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WO2024238807A2 - Aav de recombinaison à tropisme et spécificité améliorés - Google Patents

Aav de recombinaison à tropisme et spécificité améliorés Download PDF

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Publication number
WO2024238807A2
WO2024238807A2 PCT/US2024/029714 US2024029714W WO2024238807A2 WO 2024238807 A2 WO2024238807 A2 WO 2024238807A2 US 2024029714 W US2024029714 W US 2024029714W WO 2024238807 A2 WO2024238807 A2 WO 2024238807A2
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WO
WIPO (PCT)
Prior art keywords
capsid protein
seq
aav capsid
independently selected
amino acid
Prior art date
Application number
PCT/US2024/029714
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English (en)
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WO2024238807A3 (fr
Inventor
Charles Francis ALBRIGHT
Kevin C. OLIVIERI
Xiaohong CAO
Jie Tan
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Affinia Therapeutics Inc.
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Publication date
Priority claimed from PCT/US2023/072424 external-priority patent/WO2024040193A2/fr
Application filed by Affinia Therapeutics Inc. filed Critical Affinia Therapeutics Inc.
Publication of WO2024238807A2 publication Critical patent/WO2024238807A2/fr
Publication of WO2024238807A3 publication Critical patent/WO2024238807A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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

  • Adeno-associated virus has become the vector system of choice for in vivo gene therapy.
  • a growing variety of recombinant AAVs (rAAVs) engineered to deliver therapeutic nucleic acids have been developed and tested in nonhuman primates and humans, and the FDA has recently approved two rAAV gene therapy products for commercialization.
  • rAAV vectors are safer and less inflammatory than other viruses, toxicities have occurred following administration of high doses of rAAVs for gene therapy.
  • local administration of rAAVs to a target tissue or organ has been used to improve targeting and reduce systemic toxicity.
  • various natural and synthetic AAV variants have been tested to develop an AAV vector with desired tropism and specificity.
  • the capsid is thought to be the primary determinant of infectivity and host- vector related properties such as adaptive immune responses, tropism, specificity, potency, and bio-distribution. Indeed, several of these properties are known to vary between natural serotypes and engineered AAV variants.
  • novel synthetic AAV variants have been developed by using a variety of capsid engineering techniques, one of which is the insertion of small, 7 amino acid-long, peptides into an exposed loop of the capsid protein, called variable region VIII (VRVIII).
  • VRVIII variable region VIII
  • the insertion of a novel peptide into a wild type capsid changes the tropism of the variant.
  • Atty Docket No.: 38053-57988/WO (112WO) peptide having the sequence RGDLGLS (SEQ ID NO: 156) into the capsid of AAV9 was found to increase infection of astrocytes (see PhD thesis of Eike Kienle, Ruprecht-Karls- Universitat Heidelberg, 2014) and primary breast cancer cells (Michelfelder et al. (2009)). [0006] To date, however, there is little understanding as to how these changes on the capsid functionally alter these properties. Additionally, AAV vectors with a desired tropism and specificity to common therapeutic targets, such as muscles, have not yet been available. 4.
  • a modified AAV capsid protein with the preferred tropism comprises (i) a targeting peptide at a site within variable region VIII (VR VIII); or (ii) a peptide segment within variable region I (VR I), wherein the targeting peptide has a sequence of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 , wherein X 1 , X 2 , X 3 , X 7 , X 8 , X 9 and X 10 are independently selected from any amino acid residue, wherein the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8NDNP12 and P1, P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue
  • the modified AAV capsid protein comprises both (i) the targeting peptide and (ii) the peptide segment. In some cases, the AAV capsid protein comprises the targeting peptide but not the peptide segment. In some cases, the AAV capsid protein comprises the peptide segment but not the targeting peptide.
  • the resulting rAAVs containing the modified AAV capsid protein comprising (i) a targeting peptide at a site within variable region VIII (VR VIII); and (ii) a peptide segment within variable region I (VR I) demonstrated better targeting with more specific expression of a transgene in the target tissue, e.g., muscles, and also exhibited reduced liver tropism when systemically administered to a mammalian subject.
  • this disclosure features a modified adeno-associated virus (AAV) capsid protein, comprising: (i) a targeting peptide within VR VIII; and (ii) a peptide segment within VR I, wherein the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X 1 , X 2 , X 3 , X 7 , X 8 , X 9 and X 10 are independently selected from any amino acid residue, and wherein the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8NDNP12 and P1, P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue.
  • AAV adeno-associated virus
  • X7X8X9X10 is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656), or X7X8X9X10 is FNNL and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is SSTAGGASNDNA (SEQ ID NO: 47128).
  • X1 is independently selected from S, N, T, A, or Q
  • X2 is independently selected from G, S, or A
  • X3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M.
