of a capsid protein molecule described herein, and retains (or substantially retains) biological function of the capsid protein and the inserted peptide from a heterologous protein or domain thereof. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of the AAV8 capsid protein (SEQ ID NO: 33 and see FIG. 1), while retaining (or substantially retaining) biological function of the AAV8 capsid protein and the inserted peptide.
[0088] The capsid protein, coat, and rAAV particles may be produced by techniques known in the art. In some embodiments, the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector. In some embodiments, the viral genome further comprises a cap gene and/or a rep gene for expression and splicing of the cap gene. In other embodiments, the cap and rep genes are provided by a packaging cell and not present in the viral genome. [0089] In some embodiments, the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap helper plasmid in place of the existing capsid gene. When introduced together into host cells, this plasmid helps package a rAAV genome into the engineered capsid protein as the capsid coat. Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Nonlimiting examples include 293 cells or derivatives thereof, HELA cells, or insect cells.
[0090] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See. e.g.. Sambrook et al.. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection w ith, and the laboratory procedures and techniques of. analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and deliver}', and treatment of patients. Nucleic acid sequences of AAV-based viral vectors, and methods of making recombinant AAV and AAV capsids, are taught, e.g.. in US 7,282.199; US 7.790,449; US 8,318,480; US 8,962,332; and PCT/EP2014/076466, each of which is incorporated herein by reference in its entirety.
[0091] In some embodiments, the rAAVs provide transgene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below'. In some embodiments, the rAAV vector also includes regulatory control elements known to one skilled in the art to influence the expression of the RNA and/or protein products encoded by nucleic acids (transgenes) within target cells of the subject. Regulator}' control elements and may be tissue-specific, that is, active (or substantially more active or significantly more active) only in the target cell/tissue. In specific embodiments, the AAV vector comprises a regulatory sequence, such as a promoter, operably linked to the transgene that allows for expression in target tissues. The promoter may be a constitutive promoter, for example, the CB7 promoter. Additional promoters include: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65 promoter, opsin promoter, the TBG (Thyroxine-binding Globulin) promoter, the APOA2 promoter, SERPINA1 (hAAT) promoter, or MIR122 promoter. In some embodiments, particularly where it may be desirable to turn off transgene expression, an inducible promoter is used, e.g.. hypoxia-inducible or rapamycin-inducible promoter.
[0092] Provided in embodiments are AAV8 vectors comprising a viral genome comprising an expression cassette for expression of the transgene, under the control of regulatory elements, and flanked by ITRs and an engineered viral capsid as described herein or is at least 95%, 96%, 97%. 98%. 99% or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (see FIG. 1), while retaining the biological function of the engineered AAV8 capsid. In certain embodiments, the encoded AAV8 capsid has the sequence of wild type AAV8, with the peptide insertion as described herein, with, in addition, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24. 25. 26, 27, 28, 29, or 30 amino acid substitutions with respect to the wild type AAV sequence and retains biological function of the AAV8 capsid. Also provided are engineered AAV vectors other than AAV8 vectors, such as engineered AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVhu.31, AAVhu.32, AAVhu.37, AAVrh39, or AAVrh74 vectors, with the peptide insert as described herein and 1, 2, 3. 4, 5, 6. 7, 8, 9. 10. 1 1. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions relative to the wild type or unengineered sequence for that AAV type and that retains its biological function.
[0093] The recombinant adenovirus can be a first-generation vector, with an El deletion, with or without an E3 deletion, and with the expression cassette inserted into either deleted region. The recombinant adenovirus can be a second-generation vector, which contains full or partial deletions of the E2 and E4 regions. A helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (phi). The transgene generally is inserted between the packaging signal and the 3 TTR, with or without stuffer sequences to keep the genome close to wild-type size of approximately 36 kb. An exemplary protocol for production of adenoviral vectors may be found in Alba et al., 2005, “Gutless adenovirus: last generation adenovirus for gene therapy,'’ Gene Therapy 12:S18-S27, which is incorporated by reference herein in its entirety
[0094] The rAAV vector for delivering the transgene to target tissues, cells, or organs, has a tropism for that particular target tissue, cell, or organ. Tissue-specific promoters may also be used. The construct further can include expression control elements that enhance expression of the transgene driven by the vector (e.g., introns such as the chicken P-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), P-globin splice donor/immunoglobulin heavy chain spice acceptor intron, adenovirus splice donor /immunoglobulin splice acceptor intron, SV40 late splice donor /splice acceptor (19S/16S) intron, and hybrid adenovirus splice donor/IgG splice acceptor intron and polyA signals such as the rabbit P-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal. See, e.g., Powell and Rivera-Soto, 2015. Discov. Med., 19(102):49-57.
[0095] In certain embodiments, nucleic acids sequences disclosed herein may be codon- optimized, for example, via any codon-optimization technique known to one of skill in the art (see, e.g., review7 by Quax et al., 2015, Mol Cell 59:149-161).
[0096] In a specific embodiment, the constructs described herein comprise the following components: (1) AAV8 inverted terminal repeats that flank the expression cassette; (2) control elements, which include a) the CB7 promoter, comprising the CMV enhancer/chicken P-actin promoter, b) a chicken p-actin intron and c) a rabbit P-globin poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid or protein product of interest. In a specific embodiment, the constructs described herein comprise the following components: (1) AAV 8 inverted terminal repeats that flank the expression cassette; (2) control elements, which include a) a hypoxia-inducible promoter, b) a chicken p-actin intron and c) a rabbit P-globin poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid or protein product of interest.
[0097] The viral vectors provided herein may be manufactured using host cells, e.g., mammalian host cells, including host cells from humans, monkeys, mice, rats, rabbits, or hamsters. Nonlimiting examples include: A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC I, BSC 40, BMT 10, VERO. W138. HeLa. 293, Saos. C2C12, L. HT1080, HepG2. primary fibroblast, hepatocyte, and myoblast cells. Typically, the host cells are stably transformed with the sequences encoding the transgene and associated elements (i.e., the vector genome), and genetic components for producing viruses in the host cells, such as the replication and capsid genes (e.g. , the rep and cap genes of AAV). For a method of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the Detailed Description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference in its entirety. Genome copy titers of said vectors may be determined, for example, by TAQMAN® analysis. Virions may be recovered, for example, by CsCh sedimentation. Alternatively, baculovirus expression systems in insect cells may be used to produce AAV vectors. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045-1054, which is incorporated by reference herein in its entirety for manufacturing techniques.
[0098] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from a vector described herein, thus indicating, e.g., potency of the vector. For example, the PER.C6® Cell Line (Lonza), a cell line derived from human embryonic retinal cells, or retinal pigment epithelial cells, e.g., the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®), can be used to assess transgene expression. Alternatively, cell lines derived from liver or other cell types may be used, for example, but not limited, to HuH-7, HEK293, fibrosarcoma FIT-1080, HKB-11, and CAP cells. Once expressed, characteristics of the expressed product (i.e., transgene product) can be determined, including determination of the glycosylation and tyrosine sulfation patterns, using assays known in the art.
5.4. Therapeutic and Prophylactic Uses
[0099] Another aspect relates to therapies which involve administering a transgene via a rAAV vector according to the invention to a subject in need thereof, for delaying, preventing, treating, and/or managing a disease or disorder, and/or ameliorating one or more symptoms associated therewith. A subject in need thereof includes a subject suffering from the disease or disorder, or a subject pre-disposed thereto, e.g, a subject at risk of developing or having a recurrence of the disease or disorder. Generally, a rAAV carrying a particular transgene will find use with respect to a given disease or disorder in a subject where the subject’s native gene, corresponding to the transgene, is defective in providing the correct gene product, or correct amounts of the gene product. The transgene then can provide a copy of a gene that is defective in the subject.
[00100] Generally, the transgene comprises cDNA that restores protein function to a subject having a genetic mutation(s) in the corresponding native gene. In some embodiments, the cDNA comprises associated RNA for performing genomic engineering, such as genome editing via homologous recombination. In some embodiments, the transgene encodes a therapeutic RNA, such as a shRNA, artificial miRNA, or element that influences splicing.
[00101] In aspects, the rAAVs of the present invention find use in delivery to target tissues, or target cell types, including cell matrix associated with the target cell ty pes, associated with the disorder or disease to be treated/pre vented. A disease or disorder associated with a particular tissue or cell type is one that largely affects the particular tissue or cell type, in comparison to other tissue of cell types of the body, or one where the effects or symptoms of the disorder appear in the particular tissue or cell type. Methods of delivering a transgene to a target tissue of a subject in need thereof involve administering to the subject tan rAAV where the peptide insertion is a homing peptide. In the case of ocular disorders, for example, a rAAV vector comprising a peptide insertion that directs the rAAV to target the eye or ocular tissues of the subject, in particular, where the peptide insertion facilitates the rAAV in transducing ocular tissue with high efficiency, including satellite cells, yet results in lower transduction of liver cells.
[00102] For a disease or disorder associated with the retina or eye, rAAV vectors can be selected from the libraries herein that comprise a peptide insertion that directs ocular tissue transduction, relative to the parental rAAV vector without a peptide insertion.
[00103] Diseases/disorders associated with the eye or retina are referred to as “ocular diseases/’ Nonlimiting examples of ocular diseases include anterior ischemic optic neuropathy; acute macular neuroretinopathy; Bardet-Biedl syndrome; Behcet's disease; branch retinal vein occlusion; central retinal vein occlusion; choroideremia; choroidal neovascularization; chorioretinal degeneration; cone-rod dystrophy; color vision disorders (e.g., achromatopsia, protanopia. deuteranopia, and tritanopia); congenital stationary night blindness; diabetic uveitis; epiretinal membrane disorders; inherited macular degeneration; histoplasmosis; macular degeneration (e.g., acute macular degeneration, non-exudative age related macular degeneration, exudative age related macular degeneration, dry age related macular degeneration (dry' AMD) or dry' age related macular degeneration with geographic atrophy (GA)); diabetic retinopathy; edema (e.g., macular edema, cystoid macular edema, diabetic macular edema); glaucoma; Leber congenital amaurosis; Leber's hereditary optic neuropathy; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal dysfunction; ocular trauma; ocular tumors; proliferative vitreoretinopathy (PVR); retinopathy of prematurity; retinoschisis; retinitis pigmentosa; retinal arterial occlusive disease, retinal detachment, Stargardt disease (fundus flavimaculatus); sympathetic opthalmia; uveal diffusion; uveitic retinal disease; Usher syndrome; Vogt Koyanagi -Harada (VKH) syndrome; nAMD (wet AMD); dry' AMD; dry' AMD with GA; retinal vein occlusion (RVO); mucopolysaccharidosis type IVA (MPS IVA); mucopolysaccharidosis type I (MPS I); mucopolysaccharidosis type II (MPS II); familial hypercholesterolemia (FH); homozygous familial hypercholesterolemia (HoFH); coronary artery' disease; cerebrovascular disease; Duchenne muscular dystrophy, Limb Girdle muscular dystrophy; Becker muscular dystrophy and sporadic inclusion body myositis: kallikrein-related disease; or a posterior ocular condition associated with ocular laser or photodynamic therapy.
[00104] For a disease or disorder associated with the retina or eye, the rAAV vector has a capsid with ocular tropism, directing the rAAV to target the eye or ocular tissues of the subject. The term “retinal celf’ refers to one or more of the cell types found in or near the retina, including amacrine cells, bipolar cells, horizontal cells, Muller glial cells, photoreceptor cells (e.g., rods and cones), retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retina ganglion cells, and photosensitive ganglion cells), retinal pigmented epithelium, endothelial cells of the inner limiting membrane, and the like. Ocular tissues include anterior segment tissues, including the iris, cornea, lens, ciliary body, Schlemnf s canal, and trabecular meshwork, and posterior segment tissues, such as the retina or RPE-choroid, and optic nerve. [00105] In embodiments, the rAAV are administered so as to target ocular tissues and may be administered by suprachoroidal administration (i.e., into the suprachoroidal space). In other embodiments, the rAAV is administered by intravitreal, intraocular, or intracameral administration.
[00106] The rAAV vectors of the invention also can facilitate delivery', in particular, targeted delivery, of oligonucleotides, drugs, imaging agents, inorganic nanoparticles, liposomes, antibodies to target cells or tissues. The rAAV vectors also can facilitate delivery, in particular, targeted delivery, of non-coding DNA, RNA, or oligonucleotides to target tissues.
[00107] The agents may be provided as pharmaceutically acceptable compositions as known in the art and/or as described herein. Also, the rAAV molecule of the invention may be administered alone or in combination with other prophylactic and/or therapeutic agents.
[00108] The dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophy tactically effective. The dosage and frequency will ty pically vary' according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of disease, the route of administration, as well as age. body weight, response, and the past medical history of the patient, and should be decided according to the judgment of the practitioner and each patient's circumstances. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician 's Desk Reference (56th ed., 2002). Prophylactic and/or therapeutic agents can be administered repeatedly. Several aspects of the procedure may vary such as the temporal regimen of administering the prophylactic or therapeutic agents, and whether such agents are administered separately or as an admixture.
[00109] The amount of an agent of the invention that will be effective can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (z.e., the concentration of the test compound that achieves a half- maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
[00110] Prophylactic and/or therapeutic agents, as well as combinations thereof, can be tested in suitable animal model systems prior to use in humans. Such animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in the art may be used. Such model systems are widely used and well known to the skilled artisan. In some embodiments, animal model systems for an ocular condition are used that are based on rats, mice, or other small mammal other than a primate. [00111] Once the prophylactic and/or therapeutic agents of the invention have been tested in an animal model, they can be tested in clinical trials to establish their efficacy. Establishing clinical trials will be done in accordance with common methodologies known to one skilled in the art, and the optimal dosages and routes of administration as well as toxicity profiles of agents of the invention can be established. For example, a clinical trial can be designed to test a rAAV molecule of the invention for efficacy and toxicity in human patients.
[00112] Toxicity and efficacy of the prophylactic and/or therapeutic agents of the instant invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Prophylactic and/or therapeutic agents that exhibit large therapeutic indices are preferred. While prophylactic and/or therapeutic agents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. [00113] A rAAV molecule of the invention generally will be administered for a time and in an amount effective for obtain a desired therapeutic and/or prophylactic benefit. The data obtained from the cell culture assays and animal studies can be used in formulating a range and/or schedule for dosage of the prophylactic and/or therapeutic agents for use in humans. The dosage of such agents lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
[00114] A therapeutically effective dosage of an rAAV vector for patients is generally from about 0.1 ml to about 100 ml of solution containing concentrations of from about IxlO9 to about IxlO16 genomes rAAV vector, or about IxlO10 to about IxlO13, about IxlO12 to about IxlO16, or about IxlO14 to about IxlO16 rAAV genomes. Levels of expression of the transgene can be monitored to determine/adjust dosage amounts, frequency, scheduling, and the like.
[00115] Treatment of a subject with a therapeutically or prophy lactically effective amount of the agents of the invention can include a single treatment or can include a series of treatments. For example, pharmaceutical compositions comprising an agent of the invention may be administered once a day, twice a day, or three times a day. In some embodiments, the agent may be administered once a day, every other day, once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, or once per year. It will also be appreciated that the effective dosage of certain agents, e.g., the effective dosage of agents comprising a dual antigen-binding molecule of the invention, may increase or decrease over the course of treatment.
[00116] In some embodiments, ongoing treatment is indicated, e.g. , on a long-term basis, such as in the ongoing treatment and/or management of chronic diseases or disorders. For example, in embodiments, an agent of the invention is administered over a period of time, e.g., for at least 6 months, at least one year, at least tw o years, at least five years, at least ten years, at least fifteen years, at least twenty years, or for the rest of the lifetime of a subject in need thereof.
[00117] The rAAV molecules of the invention may be administered alone or in combination with other prophylactic and/or therapeutic agents. Each prophylactic or therapeutic agent may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect. Each therapeutic agent can be administered separately, in any appropriate form and by any suitable route. [00118] In various embodiments, the different prophylactic and/or therapeutic agents are administered less than 1 hour apart, at about 1 hour apart, at about 1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, no more than 24 hours apart, or no more than 48 hours apart. In certain embodiments, two or more agents are administered within the same patient visit.
[00119] Methods of administering agents of the invention include, but are not limited to, parenteral administration (e.g, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection), epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral mucosa, rectal, and intestinal mucosa, etc.).
[00120] In certain embodiments, provided herein is a method of suprachoroidal administration for treating a pathology of the eye, comprising administering to the suprachoroidal space in the eye of a human subj ect in need of treatment a recombinant viral vector comprising a nucleotide sequence encoding a therapeutic product such that the therapeutic product is expressed and results in treatment of the pathology of the eye. In certain embodiments, the administering step is by injecting the recombinant viral vector into the suprachoroidal space using a suprachoroidal drug delivery' device. In some embodiments, the suprachoroidal drug delivery device comprises a microneedle. In certain embodiments, the suprachoroidal drug delivery device is a microinjector.
[00121] Currently available technologies for suprachoroidal space (SCS) delivery exist. SC injections have been achieved with scleral flap technique, catheters and standard hypodermic needles, as well as with microneedles. A hollow-bore microneedle with a length matched to the thickness of the sclera, for example a 900 pm-long or 1100 pm-long microneedle (SCS Microinjector®, Clearside Biomedical. Inc.), inserted in the eye has been shown to deliver therapeutic agents to the posterior segment of the eye via the suprachoroidal space (Rai UDJ, et al. The suprachoroidal pathway: a new drug delivery' route to the back of the eye. Drug Discov Today. 2015;20(4):491-495; Moisseiev E, et al. The suprachoroidal space: from potential space to a space with potential. Clin Ophthalmol. 2016:10: 173-178; Chiang B. et al. The suprachoroidal space as a route of administration to the posterior segment of the eye. Adv Drug Deliv Rev. 2018;126:58-66). The SCS Microinjector has been approved for suprachoroidal use clinically with XIPERE®.
[00122] Recombinant AAV vector compositions, such as the rAAV compositions described herein, achieve delivery of the transgene to the posterior segment of the eye, including retinal cells. RPE and photoreceptor cells, via suprachoroidal administration (Ewing, TM. et al. 2022 Retinal Physician 19:24-26). In some embodiments, a method of administering an rAAV vector composition comprises inserting a needle, e.g. a microneedle, into the suprachoroidal space of the eye without the needle penetrating through the choroid (“suprachoroidal administration or injection’'). rAAV vector compositions can be injected into the eye using a microneedle, ty pically locating the needle approximately 4-4.5 mm from the limbus, into the pars plana. By way of non-limiting example, the microneedle is inserted into the sclera at a perpendicular angle, then the needle hub is depressed to create a sealing gasket effect, then the injection of a pharmaceutical composition comprising the rAAV vector is performed over 5-10 seconds. In some embodiments, the administration step is performed under local anesthesia (e.g. anesthesia administered before the suprachoroidal injection). In other embodiments, topical steroid or other anti-inflammatory may be applied to the eye before or after insertion of the needle.
[00123] Oxular Limited is developing a SCS device and delivery system (Oxulumis) that advances an illuminated cannula in the suprachoroidal space. The Orbit device (Gyroscope) is a specially-designed system enabling cannulation of the suprachoroidal space with a flexible cannula (for example, the flexible cannula is inserted between the sclera and the choroid). A microneedle inside the cannula is advanced into the subretinal space to enable targeted dose delivery. Ab intemo access to the SCS can also be achieved using micro-stents, which serve as minimally-invasive glaucoma surgery (MIGS) devices. Examples include the CyPass® MicroStent (Alcon, Fort Worth, Texas, US) and iStent® (Glaukos), which are surgically implanted to provide a conduit from the anterior chamber to the SCS to drain the aqueous humor without forming a filtering bleb.
[00124] In some embodiments, the suprachoroidal drug delivery device is a syringe with a 1 millimeter 30 gauge needle. In some embodiments, the syringe has a larger circumference (e.g., 29 gauge needle). During an injection using this device, the needle pierces to the base of the sclera and fluid containing drug enters the suprachoroidal space, leading to expansion of the suprachoroidal space. As a result, there is tactile and visual feedback during the injection. Following the injection, the fluid flows posteriorly and absorbs dominantly in the choroid and retina. This results in the production of transgene protein from all retinal cell layers and choroidal cells. Using this type of device and procedure allows for a quick and easy in-office procedure with low risk of complications.
[00125] In some embodiments, a microneedle or syringe is used to administer the rAAV compositions described herein. In some embodiments, a microneedle or syringe comprises a needle having an effective length of about 2000 microns or less. In other embodiments, a microneedle or syringe comprises a needle having an effective length between about 500 microns to about 2000 microns, or between about 800 microns to about 1200 microns. In some embodiments, a microneedle or syringe comprises a needle having an effective length of about 800 pm, about 850 pm, about 900 pm. about 950 pm, about 1000 pm. about 1100 pm, or about 1200 pm. In some embodiments, a microneedle or syringe is selected based on the viscosity of a pharmaceutical composition (e.g., liquid formulation). In some embodiments, a microneedle is selected based on the pressure resulted in the eye (e.g., in the SCS) when a pharmaceutical composition (e.g.. liquid formulation) is administered. For example, a pharmaceutical composition (e.g., liquid formulation) having medium or high viscosity may benefit from the use of a wider microneedle for injection. In some embodiments, the pressure in the SCS is lower when a wider microneedle is used as compared to the pressure obtained when a narrower microneedle is used. In some embodiments, 10 gauge needle, 11 gauge needle, 12 gauge needle. 13 gauge needle. 14 gauge needle. 15 gauge needle. 16 gauge needle, 17 gauge needle, 18 gauge needle, 19 gauge needle, 20 gauge needle, 21 gauge needle, 22 gauge needle, 23 gauge needle, 24 gauge needle, 25 gauge needle, 26 gauge needle, 27 gauge needle, 28 gauge needle, 29 gauge needle, 30 gauge needle, 31 gauge needle, 32 gauge needle, 33 gauge needle, or 34 gauge needle is used. In some embodiments, a 27 gauge needle is used. In some embodiments, a 28 gauge needle is used. In some embodiments, a 29 gauge needle is used. In some embodiments, a 30 gauge needle is used. In some embodiments, a 31 gauge needle is used. In some embodiments, a gauge that is smaller than a 27 gauge needle is used. In some embodiments, a gauge that is larger than a 27 gauge needle is used. In some embodiments, a gauge that is smaller than a 28 gauge needle is used. In some embodiments, a gauge that is smaller than a 30 gauge needle is used. In some embodiments, a gauge that is higher than a 30 gauge needle is used.
[00126] Devices used to carry out the methods described herein comprise any one of the devices disclosed in International Publication No. WO2011139713, International Publication No. W02014036009, International Publication No. WO2014074823, International Publication No. WO2014179698, International Publication No. WO2015126694, International Publication No. W02016044404, International Publication No. W02016040635, International Publication No. WO2017156227, International Publication No. WO2017218613, International Publication No. WO2019053466, International Publication No. W02019202603, International Publication No. WO2021188803, and US Patent No. 11.273,072 each of which are hereby incorporated by reference in their entireties.
[00127] In some embodiments, a pharmaceutical composition or a reference pharmaceutical composition provided herein is suitable for administration by one, two or more routes of administration (e.g., suitable for suprachoroidal and subretinal administration).
[00128] In certain embodiments, the agents of the invention are administered intravenously and may be administered together with other biologically active agents.