  • X 1 is independently selected from T, S, Q, A, D, or N
  • X 2 is independently selected from F, W, A, K, Q, N, R, S, Y, M, or T
  • X 3 is independently selected from N, S, Q, T, Y, G, E, F, M, or A.
  • X 1 is independently selected from N, S, T, A, or Q
  • X 2 is independently selected from N, S, Q, T, M, A, Y, or L
  • X 3 is independently selected from I, M, L, T, V, Q, or Y.
  • X1 is independently selected from E, Q, A, S, T, N, or D
  • X2 is independently selected from H, Y, W, R, K, F, or N
  • X3 is independently selected from H, R, S, A, T, M, G, K, Q, N, or I.
  • X1 is independently selected from S, N, T, A, or Q
  • X2 is independently selected from G, S, or A
  • X3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M
  • X 7 is F
  • X 8 is independently selected from N or Q
  • X 9 is N
  • X 10 is independently selected from T or L.
  • X 1 is independently selected from T, S, Q, A, D, or N;
  • X2 is independently selected from F, W, A, K, Q, N, R, S, Y, M, or T;
  • X 3 is independently selected from N, S, Q, T, Y, G, E, F, M, or A;
  • X 7 is F;
  • X 8 is independently selected from N or Q;
  • X 9 is N; and
  • X 10 is independently selected from T or L.
  • X1 is independently selected from N, S, T, A, or Q
  • X2 is independently selected from N, S, Q, T, M, A, Y, or L
  • X 3 is independently selected from I, M, L, T, V, Q, or Y
  • X 7 is F
  • X 8 is independently selected from N or Q
  • X 9 is N
  • X 10 is independently selected from T or L.
  • X1 is independently selected from E, Q, A, S, T, N, or D;
  • X2 is independently selected from H, Y, W, R, K, F, or N;
  • X3 is independently selected from H, R, S, A, T, M, G, K, Q, N, or I;
  • X 7 is F;
  • X 8 is independently selected from N or Q;
  • X9 is N; and
  • X10 is independently selected from T or L.
  • X7X8X9X10 is selected from RSQT, RNVV, RGQI, RGVV, and RSVV and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X 1 is independently selected from S, N, T, A, or Q
  • X 2 is independently selected from G, S, or A
  • X3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M.
  • X 1 is independently selected from T, S, Q, A, D, or N;
  • X 2 is independently selected from F, W, A, K, Q, N, R, S, Y, M, or T;
  • X 3 Atty Docket No.: 38053-57988/WO (112WO) is independently selected from N, S, Q, T, Y, G, E, F, M, or A.
  • X1 is independently selected from N, S, T, A, or Q;
  • X2 is independently selected from N, S, Q, T, M, A, Y, or L;
  • X 3 is independently selected from I, M, L, T, V, Q, or Y.
  • X1 is independently selected from S, N, T, A, or Q
  • X2 is independently selected from G, S, or A
  • X 3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M
  • X 7 is R
  • X 8 is independently selected from S, G, or N
  • X 9 is independently selected from V or Q
  • X10 is independently selected from V, T, or I.
  • X 1 is independently selected from T, S, Q, A, D, or N;
  • X 2 is independently selected from F, W, A, K, Q, N, R, S, Y, M, or T;
  • X 3 is independently selected from N, S, Q, T, Y, G, E, F, M, or A;
  • X7 is R;
  • X8 is independently selected from S, G, or N;
  • X9 is independently selected from V or Q; and
  • X10 is independently selected from V, T, or I.
  • X 1 is independently selected from N, S, T, A, or Q
  • X 2 is independently selected from N, S, Q, T, M, A, Y, or L
  • X3 is independently selected from I, M, L, T, V, Q, or Y
  • X7 is R
  • X8 is independently selected from S, G, or N
  • X9 is independently selected from V or Q
  • X 10 is independently selected from V, T, or I.
  • X 7 X 8 X 9 X 10 is selected from VRTL, RQGI, ARTL, and RTNL and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X 1 is independently selected from S, N, T, A, or Q;
  • X 2 is independently selected from G, S, or A; and
  • X 3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M.
  • X 1 is independently selected from S, N, T, A, or Q;
  • X 2 is independently selected from G, S, or A;
  • X 3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M;
  • X7 is independently selected from R, V, or A;
  • X8 is independently selected from R, T, or Q;
  • X9 is independently selected from T, G, or N; and
  • X10 is independently selected from L or I.