[00129] In another specific embodiment, agents of the invention may be delivered in a sustained release formulation, e.g., where the formulations provide extended release and thus extended half-life of the administered agent. Controlled release systems suitable for use include, without limitation, diffusion-controlled, solvent-controlled, and chemically-controlled systems. Diffusion controlled systems include, for example reservoir devices, in which the molecules of the invention are enclosed within a device such that release of the molecules is controlled by permeation through a diffusion barrier. Common reservoir devices include, for example, membranes, capsules, microcapsules, liposomes, and hollow fibers. Monolithic (matrix) device are a second type of diffusion controlled system, wherein the dual antigenbinding molecules are dispersed or dissolved in a rate-controlling matrix (e.g., a polymer matrix). Agents of the invention can be homogeneously dispersed throughout a rate-controlling matrix and the rate of release is controlled by diffusion through the matrix. Polymers suitable for use in the monolithic matrix device include naturally occurring polymers, synthetic polymers and synthetically modified natural polymers, as well as polymer derivatives.
[00130] Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more agents described herein. See. e.g. U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al.. “Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained- Release Gel,’’ Radiotherapy & Oncology, 39: 179 189, 1996; Song et al., “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,’' PDA Journal of Pharmaceutical Science & Technology, 50:372 397, 1995; Cleek et al., “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Inti. Symp. Control. Rel. Bioact. Mater., 24:853 854, 1997; and Lam et al., “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24:759 760, 1997, each of which is incorporated herein by reference in its entirety. In one embodiment, a pump may be used in a controlled release system (see Langer, supra, Sefton, CRC Crit. Ref. Biomed. Eng., 14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J. Med., 321 :574, 1989). In another embodiment, polymeric materials can be used to achieve controlled release of agents comprising dual antigen-binding molecule, or antigen-binding fragments thereof (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance. Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem., 23:61, 1983; see also Levy et al., Science, 228: 190, 1985; During et al., Aww. Neurol., 25:351, 1989; Howard et al., J. Neurosurg., 7 1: 105, 1989); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989.463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target (e.g., an affected joint), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)). Other controlled release systems are discussed in the review by Langer, Science, 249: 1527 1533, 1990.
[00131 ] In addition, rAAVs can be used for in vivo delivery of transgenes for scientific studies such as optogenetics, gene knock-down with miRNAs, recombinase delivery for conditional gene deletion, gene editing with CRISPRs, and the like.
5.5. Pharmaceutical Compositions and Kits
[00132] The invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an agent of the invention, said agent comprising a rAAV molecule of the invention. In some embodiments, the pharmaceutical composition comprises rAAV combined with a pharmaceutically acceptable carrier for administration to a subject. In one embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions and aqueous dextrose and glycerol solutions that can also be employed as liquid carriers, particularly for injectable solutions.
[00133] In certain embodiments of the invention, pharmaceutical compositions are provided for use in accordance with the methods of the invention, said pharmaceutical compositions comprising a therapeutically and/or prophylactically effective amount of an agent of the invention along with a pharmaceutically acceptable carrier.
[00134] In certain embodiments, the agent of the invention is substantially purified (/.<?., substantially free from substances that limit its effect or produce undesired side-effects). In a specific embodiment, the host or subject is an animal, e.g., a mammal such as non-primate (e.g, cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey such as, a cynomolgus monkey and a human). In a certain embodiment, the host is a human.
[00135] The invention provides further kits that can be used in the above methods. In one embodiment, a kit comprises one or more agents of the invention, e.g., in one or more containers. In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for the treatment of a condition, in one or more containers.
[00136] The invention also provides agents of the invention packaged in a hermetically sealed container, such as a clear glass vial or polymer vial, such as a cyclo olefin polymer (COP) vial or Crystal Zenith® (Daikyo) vial. In an alternative embodiment, an agent of the invention is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of agent or active agent.
[00137] The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) as well as pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient). Bulk drug compositions can be used in the preparation of unit dosage forms, e.g., comprising a prophylactically or therapeutically effective amount of an agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier.
[00138] The invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the agents of the invention. Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of the target disease or disorder can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.
[00139] Generally, the ingredients of compositions of the invention are supplied in unit dosage form, for example, as a sterile liquid or dry lyophilized powder in a hermetically sealed vial indicating the quantity of agent or active agent.
6. EXAMPLES
[00140] The following examples report a method of making rAAV libraries containing numerous rAAV capsids having surface-exposed peptides inserted at designated insertion sites of the capsid. The recombinantly engineered capsids are screened to identify candidates for particularly properties, such as tissue tropism. The invention is illustrated by way of examples, describing the construction of rAAV capsids engineered to contain 7-mer or 9-mer peptides, wherein the method is designed to increase library diversity by reducing the parental capsid formation during manufacture, thereby reducing its abundance and therefore its representation in the library, thereby enhancing screening and identifying capsids with peptide inserts relative to the parental capsid. Several libraries of peptide insertion mutants were constructed by this method and the pooled mutants were screened for viable capsid assembly, titer, and biodistribution. The top candidates were then further evaluated for use to re-target rAAV vectors to tissues of interest. Further examples, demonstrate the increased transduction and tissue tropism for certain of the modified AAV capsids described herein.
6.1.Example 1 - Construction of rAAV libraries
[00141] For generation of rAAV libraries, a custom Rep-only trans-plasmid was created in which the Cap sequence was removed. A custom cis-plasmid was created in which the CMV enhancer-promoter and Rep intron precedes the AAV Cap gene of interest, followed by the RBG poly A. A stop codon was inserted into the Cap gene variable region where the members of the peptide library would be later inserted to reduce expression of wildtype (or parental) sequence in the library' and provide capsids capable of “intelligent, guided adaptation” (Novel AAV Vector Intelligent Guided Adaptation Through Evolution = NAVIGATE.)
[00142] Nucleotide sequence encoding the AAV8 parental capsid (SEQ ID NO: 33) was used as a starting template for i) producing an AAV 8 vector library having random peptide insertions after amino acid residue G455, to generate a AAV8.VR4 vector library, and ii) producing an AAV8 vector library having random peptide insertions after amino acid residue Q589 plus the amino acid glycine (G) 5’ of the insert and the amino acid alanine (A) 3’ of the 7-mer insert, to generate a AAV8.VR8 vector library, using the methods herein.
[00143] Libraries were produced by generating a randomized 7-mer peptide insertion library synthesized in a chip-based format (Genscript Biotech) in which each amino acid except cysteine was represented by a single codon to minimize redundancy and stop codons. The synthesized insert was cloned into plasmid i) pRGX.CMV. rep-intron. AAV8.456. stopcodon or ii) pRGX.CMV. rep-intron.AAV8.590.stopcodon (FIG. 3), thereby replacing the stop codon at position 456 or 590 of the cap gene for AAV8, respectively. Following transformation of Endura competent cells (Lucigen), colony counts on dilution plates were used to determine library size. The library E. coli culture was scaled up to prepare 0.5 mg of library cA-plasmid. Sequence diversity of the library insert and amino acid evenness was confirmed by NGS. 7ro -plasmid pRGX. rep-only was generated by removing the cap gene from a standard repcap /ra s -plasmid. Next-generation sequencing of the libraries was used to characterize library diversity and the wildtype fraction. In AAV8 having the insertion library cloned into position 456 in VR-IV the diversity’ was 4.32 x!07. See also Table 1. This site occurs at the top of the VR-IV flexible loop and has previously been shown to tolerate peptide insertions (PCT International Publication No. W02020206189A1, which is hereby incorporated by reference in its entirety). The AAV8 library having the insertions at position 590 in VR-VIII had a diversity of 1.15 xl 07. FIGs. 1 and 2 depict analysis of variable region four or eight of the adeno-associated virus type 8 (AAV8 VR-IV or AAV8 VR-VIII) by amino acid sequence comparison to that of other AAVs VR-IV (FIG. 1), and an AAV9 protein model (FIG. 2) depicting the potential interactions between VR-IV, VR-V (three asparagines) and VR-VIII. AAV8 has similar regions and interactions. Nam, H.-J. et al. (2007) reported similar three- dimensional structures for residues 454 to 457 (AAV8 VP1 numbering), which are located in the large loop region between beta-strands G and H (referred to as the GH loop) for AAV8 in comparison to AAV2 (PDB ID: 2QA0; Nam, H.-J. et al. (2007) J Virol 81: 12260-12271).
[00144] To produce the library vector, a custom triple transfection of suspension-adapted HEK293 cells (NAVXCell ". REGENXBIO Inc.) was used in which the library crs-plasmid was limited to 100-copies per viable cell (typically -3,000) to reduce the likelihood of variant cross-packaging and capsid mosaicism. Three days post-transfection, cells were harvested, lysed, clarified, PEG-precipitated, and purified using lodixanol-gradient ultracentrifugation (UC). [00145] Vector titer was quantified using digital PCR with poly A primers/probe. Library variant diversity and parental/wildtype fraction following AAV packaging was characterized by next-generation sequencing (NGS) and LC-MS of the VP3 protein (see FIGs. 5A-5F).
[00146] Each library’ was produced at 20L scale, as described above, by the Vector Core group at REGENXBIO Inc. Library diversity and titer measurements determined a high level of diversity and the production lots had final BDS titer ranging from 3.5E13-8.2E13 (Table 1 and FIG. 4). Using deep NGS techniques (NovaSeq™, Illumina) to understand the input variant distribution, NGS testing obtained approximately 300 million reads per library.
Table 1
[00147] NGS analysis of initial vector libraries had high parental vector levels, as indicated for example in an similarly constructed AAV5.VR8 library’ without a stop codon, by the larger representation of parental vector following production compared to the percent parental plasmid in the initial preparation of the library (FIG. 5A). However, by inserting the stop codon into the template plasmid prevented wildtype (parental) vector production from carryover template present in the production culture after library' cloning. The drop-in vector stop codon abundance levels indicate selection against cross-packaging (FIG. 5B). Capsid crosspackaging and chimerism can be an issue in AAV library production. Library cross-packaging refers to the packaging of vector genomes within capsids whereby the sequence of capsid and genome are mismatched. Chimerism occurs when capsids are assembled from VP proteins of multiple different variants. These phenomena occur during AAV library' production due to the entrance of multiple cap gene variants in each cell during triple transfection. To address these issues, we optimized the amount of cA-plasmid during triple transfection and found that at a ratio of 100 cis-plasmids per cell, high production titers could be retained while minimizing cross-packaging. It has been previously described that ~1% of transfected DNA reaches the cell nucleus, therefore it is estimated that we achieve approximately one library variant per cell on average (Nguyen. Tam N.T. et al. 2021 Molecular Therapy: Methods & Clinical Development 21 : 642-655).
[00148] In the AAV5 example, the parental fraction of AAV5 in an AAV5 library following AAV packaging was characterized by LC-MS for detection of the VP3 protein. Following generation of such an AAV5 vector library with no stop codon in the template, the parental VP3 theoretical wildtype mass is 59,551.64 Da and AAV5 VP3 with a peptide insertion is 59,951 to 60,855 Da. FIG. 5A and FIG. 5B illustrate (by LC-MS analysis of VP3 proteins in each version of the library) that the addition of stop codon (FIG. 5B) significantly reduces the packaging of parental vector thereby reducing its overrepresentation in the library. As a validation of selection against cross-packaging using the stop codon method in a different library composed of AAV9 serotype capsids, it was observed that the expected wildtype AAV9 VP3 mass is 59,822 Da, and with the random peptide insertion the expected mass range is 60,239 to 61143 Da. By including a stop codon, little to no wildtype AAV9 VP3 was detected by mass spectrometry (FIG. 5C). NGS analysis of non-mutated sequence in the plasmid and vector library showed a 5-fold increase in non-stop codon sequence between the plasmid and vector, and a 22 -fold reduction (1.4% to 0.066%) in the abundance of the unmutated stop codon sequence between the library plasmid and library vector genomes was achieved (FIG. 5D). If cross-packaging was highly abundant, one would expect a similar level of the stop codon sequence in both library plasmid and vector genomes.
[00149] Having applied the stop codon method to the AAV8 library construction described herein, mass range for the AAV8.456 library VP3 by LC-MS was -60509 Da to -60711 Da and there was no detected signal for wildtype AAV8 VP3 mass species (at around 59800 Da) (FIG. 5E). Mass range for AAV 8.590 library VP3 was -60607 Da to -60868 Da with no detected signal for wildtype AAV8 VP3 mass species (at around 59800 Da) (FIG. 5F).
[00150] LC/MS analysis of vector libraries: The LC/MS analysis of AAV capsid proteins was performed on a Vanquish™ UHPLC (Thermo Fisher Scientific Inc.) coupled with a Q Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.). The separation was performed on a Waters UPLC C4 column using reversed phase chromatography over a 35 minute reversed-phase gradient. Proteins eluting from the column were detected by high resolution mass spectrometer. Biopharma Finder 4.0 software is used for mass deconvolution.
[00151] Differential scanning fluorimetry analysis of vector thermal stability: Vectors were diluted to ~5xl012 GC/mL in IxPBS with 0.001% Poloxamer 188 and loaded into Standard nanoDSF capillaries. The melting temperature was then determined by the Prometheus NT using a starting temperature of 20 °C and ending temperature of 95 °C with ramping temperature set at 1 °C/min.
[00152] Bioinformatic library sequence analysis: A Linux-based bioinformatics pipeline was developed to analyze the sequence composition of AAV libraries before and after in vivo administration (from collected tissues, see below) from raw fastq files generated by the Illumina MiSeq or HiSeq. The pipeline used the cutadapt tool to trim sequences flanking the peptide insertion and discard reads that are too long or missing the flanking sequences. The starcode tool was then used to cluster sequences within a Levenstein distance of 2 to reduce sequencing errors. DNA sequences were then converted to amino acid sequences, identical sequences aggregated and sequences with stop codons discarded. For each peptide sequence the total counts, relative abundance in the sample, and enrichment score was reported (relative abundance in sample divided by relative abundance in the input library).
6.2.Example 2 - Evaluation and selection of capsids from various libraries
[00153] Samples of vector libraries that were made according to the above methods, and listed in Table 2, were prepared for dosing to the suprachoroidal space (SCS) of the eyes of several non-human primates (NHPs). As such, two animals were dosed (left eye for AAV8.VR4) and right eye for AAV8.VR8) at 9.9el l to 2.1el2 GC/eye by SCS injection as indicated in Table
2.
Table 2
[00154] Animal studies complied with all applicable sections of the current version of the Final Rules of the Animal Welfare Act Regulations (9 CFR), and the Guide for the Care and Use of Laboratory Animals. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 8th edition.
[00155] Cynomolgus monkeys (Macaca fascicularis) between 2 and 4 years of age were quarantined for four weeks at the study facility with daily observations. Serum was collected and saved for possible herpes B virus evaluation, two negative intradermal tuberculosis tests were conducted, and fecal flotation for parasites was performed. The animals were housed individually in stainless steel cages. The housing was in compliance with the Guide for the Care and Use of Laboratory Animals, DHHS, (NIH) No. 86-23, and the Animal Welfare Act (9 CFR 3). [00156] Blood was collected immediately prior to dosing, and serum collected and processed. Total antibodies against AAV8 in NHP serum was tested using the Meso Scale Discovery (MSD) electrochemiluminescence immunoassay. Twenty-one (21) days following dosing, animals were euthanized and blood and target tissues (eyes) were collected. Tissues were examined, weighed, and then placed into RNAse/DNase-free cryovials and flash frozen in liquid nitrogen. In this round 1 study, whole retina and RPE were collected from 2 eyes each animal and frozen (later lysed and aliquoted for analysis). Tissue samples were collected using aseptic technique and RNAse-free instruments and workspaces, with care taken to ensure no cross contamination between tissues. Tissues were flash frozen in liquid nitrogen and then maintained on dry ice prior to storage at -70 to -90 °C. DNA and RNA was extracted from all tissues by standard techniques and next generation sequencing (NGS) and quantitative PCR (mRNA transcript expression) was performed, respectively. A custom bioinformatics platform is described above for analyzing cDNA counts and RNA-seq data to determine relative abundance in the samples, and thus enrichment scores. Analysis of the vector repertoire in multiple ocular tissues revealed that many peptides in the AAV8.VR4 library or AAV8.VR8 library7 were detected in one or more of these ocular tissues such as retina and RPE-choroid, e.g. tissues that would provide a desirable depot of vector thus contributing to transgene product expression. The pool of top hits from these libraries was further evaluated in a similar NHP study. For example, in round 2 studies, eyes were collected and tissue punches of 2-15 mg of tissue from retina, RPE-choroid, sclera, cornea, iris-ciliary7 body, trabecular meshwork, and remaining tissues were frozen and later lysed and aliquoted for analysis.
6.3.Example 3 - Evaluation and selection of capsids transducing ocular tissues from AAV8 peptide insertion libraries
[0001] A. AAV8.VR4 Libraries: Following the analy sis of a vector repertoire containing a diverse set of peptides having tropism for one or more ocular tissue samples in the first round of animal testing (Round 1), a large pool from AAV8.VR4 was further analyzed by dosing NHPs at 1.52el3 GC/kg (pooled capsids). The bioinformatics tools were again used to determine vector performance ranking based on readouts such as relative abundance (RA), ES counts, etc. (e.g. using Dunnetfs multiple comparison test p-values) to determine an enrichment score. A representative list from Round 2 of more than 10 hits (peptides) emerged, as in Table 3A for example, based on selection in retina, compared to the parental spiked-in vector control (AAV8). Table 3A: AAV8.VR4 Peptide Inserts
[00157] Following assessment using the bioinformatics tools described, several representative AAV.456 capsids from the Round 2 library' reveal that the relative abundance of variant AAV8 capsids in retina is greatly enriched. >40-fold higher, compared to the parental AAV8 capsid (shown as fold-change compared to control capsid spiked-in to the pool) (FIG. 6A).
[00158] B. AAV8.VR8 Libraries: A large pool from AAV8.VR8 (Round 1 NHP study) was then further analyzed by dosing NHPs at 1.52el3 GC/kg (pooled capsids). The bioinformatics tools were then used analogously to determine vector performance ranking based on readouts such as relative abundance (RA to determine an enrichment score from the Round 2 study, as described hereinabove. A representative list of 10 hits (peptides) emerged, as in Table 3B for example, based on selection in retina compared to the parental spiked-in vector control (AAV8).
Table 3B: AAV8.VR8 Peptide Inserts [00159] Following assessment using the bioinformatics tools described, representative AAV.590 capsids from the Round 2 library' reveal that the relative abundance of variant AAV8 capsids in retina is greatly enriched. >40-fold higher, compared to the parental AAV8 capsid (shown as fold-change compared to control capsid spiked-in to the pool) (FIG. 6B).
[00160] Round 3 Design: Individual vectors with capsids having the insertions listed above were produced with barcoded CAG.TdTomato transgene (Vector Core facility at REGENXBIO Inc., Rockville, MD) at a 0.2 mL (AAV 8) scale. Clarified lysates from these preps were pooled into six batches A-F (along with AAV3B batches) and purified by UC. Purified BDS was then mixed into three pools AB, CE, and DF and formulated into test articles SCS5, SCS6, SCS7 such that each capsid was represented at a high dose in one test article (TA) (e.g. 80%) and a low dose in the second test article (e.g. 20%) and included parental AAV controls. AAV3B libraries (not described herein) comprised the AB and CE batches (SCS5 and SCS6, respectively) and the AAV8 libraries containing capsids of Tables 3A and 3B comprised the DF batch (represented in SCS6 and SCS7).
[00161] Each test article (SCS5, 6 or 7) was dosed in three eyes by SCS administration at a dose of 3E12 GC per eye as described in Table 4.
Table 4: Test article SCS5, SCS6, or SCS7 (pool) compositions na = not applicable
[00162] All study animals were screened for the presence of AAV2, AAV8 and AAV9 neutralizing antibodies in serum by in vitro neutralizing antibody assay (Precision for Medicine) and only animals with titers below a defined threshold were enrolled in the study. Pre-dose blood collections were taken (following ketamine sedation) and placed into serum separator tubes. After a minimum 15 minutes at ambient temperature, blood was centrifuged (2500 x g for 10 minutes) and serum harvested.
[00163] Collected tissues were examined, weighed, and then placed into RNAse/DNase-free cryovials. Generally, tissue punches of 2-15 mg of tissue from retina, RPE-choroid, sclera, cornea, iris-ciliary body, trabecular meshwork, and remaining tissue samples were collected using aseptic technique and RNAse-free instruments and workspaces. Tissues were flash frozen in liquid nitrogen and then maintained on dry ice prior to storage at -70 to -90 °C until analyzed.
[00164] DNA (Kingfisher DNA) and mRNA (Dynabeads mRNA direct) were extracted from the tissues and the vector barcode sequences were amplified by PCR and then detected by NGS on the Illumina Mis eq. Units of “nRAAFI”, normalized relative abundance adjusted for input, were calculated. Essentially, the relative abundance of each barcode was divided by the input relative abundance and then normalized to a sum of one in each sample to identify the nRAAFI for each barcoded transgene.
[00165] Abundance of each vector in the pool measured by NGS in retina and RPE-choroid tissue for each insertion capsid compared to parental capsid is shown in FIG. 7A-B, represented as fold change RA relative to the control parental vector, and allowed assessment of the pool input composition. Notably, each capsid was represented similarly whether in the 80% DF pool and the 20% DF pool.
[00166] Several evolved AAV8 peptide insertion variants from the libraries mediated greater than 100-fold increased RNA expression in retina and RPE compared to parental AAV8 by SCS administration. FIGs. 8A-B show that several AAV8.456 and AAV8.590 peptide insertion capsids mediated about a 100-fold increase in mRNA expression in RPE-choroid (CHR) and retina (RER) in the 80% DF pool. Some of these capsids also mediated up to a 10- fold increase in mRNA expression of transgene in sclera (SCR)(FIG. 8C). These patterns were highly similar for the low dose pool (20% DF)(FIGs. 8D-F). Interestingly, the same evolved AAV8 peptide insertion variants mediated about 10-fold increased vector genome (vg) copies in retina and RPE compared to wild-type AAV8 by SCS administration (data not shown).
[00167] All of the round 3 engineered capsids had an average relative abundance adjusted for input (RAAFI) greater than parental AAV8 in the AAV8 VR-IV library, for example, retina: 12/12 (100%) and RPE-C: 12/12 (100%). Table 5A.
Table 5A
*duplicate capsid with insert in pool (with unique barcode)
[00168] All of the engineered of capsids showed an average abundance (RAAFI) greater than parental AVV8 in the AAV8 VR-VIII library, as such 10/10 (100%) in retina and 10/10 (100%) in RPE-C tissue. Table 5B.
Table 5B
[00169] FIGs. 9A-9B also illustrate the enrichment of SCS-delivered vectors having engineered capsids packaging a fluorescent protein, showing the majority of the tested capsids resulted in greater than 1 OX abundance over parental AAV 8 capsid. [00170] The data from the present NHP study for AAV8.456 and AAV8.590 mRNA distribution in ocular tissues shows that RNA expression of transgene from these variant capsids is greatly enriched and transduction of retina and RPE-choroid tissue over scleral tissue is better than wildtype AAV8 upon SCS administration which may be desirable for certain transgenes.
6.4.Example 4 - Assessment of Selected Capsids in vivo
[00171] AAV capsid sequences modified by peptide insertions with (AAV8.590 library' hits) or without additional substitutions (AAV8.456 library' hits) were further evaluated in in vivo for bio-distribution in test animals using next generation sequencing (NGS) and quantitative PCR.