  • X7X8X9X10 is selected from VRTL, RQGI, ARTL, and RTNL and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390), or X7X8X9X10 is selected from RSQT, RNVV, RGQI, RGVV, and RSVV and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is NSTSGASTNDNA (SEQ ID NO: 48390), or X 7 X 8 X 9 X 10 is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656), or X 7 X 8 X 9 X 10 is FNNL and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is SSTAG
  • X1X2X3 is selected from the group consisting of: AAG, AAN, AAQ, AAS, AGA, AGG, AGH, AGM, AGN, AGP, AGQ, AGS, AGT, AGV, AKD, ANG, ASA, ASG, ASN, ASP, ASQ, ASS, EAY, EQY, NAG, NGA, NGE, NGF, NGG, NGI, NGL, NGM, NGN, NGP, NGQ, NGS, NGT, NGV, NGW, NGY, NNG, NNN, NQG, NQN, NRD, NSG, NWN, NWS, NYN, QAG, QAQ, QAS, QGA, QGG, QGH, QGM, QGN, QGP, QGQ, QGS, QGT, QGV, QKD, QSG, QSN, QSQ, QSS, SAG, SFG, SFN, SGA, SGE, SGG, SGH, S
  • X 7 X 8 X 9 X 10 is selected from RSQT, RNVV, RGQI, RGVV, and RSVV and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390), or X7X8X9X10 is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656), or X 7 X 8 X 9 X 10 is FNNL and P1P2P3P4P5P6P7P8NDNP12 is SSTAGGASNDNA (SEQ ID NO: 47128).
  • X1X2X3 is selected from the group consisting of: AAA, AAH, AAY, AFG, AFN, AFQ, AFS, AFT, AHN, AKE, AMN, AMQ, ANN, ANS, AQQ, AQS, ARE, ATS, AWM, AWN, AWQ, AWT, AYN, DIR, DKA, DKG, DKI, DKM, DKN, DKQ, DKS, DKT, DRA, DRM, DRQ, DRS, DRT, DRV, DSM, DSS, DSV, DTR, DVR, ENN, ENS, ENT, ERM, NAN, NFN, NFQ, NFS, NFT, NGH, NKD, NMT, NQS, NSN, NSQ, NVN, NWA, NWQ, NWV, QAN, QAT, QAY, QFN, QFQ, QFS, QFT, QKE, QLN, QL
  • X1X2X3 is selected from the group consisting of: AAI, AAL, AAM, AAT, AAV, ADL, ADR, AER, AGF, AGI, AGL, AGY, AHI, ALI, ALM, ALV, AMH, AMI, AML, AMM, AMT, ANA, ANF, ANI, ANM, ANQ, ANT, ANV, ANY, AQF, AQI, AQL, AQM, AQT, AQV, ASF, ASI, ASL, ASM, AST, ASV, ASY, ATI, ATL, ATM, ATQ, ATT, ATV, AWI, AYH, AYL, AYQ, DGR, DMR, DQR, DWK, EGR, NAF, NAI, NAL, NAM, NAQ, NAS, NAT, NAV, NAY, NDK, NEK, NER, NFI, NFL, NFM, NHI, NHL,
  • X7X8X9X10 is selected from FNNL, FNNT, and FQNT, and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is SGTTGGSSNDNT (SEQ ID NO: 46656), or X 7 X 8 X 9 X 10 is FNNL and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is SSTAGGASNDNA (SEQ ID NO: 47128).
  • X1X2X3 is selected from the group consisting of: ADK, AFA, AFH, AFM, AGK, AHF, AHG, AHM, AHQ, AHS, AHT, AHY, AKY, ANH, ANP, AQH, ARI, ASH, ATA, ATG, AWA, AWP, AWS, AYS, AYT, DAR, DHA, DHS, DHT, DIS, DNK, DRG, DRH, DSR, DWR, DYN, DYR, DYS, EAR, EFR, EHH, EHK, EHN, EHR, EIR, EKA, EKE, EKG, EKH, EKI, EKL, EKM, EKN, EKQ, EKS, EKT, EKV, ELR, EMK, EMR, ENK, ENR, EQR, ERA, ERH, ERI, ERL, ERN, ERQ, ERS, ERT
  • X7X8X9X10 is selected from LDEL, QSTL, ISRT, LLLS, IHNL, LIGR, FVQR, HVNL, RQGI, KERF, RSVV, ARTL, VRTL, RNVV, RSQT, RGVV, RGQI, and RTNL
  • P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X1X2X3 is selected from the group consisting of: AAP, AAR, AER, AFK, AFR, AGA, AGR, AIR, AKP, AKQ, AKR, ALR, AMK, AMR, ANK, ANR, AQG, AQK, AQR, ARK, ARP, ARR, ARS, ASG, ASP, ASR, ATR, AVR, AWK, AWR, DSG, EKP, NAG, NAK, NAR, NER, NFK, NFR, NGA, NGD, NGH, NGI, NGK, NGL, NGN, NGQ, NGR, NGS, NGT, NGV, NGY, NIK, NIR, NKI, NKP, NKT, NKV, NLK, NLR, NMK, NMR, NNK, NNR, NPR, NQG, NQK, NQR, NRK, NRN, NRP, N
  • X1X2X3 is selected from the group consisting of: ADL, ADN, ADS, ADT, AEA, AED, AEE, AHE, AHL, AIW, ALE, APE, APL, ARW, AWA, AYE, DAD, DAE, DAI, DAL, DAM, DAT, DAV, DDD, DDE, DDF, DDH, DDL, DDM, DDP, DDQ, DDT, DDV, DDY, DED, DEM, DEN, DEP, DEW, DFA, DFD, DFF, DFI, DFM, DFN, DFP, DFV, DFW, DGD, DGI, DHD, DHE, DHL, DHW, DHY, DIE, DII, DIL, DIM, DIN, DIP, DIS, DIT, DIV, DKA, DKD, DKE, DKH, DKQ, DKW, DLA, DLF, DLG, DLH, DLI
  • X 7 X 8 X 9 X 10 is selected from FNNL, FNNT, and FQNT and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is SGTTGGSSNDNT (SEQ ID NO: 46656).
  • X1X2X3 is not selected from the group consisting of: ADK, AEK, AGL, AGM, AHG, AHK, AIK, AKA, AKD, AKE, AKF, AKH, AKI, AKK, AKL, AKM, AKN, AKS, AKT, AKV, AKY, ALK, ANG, ANN, ANT, APK, AQI, AQT, ARA, ARE, ARF, ARG, ARI, ARN, ARQ, ART, ARV, ARY, ASA, ASK, ASN, ATG, ATK, ATY, AWN, AYH, AYK, AYP, AYR, DKK, DKR, DNR, DRR, DWR, DYR, EGR, EIR, EKK, EKM, EKR, ENK, EQK, ERK, ERM, ERP, ERT, ESR, EVR, NAQ, NAT, NEG,
  • X 1 X 2 X 3 is selected from the group consisting of: AAP, AAR, AER, AFK, AFR, AGA, AGR, AIR, AKP, AKQ, AKR, ALR, AMK, AMR, ANK, ANR, AQG, AQK, AQR, ARK, ARP, ARR, ARS, ASG, ASP, ASR, ATR, AVR, AWK, AWR, DSG, EKP, NAG, NAK, NAR, NER, NFK, NFR, NGA, NGD, NGH, NGI, NGK, NGL, NGN, NGQ, NGR, NGS, NGT, NGV, NGY, NIK, NIR, NKI, NKP, NKT, NKV, NLK, NLR, NMK, NMR, NNK, NNR, NPR, NQG, NQK, NQR, NRK, NRN,
  • X 1 X 2 X 3 is selected from the group consisting of: ADL, ADN, ADS, ADT, AEA, AED, AEE, AHE, AHL, AIW, ALE, APE, APL, ARW, AWA, AYE, DAD, DAE, DAI, DAL, DAM, DAT, DAV, DDD, DDE, DDF, DDH, DDL, DDM, DDP, DDQ, DDT, DDV, DDY, DED, DEM, DEN, DEP, DEW, DFA, DFD, DFF, DFI, DFM, DFN, DFP, DFV, DFW, DGD, DGI, DHD, DHE, DHL, DHW, DHY, DIE, DII, DIL, DIM, DIN, DIP, DIS, DIT, DIV, DKA, DKD, DKE, DKH, DKQ, DKW, DLA, DLF, DLG, DLH,
  • X7X8X9X10 is FNNT and P1P2P3P4P5P6P7P8NDNP12 is SSTAGGATNDNA (SEQ ID NO: 47131), or X 7 X 8 X 9 X 10 is FNNL and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is SSTAGGASNDNA (SEQ ID NO: 47128).
  • X1X2X3 is selected from the group consisting of: ADL, ADN, ADS, ADT, AEA, AED, AEE, AHE, AHL, AIW, ALE, APE, APL, ARW, AWA, AYE, DAD, DAE, DAI, DAL, DAM, DAT, DAV, DDD, DDE, DDF, DDH, DDL, DDM, DDP, DDQ, DDT, DDV, DDY, DED, DEM, DEN, DEP, DEW, DFA, DFD, DFF, DFI, DFM, DFN, DFP, DFV, DFW, DGD, DGI, DHD, DHE, DHL, DHW, DHY, DIE, DII, DIL, DIM, DIN, DIP, DIS, DIT, DIV, DKA, DKD, DKE, DKH, DKQ, DKW, DLA, DLF, DLG, DLH, DLI
  • X 1 X 2 X 3 is not selected from the group consisting of: ADK, AEK, AGL, AGM, AHG, AHK, AIK, AKA, AKD, AKE, AKF, AKH, AKI, AKK, AKL, AKM, AKN, AKS, AKT, AKV, AKY, ALK, ANG, ANN, ANT, APK, AQI, AQT, ARA, ARE, ARF, ARG, ARI, ARN, ARQ, ART, ARV, ARY, ASA, ASK, ASN, Atty Docket No.: 38053-57988/WO (112WO) ATG, ATK, ATY, AWN, AYH, AYK, AYP, AYR, DKK, DKR, DNR, DRR, DWR, DYR, EGR, EIR, EKK, EKM, EKR, ENK, EQK, ERK, E
  • X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 has a sequence selected from SEQ ID NOs: 98944 to 157056.