[00172] Studies were performed where selected vectors from the library were produced by methods described in these Examples, or by standard methods of triple transfection utilizing Rep/cap trans plasmid (containing the engineered capsid gene sequence), cis plasmid encoding a transgene, and helper gene plasmid. The engineered vectors of interest were injected individually into test animals, e.g. NHPs, mice or rats, and DNA and RNA determinations from extracted tissues are compared to the parental vectors. Quantitative PCR (qPCR) can be done on a QuantStudio 5 (Life Technologies, Inc.) or any standard method such as ddPCR, using primer-probe combinations specific for the transgene.
[00173] From the studies where individual vectors are injected into test animals for characterization, formalin fixed eyes may' be, e.g. sectioned at 40pm thickness on a vibrating blade microtome (VT1000S, Leica) and the floating sections probed with antibodies against transgene (or viewed for fluorescence if fluorescent transgene) to look at the cellular distribution of the delivered vectors.
[00174] Recent clinical advancements delivering adeno-associated viral (AAV) vectors into the suprachoroidal space (SCS) have established the feasibility' and value of this in-office route of administration in ocular gene therapy. The identification of engineered capsids optimized for SCS delivery that both efficiently transduce ocular tissue in a targeted fashion and display favorable off-target transduction profiles would enable widespread use of this route of administration.
[00175] Subsequently these variants were evaluated in additional in vivo and in vitro model systems. These results validate the utility of the NAVIGATE platform and its ability to identify AAV variants that significantly outperform parental serotypes and that have the potential to be developed as gene therapy^ treatments in ophthalmology.
A. NHP studies:
[00176] NHP Library Down-selection Summary': As described hereinabove, several high diversity peptide insertion libraries (up to 108 variants per library) were designed and produced, each encoding AAV cap under the control of the CMV promoter with randomized 7-mer insertions at VR-IV or VR-VIII of AAV8. The libraries were delivered intraocularly to nonhuman primates (NHP, cynomolgus macaques). Leveraging a directed evolution platform NAVIGATE (Novel AAV Vector Intelligent Guided Adaptation Through Evolution), 2-4k novel variants overall were identified and enriched in retina and RPE-choroid from each high diversity library, which were then re-packaged and administered to NHP via SCS delivery. Several capsid variants (12 AAV8. VR-IV and 10 AAV 8. VR-VIII) were chosen for final library' down-selection. Each variant (as well as parental control AAV8) was produced individually with genomes encoding a barcoded CAG.tdTomato cassette, pooled, and delivered to NHP, again by SCS delivery7, at a dose of 3el2 GC/eye. Briefly, Cynomolgus monkeys (“NHPs”; n=3 per test article group) were administered AAV8.456 or AAV8.590 capsids carry ing a CAG.TdTomato transgene, 1E12 GC/eye SCS administration per animal. Following 3 weeks, animals were sacrificed and eyes and several peripheral tissues were collected. TdTomato genome (cDNA) and transcripts (mRNA) were detected by digital PCR and plotted against a reference gene. Results show that relative abundance adjusted for input (RAAFI) (DNA and mRNA) was increased in retina and RPE-choroid (RPE-C) for several AAV8.456 or AAV8.590 vectors in the pool compared to parental AAV8 or wtAAV3B vector. FIGs. 10A- B. Four capsids/vector with high abundance are shown in Table 5C (with further results of the individual capsids in Tables 5D-5G).
Table 5C
A Also evaluated in the single vector NHP and mini-pig scFv cohorts
[00177] NHP Single Vector Summary: Briefly. Cynomolgus monkeys (“NHPs”: n=3 x 2 eyes per test article group) were administered 3E12 GC/eye single AAV8.456 or AAV8.590 vectors, namely AAV8 variants 8.1 (AAV8.456.RIQMGTK), 8.2 (AAV8.456.RQKNAMV), and 8.4 (AAV8.590.GRTIRGDLA) (also named PEPIN8.1, PEPIN8.2 and PEPIN8.4) packaging a therapeutic antibody transgene (scFv format) and tissues and ocular biofluids collected after 4 weeks.
[00178] PEPIN8.1, PEPIN8.2 and PEPIN8.4 produce greater than 10 times more protein in ocular tissues (retina/RPE-C) and biofluids (AH/VH) than AAV8 following SCS deliver}7 of scFvOl transgene in NHPs (n=6 eyes at 3E12 GC/eye) (FIGs. 13A-C).
Table 5D
Table 5E
[00179] All variants expressed transgene mRNA in retina more efficiently than parental serotypes as measured by average RNA relative abundance across multiple samples per eye. AAV8.VR-IV/AAV8.VR-VIII insertion variants were up to >30-fold and up to >50-fold improved, respectively, when compared to the parental serotypes. Most AAV8-based insertion variants also expressed >20-fold more transgene mRNA in RPE-choroid compared to AAV8, with the top variant producing in excess of 200-fold more mRNA than AAV8. Numerous variants with advantageous off-target transduction profiles w ere also identified. For example, the majority of AAV8.VR-IV and AAV8.VR-VIII variants that showed a greater than 10-fold increase in retinal transduction yet did not transduce liver, heart, or kidney to a greater degree than AAV 8.
B, Mini -pig study:
[00180] A variant capsid library and a subset of single vectors was assessed via SCS delivery in Yucatan mini -pigs.
[00181] Briefly, Yucatan pigs ('‘mini-pigs’’: n=3 per test article group) were administered as a single AAV8.456 or AAV8.590 capsids carrying an antibody (mAb) transgene, formatted as an scFv, at a low dose 3E11 and high dose 3E12 GC/eye SCS administration per animal. Also, pooled capsids (vectors) were administered to one cohort (2 animals, 2 eyes collected each). Following 4 weeks, animals were sacrificed and eyes and several peripheral tissues were collected (Day 15 and Day 29).
[00182] scFv genome (cDNA) and transcripts (mRNA) were detected by digital PCR and plotted against a reference gene. Aqueous humor was tested for transgene product (TP measured as ng/mL protein). FIG. 11 A.
[00183] Novel AAV8 variants from both libraries outperform AAV8 following single vector SCS delivery’ of scFvOl transgene in mini-pigs in a dose-dependent manner. AAV8 variants achieve TP concentrations up to 344ng/mg in retina, consistent with biodistribution. FIGs. 12A-B. AAV8.3 achieves >153X AAV8 expression levels if transgene product in AAV8 in AH.
Table 5F
Table 5G [00184] Results show that transgene product (TP) in aqueous humor for the AAV8.456.RIQMGTK (8.1), AAV8.456.RQKNAMV (8.2), AAV8.590.GDNTTFRRA (8.3), and AAV8.590.GRTIRGDLA (8.4) vectors were increased relative to wildtype AAV8 vector. AAV8.456.RIQMGTK (8.1) and AAV8.456.RQKNAMV (8.2) vectors exhibited about 50- fold and 150-fold change, respectively, in TP concentration relative to wtAAV8 vector at the high dose at Day 15.
[00185] Findings from the AAV8 insertion variants, were largely translatable from NHP to mini-pigs, with top hits achieving improvement relative to AAV8 of >60-fold and >100-fold transduction in RPE-choroid and retina, respectively. Variants evaluated as single vectors achieved up to 150-fold higher secreted transgene protein expression in the aqueous humor as compared to the AAV8 control. Transduction of iCell RPE cells and an ARPE-AAVR cell line with either the 63-member library (SCS5, 6, 7 pools) or a subset of top hits as single vectors established that most variants exhibit superior transduction efficiency compared to AAV8 with top variants achieving >20-fold improvement in both the library and single vector settings.
6.5. Example 5 -Evaluation of a Consensus Sequence
[00186] Highly similar sequences and motifs were found amongst the peptides that transduced eye tissues efficiently following SCS delivery' of the AAV8.VR4 or AAV8.VR8 capsid libraries.
[00187] A Uniform Manifold Approximation and Projection (UMAP) method was utilized to display and analyze the data for peptide insertion capsids in a ty pe of dimensionality reduction tool. The peptides of Table 3A and 3B and all the input peptides in the libraries that were detected in at least one tissue sample were clustered. For example, combined peptides (> 20000 peptides in total for AAV8.456 and >10000 in total for AAV8.590 detected in at least one sample) from round 2 RPE and retina samples were analyzed in several clusters where no filter was applied to the input counts. In further analysis, the enrichment scores of a peptide detected in both RPE and Retina libraries were averaged, and then the density plot of the resulting merged enrichment scores of all peptides detected in at least 1 sample and in the input library were plotted.
Table 6:
X = any natural amino acid
[00188] Accordingly, the peptide insertion sequence a) has an amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: xviii)Xi and X2 are each any amino acid; X? is R or K or Q or H; X4 is any amino acid; Xs is K or Q or S or T; Xs is S or T or Q or A or I or V; and X?is V or Q or T or P or S (SEQ ID NO: 115); xix) Xi and X2 are each any amino acid; X3 is R or K or Q; X4 is V or K; Xs is K or Q or S or T; Xs is S or T or Q or A or I or V; and X71S V or Q or T or P or S (SEQ ID NO: 116); xx) Xi is any amino acid; X2 is R or K or F or P or N; X3 and X4 are each any amino acid; Xs is R or P or Q; Xs and X7 are each any amino acid (SEQ ID NO: 117); xxi) Xi is any amino acid; X2 is R or K or F or P or N; X3 is and X4 are each any amino acid; X5 is R or Q or P; X6 is S or Q or N; X7 is S or P or A (SEQ ID NO: 1 18); xxii) Xi, X2, and X3 are each any amino acid, and X4 is R or K or M or A; Xs is any amino acid; Xs is P or T or Q; and X7 is any amino acid (SEQ ID NO: 119); xxiii) Xi is K or Q; X2,and X3 are each any amino acid; X4 is R or K or M or A; Xs is any amino acid; Xs is P or T or Q; X7 is any amino acid; (SEQ ID NO: 120); xxiv) Xi. Xzand X3 are each any amino acid; X4 is S or Q or T or G; X5 and Xe are each any amino acid; X7 is K or R or Q or T or S (SEQ ID NO: 121); xxv) Xi is R; X2,is K; X3 is any amino acid; X4 is S or Q or T or G; X5 and Xe are each any amino acid; X7 is K or R or Q or T or S (SEQ ID NO: 122): xxvi) Xi is A or G or D; X2 is V or K or A; X3 is R or Q or V; X4 is K or R or S or H; X5 is K or A or S or P or R or P; Xe is any amino acid; X7 is K or T or Q or V (SEQ ID NO: 124); xxvii) Xi and X2 are each any amino acid; X3 is R or Q or T or D or V; X4, X5 and Xe are each any amino acid; X7 is R or Q or S or N or K (SEQ ID NO: 125); xxviii) Xi is K or S or V or Q or N; X2 is G or T or K or P or N; X3 is T or S or K or A; X4 is K or R or Q or G; X5 is R or S or K or G; Xe and X7 are each any amino acid (SEQ ID NO: 126); xxix) Xi is any amino acid; X2 is R or V or Q or D or A; X3, X4, and Xs are each any amino acid; Xe is K or T or A or V; X7 is any amino acid (SEQ ID NO: 127); xxx) Xi is any amino acid; X2 is R or V or Q or D or A; X3 is R or G or A; X4, and Xs are each any amino acid; Xe is K or T or A or V; X7 is any amino acid (SEQ ID NO: 128); xxxi) Xi is any amino acid; Xi is R or V or Q or D or A; X3 is R or G or A; X4 is T or D; X5 is any amino acid; Xe is K or T or A or V; X7 is any amino acid (SEQ ID NO: 129); xxxii) Xi is any amino acid; Xi is R or V or Q or D or A; X3 is R or G or A; X4 is T or D; Xs is any amino acid; Xe is K or T or A or V; Xi is S or V (SEQ ID NO: 130); xxxiii) Xi,Xe. X3, X4, and X5 are each any amino acid; Xe is R or K or Q; Xi is any amino acid (SEQ ID NO: 131); or b) the peptide insertion sequence has an amino acid sequence of X1-X2-X3-X4-X5-X6- X7-X8-X9, wherein: xxxiv) Xi is G; Xi. Xi X4, Xs, Xe are each any amino acid; Xs is R or K; Xs is any amino acid; X9 is A (SEQ ID NO: 123).