  • P1P2P3P4P5P6P7P8NDNP12 is selected from: NSTSGASTNDNA (SEQ ID NO: 48390); SGTTGGSSNDNT (SEQ ID NO: 46656); SSTAGGASNDNA (SEQ ID NO: 47128); and SSTAGGATNDNA (SEQ ID NO: 47131).
  • X1X2X3RGDX7X8X9X10 is NYQRGDFQNT (SEQ ID NO: 145855) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656).
  • X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 is SYQRGDFQNT (SEQ ID NO: 146577) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656).
  • X1X2X3RGDX7X8X9X10 is ADRRGDFNNT (SEQ ID NO: 146987) and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is SGTTGGSSNDNT (SEQ ID NO: 46656).
  • X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 is NNQRGDFNNT (SEQ ID NO: 148096) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656).
  • X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 is SGQRGDRGQI (SEQ ID NO: 124229) and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X1X2X3RGDX7X8X9X10 is TGYRGDRGQI (SEQ ID NO: 124587) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 is QGQRGDRGQI (SEQ ID NO: 126394) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X1X2X3RGDX7X8X9X10 is SGVRGDRGVV (SEQ ID NO: 126741) and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 is TGFRGDARTL (SEQ ID NO: 99313) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 is QGIRGDRSQT (SEQ ID NO: 136490) and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • X1X2X3RGDX7X8X9X10 is SAQRGDRGVV (SEQ ID NO: 48394) and P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 is NSTSGASTNDNA (SEQ ID NO: 48390).
  • the modified AAV capsid protein has at least 90%, 95%, 98%, 99% or 99.5% sequence identity to the sequence of the reference AAV capsid protein.
  • the AAV capsid protein is selected from VP1, VP2 and VP3.
  • the reference AAV capsid protein is a capsid protein of an AAV variant selected from the group consisting of: AAV9; Anc8065; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56- B; hu.45-B; rh.57-B; rh.35
  • the reference AAV capsid protein is a capsid protein having a sequence selected from SEQ ID NOs: 54-152, 44885-44898, 44916-44917, or a fragment thereof. In some embodiments, the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 61 or a fragment thereof. In some embodiments, the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 132 or a fragment thereof. In some embodiments, the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 142 or a fragment thereof.
  • the targeting peptide is positioned between 565 and 595 within VR VIII of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV1 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein, thereby replacing residues S586, S587, and S588 of the reference capsid protein;
  • the reference AAV capsid protein is a capsid protein of AAV2 or a modification thereof and the targeting peptide is between Q584 and R588 of the reference AAV capsid protein, thereby replacing residues R585, G586, and N587 of the reference capsid protein;
  • the reference AAV capsid protein is a capsid protein of AAV3 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein, thereby replacing residues S586, S587,
  • variable region I corresponds to amino acid residues between about position 259 to about position 275 of the modified capsid protein.
  • the peptide segment is at a position between S261 and Y274 of an AAV9 capsid protein (SEQ ID NO: 61).
  • the peptide segment is at a position between S260 and Y273 of an Anc80 capsid protein (SEQ ID NO: 132).
  • the peptide segment is at a position between S260 and Y273 of an Anc80L65 capsid protein (SEQ ID NO: 142).
  • a polynucleotide encoding the modified AAV capsid protein is a polynucleotide encoding the modified AAV capsid protein.
  • a vector comprising the polynucleotide.
  • the vector further comprises a promoter operably linked to the polynucleotide.
  • a host cell comprising the modified AAV capsid protein, the polynucleotide, or the vector.
  • rAAV recombinant AAV virion
  • the rAAV virion further comprises an exogenous polynucleotide.
  • the exogenous polynucleotide comprises a template for homology directed repair. In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA. In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein. [0030] In some embodiments, the therapeutic protein is MTM1 or a fragment thereof. In some embodiments, the expressible polypeptide comprises the sequence of SEQ ID NO: 165 or a fragment thereof.
  • the expressible polypeptide comprises the sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any of SEQ ID Nos: 166-170.
  • the exogenous polynucleotide further comprises a regulatory sequence.
  • the regulatory sequence comprises expression regulatory elements (EREs).
  • the EREs comprise a CAG promoter.
  • the EREs comprise a sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ IDs NO:171-173.
  • a pharmaceutical composition comprising the modified AAV capsid protein or the AAV virion.
  • a method for treating or ameliorating or preventing a disease or condition in a subject comprising administering a therapeutically effective amount of the rAAV virion or the pharmaceutical composition.
  • the disease is a muscular disease and/or the condition is muscle degeneration.
  • the muscle is a striated muscle, preferably heart or a skeletal muscle or diaphragm.
  • the muscular disease is a muscular dystrophy, a cardiomyopathy, a myotonia, a muscular atrophy, a myoclonus dystonia, a mitochondrial myopathy, a rhabdomyolysis, a fibromyalgia, and/or a myofascial pain syndrome.
  • AAV modified adeno-associated virus
  • a recombinant AAV (rAAV) virion comprising the modified AAV capsid protein of this disclosure or the rAAV virion of this disclosure for use in treating and/or preventing a muscular disease and/or in muscle regeneration.
  • a pharmaceutical composition comprising the modified AAV capsid protein and/or the rAAV virion for use in treating and/or preventing a muscular disease and/or in muscle regeneration.
  • a method of transferring an exogenous polynucleotide into a muscle cell comprising the step of administering the rAAV virion to a subject.
  • the administration results in transfer of the exogenous polynucleotide in the muscle cell, at a muscle:liver infection ratio of greater than 1 when measured by genome copies of the rAAV virion.
  • the muscle:liver infection ratio ranges from 1 to 100.
  • the muscle:liver infection ration ranges from 1 to 10.
  • the muscle:liver infection ratio ranges from 2 to 8.
  • the administration results in expression of the exogenous polynucleotide in the muscle cell, at a muscle:liver expression ratio of greater than 10.
  • the muscle:liver expression ratio ranges from 10 to 100. In some embodiments, the muscle:liver expression ratio ranges from 20 to 80.
  • the muscle:liver expression ratio ranges from 50 to 80 when measured by mRNA transcript expression. In some embodiments, the muscle:liver expression ratio ranges from 10 to 50 when measured by protein expression. In some embodiments, the muscle cell is selected from triceps surae, biceps, heart and quadricep. [0039] In another aspect, provided herein is use of the modified AAV capsid protein and/or the rAAV virion for transferring an exogenous polynucleotide into a muscle cell. In some embodiments, the use is a non-therapeutic use, preferably wherein the use is an in vitro use.
  • the muscle cell is selected from triceps surae, biceps, heart and quadricep.
  • a method of transferring an exogenous polynucleotide into a target cell comprising the step of administering an rAAV virion comprising the modified AAV capsid protein or the rAAV virion to a subject.
  • the target cell is heart and X 7 X 8 X 9 X 10 is FVQR, IHNL, KERF, LDEL, LIGR, QSTL, RTNL, or VRTL; the target cell is muscle and X 7 X 8 X 9 X 10 is ARTL, FNNL, FNNT, FQNT, HVNL, RGQI, RGVV, RNVV, RQGI, or RSVV; or the target cell is skeletal muscle and X 7 X 8 X 9 X 10 is ISRT or RSQT.
  • the method comprises administering 1e12 vg/kg to 1e14 vg/kg dose of rAAV to the subject.
  • the method comprises administering 1.5e12 vg/kg to 1.5e13 vg/kg dose of rAAV to the subject. In some embodiments, the method comprises administering 1e13 vg/kg dose of rAAV to the subject. 5. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS [0042] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings where: [0043] FIG.1 illustrates the structure of an AAV VP1 protein with certain variable regions (VR I, VR III, VR IV) highlighted. The location of the liver toggle (mut1) in VR I and the peptide insertion (deco1) in VR VIII are indicated.
  • FIGs.2A-2C provide the sequence alignment of VP1 sequences of certain AAV variants using AAV2 VP1 as a reference. The location of residue 168, the liver toggle site, mut1 (FIG.2A), and the insertion site of a targeting peptide (FIG.2B),are indicated.
  • FIGs. 2A-2B disclose SEQ ID NOs: 55, 54, 58, 56, 64, 59, 60, 89, 111, 61, 63, 62, and 57, respectively, in order of appearance.
  • FIGs.3A-3D provide the sequence alignment of VP1 sequences of ancestral AAVs using AAV2 as a reference.
  • FIGs.3A-3D disclose SEQ ID NOs: 217-237, respectively, in order of appearance. Atty Docket No.: 38053-57988/WO (112WO) [0046]
  • FIGs.4A-4J shows immunohistochemistry data obtained from the experiment described in Example 2 below in the Example section.
  • FIG.4A Anti-GFP immunohistochemistry was performed on liver with vehicle (FIG.4A), AAV9 (FIG.4B), AAV mut1 (FIG.4C), AAV deco1 (FIG.4D), or AAV mut1-deco1 (FIG.4E); and skeletal muscle (quadriceps) tissue cross-sections of mice injected with vehicle (FIG.4F), AAV9 (FIG.4G), AAV mut1 (FIG.4H), AAV deco1 (FIG.4I), or AAV mut1-deco1 (FIG.4J).