6.6. Example 6 -Assessment of Manufacturing Suitability of Modified Capsids
[00189] During the library' screening phase, various high-throughput techniques were applied for material generation, such as transfecting with enhanced plasmid ratio or purifying mixed AAV variants using ultracentrifugation technology, which resulted in production of -300 runs and >10000 peptide variants. The AAV library material produced went through multiple rounds of selection and the biological activity data generated showed differences in folds of improvement for the novel AAV8 capsids over the AAV8 parental control. Subsequently, a panel of variants were selected at 0.25L production scale for preliminary manufacturability assessment.
[00190] Manufacturability assessment is crucial in helping to identify the suitability of a therapeutic molecule, to evaluate its fitness to a platform, and to reduce the process development effort due to unexpected results. For the initial manufacturability screening study, we used an in-house developed high throughput chromatography technology (REGENXBIO Inc.) that can simultaneously purify 12 runs, at both the capturing and polishing chromatography steps. Process differences between these novel SCS capsids were observed. While the harvest titers, analyzed by digital droplet PCR (ddPCR). were all acceptable, there is a ~2.5 fold difference between the lowest and highest harvest titers for this panel of tested variants. Several AAV8 variants, particularly 8.1, 8.2, 8.3 and 8.4 resulted in harvest yields (titer) mostly comparable to \\1AAV8. FIG. 15. The purification steps also demonstrated differences in the step yields and elution patterns for certain AAV8 variants. The polishing steps showed that the AAV8 mutants all eluted earlier than, for example, AAV3B mutants, and when compared to AAV8 and AAV3B controls. Several novel SCS capsids were selected to produce at 10L scale for a more complete manufacturability assessment, and the process profiles matched that of the smaller scale high throughput screening. The final produced material all had similar product qualities based on capsid purity, genome integrity, and percentage of full capsids present in solution, but thermostability testing showed major differences, up to ~30°C, in melting temperatures. This information in combination with earlier biological activity data helped to determine the desirable capsids for further NHP studies.
[00191] In summary, the use of a high throughput toolbox ensured optimal production efficiency to provide material for NHP studies, while swiftly discovering the potential process and product differences to better understand manufacturability for these capsid variants. The manufacturing profile has also aided in selecting the novel SCS capsids that have optimal biological activity profiles and also possess the desirable manufacturability attributes, thus saving time, cost, and effort related to potential product redesigning. 6.7. Example 7- Assessment of Selected Capsids in vitro
[00178] Transduction efficiency of several capsids was also assessed in iPSC-derived human RPE cells as well as human immortalized transgenic ARPE-AAVR cells. Briefly, iPSC-derived RPE were transduced with variant AAV8.scFV01 vectors (carrying an antibody transgene formatted to express as a scFv protein). iRPE cells were treated at an MOI of 3e5 vg/cell. Transgene product (TP) level in apical and basal compartments was measured. ARPE cells stable expressing AAV receptor (AAVR) were cultured in 96 well plates for 4 weeks prior to AAV transduction. Cells were also transduced with variant AAV8.scFv01 vectors at an MOI of 3e5 vg/cell, transgene product collected and quantitated.
[00179] It was determined that the majority of variants outperform AAV8 in vitro, achieving secreted transgene product levels approaching or exceeding those of AAV3B, which is traditionally highly potent in vitro. AAV8 variants also performed slightly better than the AAV3B variants (AAV3B.3.1 or AAV3B3.2 variants) in iPSC cells (FIG. 14A). Transduction efficiency of AAV8 variants in ARPE cells was comparable or better than AAV8 and AAV3B (FIG. 14B).
6.8.Conclusions
[00192] AAV capsid modifications performed by random peptide insertions in surface exposed loop of VR-IV were able to produce sufficient library titers in the production system described herein for analysis of transduction properties in NHP tissues. The method provided for reducing carryover parental plasmid following cloning of the random insertions and was shown to reduce overrepresentation of the parental vector in the library’ biodistribution and transduction studies.
[00193] Suprachoroidal administration of AAV8.VR4 and AAV8.VR8 modified capsids to NHPs resulted in a number of higher relative abundance engineered vectors in retina and RPE- choroid suitable for use as a gene therapy vector carry ing a gene of interest to treat ocular disorders.
Table 7. Capsid Amino Acid Sequences
7. Equivalents
[00194] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
[00195] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference in their entireties. [00196] The discussion herein provides a beter understanding of the nature of the problems confronting the art and should not be construed in any way as an admission as to prior art nor should the citation of any reference herein be construed as an admission that such reference constitutes “prior art” to the instant application.
[00197] All references including patent applications and publications cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

We claim:
1. A recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insertion of at least 4 and up to 9 contiguous amino acids, wherein the peptide insertion is immediately after an amino acid residue corresponding to one of amino acids 451 to 461 or amino acids 585-593 of a parental capsid protein, wherein the parental capsid protein is an AAV8 capsid protein having an amino acid sequence of SEQ ID NO: 33 or a capsid protein that has 90%, 95%, or 99% sequence identity thereto, wherein said peptide insertion has an amino acid sequence of one of SEQ ID NOs: 1-32, and wherein an rAAV vector comprising the capsid protein comprising the peptide insertion has enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein.
2. A recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insertion of at least 4 and up to 9 contiguous amino acids, wherein the peptide insertion is immediately after an amino acid residue corresponding to one of amino acids 451 to 461 or amino acids 585-593 of a parental capsid protein, wherein the parental capsid protein is an AAV8 capsid protein having an amino acid sequence of SEQ ID NO: 33 or a capsid protein that has 90%, 95%, or 99% sequence identity thereto, wherein said peptide insertion has an amino acid sequence of one of SEQ ID NOs: 115-131, and wherein an rAAV vector comprising the capsid protein comprising the peptide insertion has enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein.
3. The rAAV capsid protein of claim 1 or 2, wherein said parental capsid protein is serotype
8 having 496NNN/AAA498 substitutions (AAV8.AAA) and has an amino acid sequence of SEQ ID NO: 114.
4. The rAAV capsid protein of claim 3, wherein said peptide insertion occurs immediately after one of ammo acids Q451, T452. T453, G454. G455, T456. A457. N458, T459. Q460, or T461 of the parental capsid.
5. The rAAV capsid protein of claim 4. wherein said peptide insertion occurs immediately after amino acid G455 of the parental capsid.
6. The rAAV capsid protein of any one of claims 1-5 wherein the peptide insertion is 7 to
9 amino acids of one of the amino acid sequences of SEQ ID NOs: 1-12 or 115-122.
7. The rAAV capsid protein of claim 6 which has an amino acid sequence of one of SEQ
ID NOs: 50 to 61.
8. The rAAV capsid protein of claim 1 or 2, wherein the peptide insertion occurs immediately after one of amino acids 585 to 593 of the parental capsid.
9. The rAAV capsid protein of claim 8, wherein the peptide insertion occurs immediately after amino acid Q589 of the parental capsid.
10. The rAAV capsid protein of claims 8 or 9 wherein the peptide insertion is 7 to 9 amino acids of one of the amino acid sequences of SEQ ID NOs: 13-32 or 123-131.
11. The rAAV capsid protein of claim 10 which has an amino acid sequence of one of SEQ
ID NOs: 62 to 113.
12. The rAAV capsid protein of any one of the preceding claims which has enhanced tropism for retina or RPE choroid tissue relative to the parental capsid protein.
13. The rAAV capsid protein of claim 12, wherein an rAAV vector comprising the capsid protein exhibits at least about 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 25 fold or 100 fold greater transduction of retina and/or RPE choroid tissue than an rAAV vector comprising the parental capsid protein.
14. The rAAV capsid protein of any one of the preceding claims wherein, upon administration to an eye, an rAAV vector comprising the capsid protein has reduced transduction of peripheral tissues than an rAAV vector comprising the parental capsid protein.
15. The rAAV capsid protein of claim 14 wherein, upon administration to an eye, the rAAV vector comprising the capsid protein has an at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 25 fold or 40 fold lower transduction of liver than an rAAV vector comprising the parental capsid protein.
16. A nucleic acid comprising a nucleotide sequence encoding the rAAV capsid protein of any one of the preceding claims, or encoding an amino acid sequence having at least 80% identity therewith, wherein an rAAV vector comprising the rAAV capsid protein of any of the preceding claims retains enhanced tropism to ocular tissue compared to an rAAV vector comprising the parental capsid protein.
17. The nucleic acid of claim 16 which encodes the rAAV capsid protein of any one of claims 1-15.
18. A packaging cell capable of expressing the nucleic acid of claim 16 or claim 17 to produce AAV vectors comprising the capsid protein encoded by said nucleotide sequence.
19. A rAAV vector comprising the rAAV capsid protein of any one of claims 1-15.
20. The rAAV vector of claim 18, further comprising a rAAV genome comprising a transgene flanked by AAV ITR sequences, wherein the transgene encodes a therapeutic for an ocular disease.
21. A pharmaceutical composition comprising the rAAV vector of claim 19 or 20 and a pharmaceutically acceptable carrier.
22. The pharmaceutical composition of claim 21, wherein said composition is formulated for administration to the suprachoroidal space.
23. The pharmaceutical composition of claim 21, wherein said composition is formulated for administration to the suprachoroidal space with a microneedle.
24. A method of delivering a transgene to a cell, said method comprising contacting said cell with the rAAV vector of claim 19 or 20.
25. A method of delivering a transgene to ocular tissue of a subject in need thereof, said method comprising administering to said subject the rAAV vector of claim 19 or 20.
26. The method according to claim 24, wherein said rAAV vector is administered to the suprachoroidal space in the eye.
27. The method according to any of claims 23 to 25. wherein said target tissue is retinal or
RPE choroidal tissue.
28. A pharmaceutical composition for use in delivering a transgene to a cell in a subject in need thereof, wherein the pharmaceutical composition comprises the rAAV vector of claim 19 or 20.
29. The pharmaceutical composition for use in delivering a transgene to ocular tissue of a subject in need thereof, wherein the pharmaceutical composition comprises the rAAV vector of claim 19 or 20.
30. The pharmaceutical composition for use according to claim 28 or claim 29, wherein said rAAV vector is administered to the suprachoroidal space in the eye.
31. The pharmaceutical composition for use according to claim 30, wherein said rAAV vector is administered by a microneedle device or microinj ector device.
32. The pharmaceutical composition for use according to any one of claims 28-31, wherein said target tissue is retinal or RPE choroidal tissue.
33. A pharmaceutical composition for use in treating Age-Related Macular Degeneration
(AMD) in a subject or a method for treating AMD in a human subject in need thereof comprising administering a therapeutically effective amount of recombinant adeno- associated virus (rAAV) vector comprising the rAAV capsid protein of any one of claims 1 to 15.
34. The pharmaceutical composition or method for treating according to claim 33, wherein the AMD is dry AMD with geographic atrophy.
35. The pharmaceutical composition or method for treating according to claim 33 or 34, wherein the rAAV is administered suprachoroi dally.
6. The pharmaceutical composition or method for treating according to any one of claims
33 to 35, wherein the rAAV is administered by a microneedle device or microinjector device.