  • FIGs.5A-5B show mRNA expression in various tissues of C57BL/6 mice treated with different AAV vectors, as measure by RT-ddPCR.
  • Y-axis represents the ratio of copies of eGFP mRNA transcripts over RPP30 mRNA and x-axis represents AAV vectors and the dose injected into the experimental animals.
  • Each graph shows eGFP expression in liver (FIG.5A) and quadriceps (FIG.5B).
  • FIGs.6A-6E show eGFP mRNA expression in various tissues of C57BL/6 mice treated with different AAV vectors, as measure by RT-ddPCR.
  • FIGs.7A-7D show eGFP vector genome (DNA) and eGFP expression (mRNA) in liver and quad tissues of C57BL/6 mice treated with vehicle, AAV Mut1 and AAV Mut1-deco1 AAV vectors.
  • DNA data is shown in FIGs.7A and 7B with eGFP genomic copies as measured by RT-ddPCR plotted at 14 and 28 days, respectively.
  • Y-axis represents vector genome (copies per DPG) and x-axis represents vehicle and AAV vectors.
  • mRNA data is shown in FIGs.7C and 7D with eGFP expression as measured by RT-ddPCR plotted at 14 and 28 days, respectively.
  • Y-axis represents the ratio of copies of eGFP over RPP30 mRNA and x-axis represents AAV vectors.
  • FIG.8 shows eGFP mRNA expression in various tissues of BalbC mice treated with vehicle, AAV mut1 and AAV mut1-deco1 AAV vectors, as measured by RT-ddPCR.
  • Y-axis represents the ratio of copies of eGFP over RPP30 mRNA and x-axis represents AAV vectors and the dose injected into the experimental animals.
  • the graph shows eGFP expression in liver (left) and quadriceps (right).
  • FIGs.9A and 9B show exemplary IHC tissue analysis obtained from of Run 1 samples from NHPs.
  • FIG.9A Liver tissue is shown in FIG.9A, the left side shows tissue obtained from an AAV9 vector treated NHP and the right side shows tissue obtained from an Atty Docket No.: 38053-57988/WO (112WO) AAV mut1_deco1 vector treated NHP; exemplary IHC quadriceps tissue is shown in FIG.9B, obtained from AAV9 vector treated NHP on left and AAV mut1_deco1 vector treated NHP on the right.
  • FIG.10 shows the % GFP positive cells in the liver tissue (right and left side of the organ) and quadriceps tissue (right and left leg) in slides obtained from Run 1 from NHPs administered vehicle, AAV9 or AAV mut1_deco1 vector.
  • FIG.11 shows the % GFP positive cells in various skeletal muscle and liver tissue (average from Runs 1 and 2) in slides obtained from NHPs administered vehicle, AAV9 or AAV mut1_deco1 vector.
  • FIG.12 shows the % GFP positive cells per animal in various skeletal muscle and liver tissue (average from Runs 1 and 2) in slides obtained from NHPs administered vehicle, AAV9 or AAV mut1_deco1 vector.
  • FIG.13 shows the average combined quantification of % GFP positive cells per animal in various skeletal muscle and liver tissue (average from Runs 1 and 2) obtained from NHPs administered vehicle, AAV9 or AAV mut1_deco1 vector.
  • FIG.14 shows the % GFP positive cells in various cardiac tissues (average from Runs 1 and 2) obtained from NHPs administered vehicle, AAV9 or AAV mut1_deco1 vector.
  • FIG.15 shows the % GFP positive cells per animal in various cardiac muscle (average from Runs 1 and 2) obtained from NHPs administered vehicle, AAV9 or AAV mut1_deco1 vector.
  • FIG.16 shows the average % GFP positive cells per animal in ventricle wall, atria, inter ventr septum slides (average from Runs 1 and 2) obtained from NHPs administered vehicle, AAV9 or AAV mut1_deco1 vectors.
  • FIGs.17A-17C shows the average % GFP positive cells per NHP animal in various tissues (average from Runs 1 and 2) administered vehicle and AAV9 and AAV mut1_deco1 vectors.
  • FIG.17A shows average % GFP positive cells per animal in liver tissue.
  • FIG.17B shows average % GFP positive cells per animal in various skeletal muscle tissue.
  • FIG.17C shows average % GFP positive cells per animal in various cardiac tissue.
  • FIGs.18A-18D show the results of DNA samples analyzed for biodistribution of vector genomes in the liver and quadriceps tissue using a duplexed ddPCR method targeting the transgene (eGFP) and a reference gene (RPP30). The results are shown in FIG.18A (liver), FIG.18B (quadriceps), FIG.18C (biceps), and FIG.18D (heart) where the x-axis represents AAV vectors (wild type AAV9 on the left and AAV mut1deco1 on the right of each plot) and indicating whether the sample was taken from the left or right side of the organ/animal.