PCT/US2025/023741 2024-04-08 2025-04-08 Recombinant adeno-associated viruses and uses thereof Pending WO2025217214A2 (en)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202463631398P 2024-04-08 2024-04-08
US63/631,398 2024-04-08
US202463642635P 2024-05-03 2024-05-03
US63/642,635 2024-05-03
US202463703161P 2024-10-03 2024-10-03
US63/703,161 2024-10-03

Publications (2)

Publication Number Publication Date
WO2025217214A2 true WO2025217214A2 (en) 2025-10-16
WO2025217214A3 WO2025217214A3 (en) 2025-12-04

Family

ID=95656388

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/023741 Pending WO2025217214A2 (en) 2024-04-08 2025-04-08 Recombinant adeno-associated viruses and uses thereof

Country Status (1)

Country Link
WO (1) WO2025217214A2 (en)

Citations (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4526938A (en) 1982-04-22 1985-07-02 Imperial Chemical Industries Plc Continuous release formulations
WO1991005548A1 (en) 1989-10-10 1991-05-02 Pitman-Moore, Inc. Sustained release composition for macromolecular proteins
US5128326A (en) 1984-12-06 1992-07-07 Biomatrix, Inc. Drug delivery systems based on hyaluronans derivatives thereof and their salts and methods of producing same
WO1996020698A2 (en) 1995-01-05 1996-07-11 The Board Of Regents Acting For And On Behalf Of The University Of Michigan Surface-modified nanoparticles and method of making and using same
US5679377A (en) 1989-11-06 1997-10-21 Alkermes Controlled Therapeutics, Inc. Protein microspheres and methods of using them
WO1999015154A1 (en) 1997-09-24 1999-04-01 Alkermes Controlled Therapeutics, Inc. Methods for fabricating polymer-based controlled release preparations
WO1999020253A1 (en) 1997-10-23 1999-04-29 Bioglan Therapeutics Ab Encapsulation method
US5912015A (en) 1992-03-12 1999-06-15 Alkermes Controlled Therapeutics, Inc. Modulated release from biocompatible polymers
US5916597A (en) 1995-08-31 1999-06-29 Alkermes Controlled Therapeutics, Inc. Composition and method using solid-phase particles for sustained in vivo release of a biologically active agent
WO2003042397A2 (en) 2001-11-13 2003-05-22 The Trustees Of The University Of Pennsylvania A method of detecting and/or identifying adeno-associated virus (aav) sequences and isolating novel sequences identified thereby
WO2003052051A2 (en) 2001-12-17 2003-06-26 The Trustees Of The University Of Pennsylvania Adeno-associated virus (aav) serotype 8 sequences
US6596535B1 (en) 1999-08-09 2003-07-22 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for the preparation and use
WO2005033321A2 (en) 2003-09-30 2005-04-14 The Trustees Of The University Of Pennsylvania Adeno-associated virus (aav) clades, sequences, vectors containing same, and uses therefor
WO2006068888A1 (en) 2004-12-22 2006-06-29 Raytheon Company System and technique for calibrating radar arrays
WO2006110689A2 (en) 2005-04-07 2006-10-19 The Trustees Of The University Of Pennsylvania Method of increasing the function of an aav vector
US7456683B2 (en) 2005-06-09 2008-11-25 Panasonic Corporation Amplitude error compensating device and quadrature skew error compensating device
WO2009104964A1 (en) 2008-02-19 2009-08-27 Amsterdam Molecular Therapeutics B.V. Optimisation of expression of parvoviral rep and cap proteins in insect cells
WO2010127097A1 (en) 2009-04-30 2010-11-04 The Trustees Of The University Of Pennsylvania Compositions for targeting conducting airway cells comprising adeno-associated virus constructs
WO2011139713A2 (en) 2010-04-26 2011-11-10 Emory University Methods and devices for drug delivery to ocular tissue using microneedle
US20130224836A1 (en) 2010-10-27 2013-08-29 Jichi Medical University Adeno-Associated Virus Virion for Gene Transfer to Nervous System Cells
US8628966B2 (en) 2010-04-30 2014-01-14 City Of Hope CD34-derived recombinant adeno-associated vectors for stem cell transduction and systemic therapeutic gene transfer
WO2014036009A1 (en) 2012-08-27 2014-03-06 Clearside Biomedical, Inc. Apparatus and methods for drug delivery using microneedles
WO2014074823A1 (en) 2012-11-08 2014-05-15 Clearside Biomedical, Inc. Methods and devices for the treatment of ocular diseases in human subjects
US8734809B2 (en) 2009-05-28 2014-05-27 University Of Massachusetts AAV's and uses thereof
WO2014172669A1 (en) 2013-04-20 2014-10-23 Research Institute At Nationwide Children's Hospital Recombinant adeno-associated virus delivery of exon 2-targeted u7snrna polynucleotide constructs
WO2014179698A2 (en) 2013-05-03 2014-11-06 Clearside Biomedical, Inc. Apparatus and methods for ocular injection
US8927514B2 (en) 2010-04-30 2015-01-06 City Of Hope Recombinant adeno-associated vectors for targeted treatment
US20150023924A1 (en) 2013-07-22 2015-01-22 The Children's Hospital Of Philadelphia Variant aav and compositions, methods and uses for gene transfer to cells, organs and tissues
US20150126588A1 (en) 2012-05-09 2015-05-07 Oregon Health & Science University Adeno associated virus plasmids and vectors
WO2015126694A1 (en) 2014-02-12 2015-08-27 Ethicon Endo-Surgery, Inc. Method and apparatus for suprachoroidal administration of therapeutic agent
US9169299B2 (en) 2011-08-24 2015-10-27 The Board Of Trustees Of The Leleand Stanford Junior University AAV capsid proteins for nucleic acid transfer
US9193956B2 (en) 2011-04-22 2015-11-24 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
WO2015191508A1 (en) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Chimeric capsids
US20150374803A1 (en) 2013-03-13 2015-12-31 The Children's Hospital Of Philadelphia Adeno-associated virus vectors and methods of use thereof
WO2016040635A1 (en) 2014-09-11 2016-03-17 Ethicon Endo-Surgery, Inc. Motorized suprachoroidal injection of therapeutic agent
WO2016044404A2 (en) 2014-09-18 2016-03-24 Ethicon Endo-Surgery, Inc. Therapeutic agent delivery device
WO2016049230A1 (en) 2014-09-24 2016-03-31 City Of Hope Adeno-associated virus vector variants for high efficiency genome editing and methods thereof
US20160215024A1 (en) 2013-10-11 2016-07-28 Massachusetts Eye & Ear Infirmary Methods of Predicting Ancestral Virus Sequences and Uses Thereof
US9409953B2 (en) 2011-02-10 2016-08-09 The University Of North Carolina At Chapel Hill Viral vectors with modified transduction profiles and methods of making and using the same
US9585971B2 (en) 2013-09-13 2017-03-07 California Institute Of Technology Recombinant AAV capsid protein
US20170067908A1 (en) 2014-04-25 2017-03-09 Oregon Health & Science University Methods of viral neutralizing antibody epitope mapping
WO2017070491A1 (en) 2015-10-23 2017-04-27 Applied Genetic Technologies Corporation Ophthalmic formulations
WO2017156227A1 (en) 2016-03-09 2017-09-14 Janssen Biotech, Inc. Apparatus for subretinal administration of therapeutic agent via a curved needle
WO2017218613A2 (en) 2016-06-17 2017-12-21 Janssen Biotech, Inc. Guide apparatus for tangential entry into suprachoroidal space
US9923120B2 (en) 2015-09-26 2018-03-20 Nichia Corporation Semiconductor light emitting element and method of producing the same
WO2018075798A1 (en) 2016-10-19 2018-04-26 Adverum Biotechnologies, Inc. Modified aav capsids and uses thereof
WO2019053466A1 (en) 2017-09-15 2019-03-21 Oxular Limited Ophthalmic drug compositions
WO2019202603A1 (en) 2018-04-19 2019-10-24 Everads Therapy Ltd Device for injecting a substance into an interlayer of a body tissue or organ
WO2020206189A1 (en) 2019-04-04 2020-10-08 Regenxbio Inc. Recombinant adeno-associated viruses and uses thereof
WO2021188803A1 (en) 2020-03-19 2021-09-23 Clearside Biomedical, Inc. Compositions and methods for treating ocular disorders
US11273072B2 (en) 2017-01-13 2022-03-15 Gyroscope Therapeutics Limited Suprachoroidal injection device

Patent Citations (67)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4526938A (en) 1982-04-22 1985-07-02 Imperial Chemical Industries Plc Continuous release formulations
US5128326A (en) 1984-12-06 1992-07-07 Biomatrix, Inc. Drug delivery systems based on hyaluronans derivatives thereof and their salts and methods of producing same
WO1991005548A1 (en) 1989-10-10 1991-05-02 Pitman-Moore, Inc. Sustained release composition for macromolecular proteins
US5679377A (en) 1989-11-06 1997-10-21 Alkermes Controlled Therapeutics, Inc. Protein microspheres and methods of using them
US5912015A (en) 1992-03-12 1999-06-15 Alkermes Controlled Therapeutics, Inc. Modulated release from biocompatible polymers
WO1996020698A2 (en) 1995-01-05 1996-07-11 The Board Of Regents Acting For And On Behalf Of The University Of Michigan Surface-modified nanoparticles and method of making and using same
US5916597A (en) 1995-08-31 1999-06-29 Alkermes Controlled Therapeutics, Inc. Composition and method using solid-phase particles for sustained in vivo release of a biologically active agent
US5989463A (en) 1997-09-24 1999-11-23 Alkermes Controlled Therapeutics, Inc. Methods for fabricating polymer-based controlled release devices
WO1999015154A1 (en) 1997-09-24 1999-04-01 Alkermes Controlled Therapeutics, Inc. Methods for fabricating polymer-based controlled release preparations
WO1999020253A1 (en) 1997-10-23 1999-04-29 Bioglan Therapeutics Ab Encapsulation method
US7125717B2 (en) 1999-08-09 2006-10-24 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for their preparation and use
US6596535B1 (en) 1999-08-09 2003-07-22 Targeted Genetics Corporation Metabolically activated recombinant viral vectors and methods for the preparation and use
WO2003042397A2 (en) 2001-11-13 2003-05-22 The Trustees Of The University Of Pennsylvania A method of detecting and/or identifying adeno-associated virus (aav) sequences and isolating novel sequences identified thereby
US8524446B2 (en) 2001-11-13 2013-09-03 The Trustees Of The University Of Pennsylvania Method for detecting adeno-associated virus
US7790449B2 (en) 2001-12-17 2010-09-07 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing the same, and uses therefor
US8318480B2 (en) 2001-12-17 2012-11-27 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
US8962332B2 (en) 2001-12-17 2015-02-24 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
US7282199B2 (en) 2001-12-17 2007-10-16 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) serotype 8 sequences, vectors containing same, and uses therefor
WO2003052051A2 (en) 2001-12-17 2003-06-26 The Trustees Of The University Of Pennsylvania Adeno-associated virus (aav) serotype 8 sequences
US7906111B2 (en) 2003-09-30 2011-03-15 The Trustees Of The University Of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses therefor
WO2005033321A2 (en) 2003-09-30 2005-04-14 The Trustees Of The University Of Pennsylvania Adeno-associated virus (aav) clades, sequences, vectors containing same, and uses therefor
WO2006068888A1 (en) 2004-12-22 2006-06-29 Raytheon Company System and technique for calibrating radar arrays
WO2006110689A2 (en) 2005-04-07 2006-10-19 The Trustees Of The University Of Pennsylvania Method of increasing the function of an aav vector
US8999678B2 (en) 2005-04-07 2015-04-07 The Trustees Of The University Of Pennsylvania Method of increasing the function of an AAV vector
US7456683B2 (en) 2005-06-09 2008-11-25 Panasonic Corporation Amplitude error compensating device and quadrature skew error compensating device
WO2009104964A1 (en) 2008-02-19 2009-08-27 Amsterdam Molecular Therapeutics B.V. Optimisation of expression of parvoviral rep and cap proteins in insect cells
WO2010127097A1 (en) 2009-04-30 2010-11-04 The Trustees Of The University Of Pennsylvania Compositions for targeting conducting airway cells comprising adeno-associated virus constructs
US9284357B2 (en) 2009-05-28 2016-03-15 University Of Massachusetts AAV's and uses thereof
US8734809B2 (en) 2009-05-28 2014-05-27 University Of Massachusetts AAV's and uses thereof
WO2011139713A2 (en) 2010-04-26 2011-11-10 Emory University Methods and devices for drug delivery to ocular tissue using microneedle
US8927514B2 (en) 2010-04-30 2015-01-06 City Of Hope Recombinant adeno-associated vectors for targeted treatment
US8628966B2 (en) 2010-04-30 2014-01-14 City Of Hope CD34-derived recombinant adeno-associated vectors for stem cell transduction and systemic therapeutic gene transfer
US20130224836A1 (en) 2010-10-27 2013-08-29 Jichi Medical University Adeno-Associated Virus Virion for Gene Transfer to Nervous System Cells
US9409953B2 (en) 2011-02-10 2016-08-09 The University Of North Carolina At Chapel Hill Viral vectors with modified transduction profiles and methods of making and using the same
US9587282B2 (en) 2011-04-22 2017-03-07 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
US20160376323A1 (en) 2011-04-22 2016-12-29 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
US9458517B2 (en) 2011-04-22 2016-10-04 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
US9193956B2 (en) 2011-04-22 2015-11-24 The Regents Of The University Of California Adeno-associated virus virions with variant capsid and methods of use thereof
US9169299B2 (en) 2011-08-24 2015-10-27 The Board Of Trustees Of The Leleand Stanford Junior University AAV capsid proteins for nucleic acid transfer
US20150126588A1 (en) 2012-05-09 2015-05-07 Oregon Health & Science University Adeno associated virus plasmids and vectors
WO2014036009A1 (en) 2012-08-27 2014-03-06 Clearside Biomedical, Inc. Apparatus and methods for drug delivery using microneedles
WO2014074823A1 (en) 2012-11-08 2014-05-15 Clearside Biomedical, Inc. Methods and devices for the treatment of ocular diseases in human subjects
US20150374803A1 (en) 2013-03-13 2015-12-31 The Children's Hospital Of Philadelphia Adeno-associated virus vectors and methods of use thereof
WO2014172669A1 (en) 2013-04-20 2014-10-23 Research Institute At Nationwide Children's Hospital Recombinant adeno-associated virus delivery of exon 2-targeted u7snrna polynucleotide constructs
WO2014179698A2 (en) 2013-05-03 2014-11-06 Clearside Biomedical, Inc. Apparatus and methods for ocular injection
US20150023924A1 (en) 2013-07-22 2015-01-22 The Children's Hospital Of Philadelphia Variant aav and compositions, methods and uses for gene transfer to cells, organs and tissues
US9840719B2 (en) 2013-07-22 2017-12-12 The Children's Hospital Of Philadelphia Variant AAV and compositions, methods and uses for gene transfer to cells, organs and tissues
WO2015013313A2 (en) 2013-07-22 2015-01-29 The Children's Hospital Of Philadelphia Variant aav and compositions, methods and uses for gene transfer to cells, organs and tissues
US9585971B2 (en) 2013-09-13 2017-03-07 California Institute Of Technology Recombinant AAV capsid protein
US20160215024A1 (en) 2013-10-11 2016-07-28 Massachusetts Eye & Ear Infirmary Methods of Predicting Ancestral Virus Sequences and Uses Thereof
US20170051257A1 (en) 2013-10-11 2017-02-23 Massachusetts Eye And Ear Infirmary Methods of predicting ancestral virus sequences and uses thereof
WO2015126694A1 (en) 2014-02-12 2015-08-27 Ethicon Endo-Surgery, Inc. Method and apparatus for suprachoroidal administration of therapeutic agent
US20170067908A1 (en) 2014-04-25 2017-03-09 Oregon Health & Science University Methods of viral neutralizing antibody epitope mapping
WO2015191508A1 (en) 2014-06-09 2015-12-17 Voyager Therapeutics, Inc. Chimeric capsids
WO2016040635A1 (en) 2014-09-11 2016-03-17 Ethicon Endo-Surgery, Inc. Motorized suprachoroidal injection of therapeutic agent
WO2016044404A2 (en) 2014-09-18 2016-03-24 Ethicon Endo-Surgery, Inc. Therapeutic agent delivery device
WO2016049230A1 (en) 2014-09-24 2016-03-31 City Of Hope Adeno-associated virus vector variants for high efficiency genome editing and methods thereof
US9923120B2 (en) 2015-09-26 2018-03-20 Nichia Corporation Semiconductor light emitting element and method of producing the same
WO2017070491A1 (en) 2015-10-23 2017-04-27 Applied Genetic Technologies Corporation Ophthalmic formulations
WO2017156227A1 (en) 2016-03-09 2017-09-14 Janssen Biotech, Inc. Apparatus for subretinal administration of therapeutic agent via a curved needle
WO2017218613A2 (en) 2016-06-17 2017-12-21 Janssen Biotech, Inc. Guide apparatus for tangential entry into suprachoroidal space
WO2018075798A1 (en) 2016-10-19 2018-04-26 Adverum Biotechnologies, Inc. Modified aav capsids and uses thereof
US11273072B2 (en) 2017-01-13 2022-03-15 Gyroscope Therapeutics Limited Suprachoroidal injection device
WO2019053466A1 (en) 2017-09-15 2019-03-21 Oxular Limited Ophthalmic drug compositions
WO2019202603A1 (en) 2018-04-19 2019-10-24 Everads Therapy Ltd Device for injecting a substance into an interlayer of a body tissue or organ
WO2020206189A1 (en) 2019-04-04 2020-10-08 Regenxbio Inc. Recombinant adeno-associated viruses and uses thereof
WO2021188803A1 (en) 2020-03-19 2021-09-23 Clearside Biomedical, Inc. Compositions and methods for treating ocular disorders

Non-Patent Citations (39)

* Cited by examiner, † Cited by third party
Title
"Institute of Laboratory Animal Resources, Commission on Life Sciences", NATIONAL RESEARCH COUNCIL, article "Care and Use of Laboratory Animals"
"Medical Applications of Controlled Release", 1974, CRC PRES
"the Physician 's Desk Reference", 2002
ALBA ET AL.: "Gutless adenovirus: last generation adenovirus for gene therapy", GENE THERAPY, vol. 12, 2005, pages 18 - 27
APONTE-UBILLUS ET AL., APPL. MICROBIOL. BIOTECHNOL, vol. 102, 2018, pages 1045 - 1054
AURICCHIO ET AL., HUM. MOLEC. GENET, vol. 10, 2001, pages 3075 - 3081
BUCHWALD ET AL., SURGERY, vol. 88, 1980, pages 507
CHIANG B ET AL.: "The suprachoroidal space as a route of administration to the posterior segment of the eye", ADV DRUG DELIV REV., vol. 126, 2018, pages 58 - 66, XP093102157, DOI: 10.1016/j.addr.2018.03.001
CLEEK ET AL.: "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application", PRO. INTL. SYRNP. CONTROL. REL. BIOACT. MATER, vol. 24, 1997, pages 853 854
DUAN ET AL., J. VIROL, vol. 75, 2001, pages 7662 - 7671
DURING ET AL., ANN. NETTROL, vol. 25, 1989, pages 351
EWING, TM ET AL., RETINAL PHYSICIAN, vol. 19, 2022, pages 24 - 26
GEORGIADIS ET AL., GENE THERAPY, 2018, pages 450
GEORGIADIS ET AL., GENE THERAPY, vol. 23, 2016, pages 857 - 862
GILES ET AL., MAPPING AN ADENO-ASSOCIATED VIRUS 9-SPECIFIC NEUTRALIZING EPITOPE TO DEVELOP NEXT-GENERATION GENE DELIVERY VECTORS, 2018
GOODSON, MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, 1984, pages 115 138
HALBERT ET AL., J. VIROL, vol. 74, 2000, pages 1524 - 1532
HOWARD ET AL., J. NETTI-O,YTTRG, vol. 7 1, 1989, pages 105
ISSA ET AL., PLOS ONE, vol. 8, no. 4, 2013, pages 60361
LAM ET AL.: "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery", PROC. INT'L. SYMP. CONTROL REL. BIOACT. MATER, vol. 24, 1997, pages 759 760
LANGER, SCIENCE, vol. 249, 1990, pages 1527 1533
LEVY ET AL., SCIENCE, vol. 228, 1985, pages 190
MCCARTY ET AL., GENE THERAPY, vol. 8, no. 16, 2001, pages 1248 - 1254
MOISSEIEV E ET AL.: "The suprachoroidal space: from potential space to a space with potential", CLIN OPHTHALMOL, vol. 10, 2016, pages 173 - 178, XP055803016, DOI: 10.2147/OPTH.S89784
N. ENGL. J. MED, vol. 321, 1989, pages 574
NAM, H.-J ET AL., J VIROL, vol. 81, 2007, pages 12260 - 12271
NGUYEN, TAM N.T ET AL., MOLECULAR THERAPY: METHODS & CLINICAL DEVELOPMENT, vol. 21, 2021, pages 642 - 655
NING ET AL.: "Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel", RADIOTHERAPY & ONCOLOGY, vol. 39, 1996, pages 179 189
POWELLRIVERA-SOTO, DISCOV. MED, vol. 19, no. 102, 2015, pages 49 - 57
PUZZO ET AL., SCI. TRANSL. MED, vol. 29, no. 9, 2017, pages 418
QUAX ET AL., MOL CELL, vol. 59, 2015, pages 149 - 161
RAI UDJ ET AL.: "The suprachoroidal pathway: a new drug delivery route to the back of the eye", DRUG DISCOV TODAY, vol. 20, no. 4, 2015, pages 491 - 495, XP055629994, DOI: 10.1016/j.drudis.2014.10.010
RANGERPEPPAS, J, MACROMOL. SCI. REV. MACROMOL. CHEM, vol. 23, 1983, pages 61
SEFTON, CRC CRIT. REF. BIOMED. ENG, vol. 14, 1987, pages 20
SHEN ET AL., GLYCAN BINDING AVIDITY DETERMINES THE SYSTEMIC FATE OF ADENO-ASSOCIATED VIRUS TYPE, vol. 9, 2012
SONG ET AL.: "Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA JOURNAL OF PHARMACEUTICAL SCIENCE & TECHNOLOGY, vol. 50, 1995, pages 372 397
WU, HUMAN GENE THERAPY, vol. 18, no. 2, 2007, pages 171 - 82
ZINN ET AL., CELL REP, vol. 12, no. 6, 2015, pages 1056 - 1068
ZOLOTUKHIN ET AL., METHODS, vol. 28, 2002, pages 158 - 167

Similar Documents

Publication Publication Date Title
JP7619958B2 (en) Recombinant adeno-associated viruses and uses thereof
US11565001B2 (en) Adeno-associated virus virions with variant capsid and methods of use thereof
US20230381341A1 (en) Adeno-associated viruses for ocular delivery of gene therapy
WO2022076750A2 (en) Recombinant adeno-associated viruses for cns or muscle delivery
US20250001006A1 (en) Recombinant adeno-associated viruses for targeted delivery
WO2022222869A1 (en) Recombinant adeno-associated virus and application thereof
JP2025527658A (en) Recombinant adeno-associated viruses and uses thereof
WO2025217214A2 (en) Recombinant adeno-associated viruses and uses thereof
WO2023201277A1 (en) Recombinant adeno-associated viruses for cns tropic delivery
JP2023545384A (en) Recombinant adeno-associated virus for central nervous system or muscle delivery
CN116568815A (en) Adeno-associated virus for ocular delivery of gene therapy
WO2024238853A1 (en) Adeno-associated viruses for ocular delivery of gene therapy
HK40051378A (en) Adeno-associated virus virions with variant capsid and methods of use thereof