  • AAV vectors wild type AAV9 on the left and AAV mut1deco1 on the right of each plot
  • FIGs.19A-19D show the results of mRNA transcript analysis measured by eGFP copies of eGFP over RPP30 mRNA.
  • FIG.19A liver
  • FIG.19B quaddriceps
  • FIG.19C biceps
  • FIG.19D heart
  • the x-axis represents AAV vectors (wild type AAV9 on the left and AAV mut1deco1 on the right) and indicating whether the sample was taken from the left or right side of the organ/animal.
  • FIG.20 shows human MTM1 protein expression in RD cells. The expression level of human MTM protein was determined by automated JESS-ProteinSimple instrument.
  • FIG.21 provides a study design to assess distribution patterns of Anc80 variants in non-human primates, as described in Example 8.
  • FIG.22 provides a scatter plot of Anc80 variant counts from an Anc80 library showing each variant’s enrichment in muscle (y-axis) and the liver (x-axis).
  • FIG.23 shows negative log fold changes in tissues compared to the test article input (Anc80 variants of Anc80 library) on day 28 in liver-off variants (variants with “A” at P3) of Anc80 library (left) and liver-on variants (variants with “G” at P3) of Anc80 library (right).
  • the test article, used herein, refers to the pool of vectors administered.
  • FIG.24 shows the effect of P3 on log fold-change of tissue enrichment in primate G62N of Group 2 in day 28 quadriceps. A significant difference of Log fold-change of tissue enrichment in the quadriceps was found between “liver off” “0” and “liver on” “1” Anc80 variants.
  • FIG.25 shows the effect of P3 on log fold-change of tissue enrichment in the quadriceps in primate G66E of Group 2 in day 28 quadriceps. A significant difference of log fold-change of tissue enrichment in the quadriceps was found between “liver off” “0” and “liver on” “1” Anc80 variants. On the x-axis, “0” represents amino acid residue “G”, and “1.0” represents amino acid “A”.
  • FIG.26 shows the effect of P3 on average log fold-change of tissue enrichment in the quadriceps in both primates of Group 2 on day 28 quadriceps. A significant difference of log fold-change of tissue enrichment in the quadriceps was found between “liver off” “0” and “liver on” “1” Anc80 variants. On the x-axis, “0” represents amino acid residue “G”, and “1.0” represents amino acid “A”. [0069] FIG.27 shows a quadrant liver plot of G62N primate on day 28. The upper left (UL) quadrant had higher biodistribution of 74 Anc80 variants in the quadricep muscle with liver toggle on “liver on”.
  • FIG.28 shows a quadrant liver plot of G66E primate on day 28.
  • the upper left (UL) quadrant had higher biodistribution of 66 Anc80 variants in the quadricep muscle with liver toggle on “liver on”.
  • FIG.29 provides a flow chart representing comparative data amongst animals in each of Group 1 and Group 2. When the tissue enrichment data for 2 primates for each group was averaged, 13 variant pairs were found to have an effect on liver toggle. Variability in AAV transduction is common, particulary at low doses in primates.
  • FIG.30 shows an average of the liver toggle data between both non-primate animals G62N and G66E of Group 2.
  • the upper left (UL) quadrant had higher biodistribution of 69 Atty Docket No.: 38053-57988/WO (112WO) Anc80 variants in the quadricep muscle with liver toggle on “liver on”.
  • the upper right (UR) quadrant had high biodistribution of 13 Anc80 variants in the quadricep muscle with liver toggle on and off.
  • FIG.31 shows a fingerprint plot for the top 100 Anc80 variant positions P1 (amino acid 168 of Anc80 capsid), P2 (amino acid 205), P3 (amino acid 266), P4, ... P10 (amino acid 587), P11 (amino acid 609) on Anc80 scaffold.
  • the Anc80 variants are color coded by the toggle amino acid at that particular position (P1-P11).
  • FIG.32 shows linear modeling of a position pattern evaluation on average log fold- change of P1-P11 variant pairs.
  • the position pattern evaluation provides tissue enrichment scores versus average log fold changes at every amino acid position (P1-P11).
  • the analysis shows that variations at P3, P5, P6 and P10 have some pattern impact on the average log fold changes (log(FC)) to the quadricep muscle.
  • FIG.33 provides binary codes described in FIG.31 for liver toggle variant pairs in the Anc80 library of Example 8.
  • P3 represents liver toggle variant pairs of “liver off” and “liver on”.
  • some Anc80 variants are more present in muscle only with “liver off” (LR) which was dependent on liver toggle, while other variants worked well in both states “liver on” and “liver off” (UR).
  • FIG.34 provides amino acid positions of 50,625 RGD targeting peptides in the RGD targeting peptide library of as described in Table 27. “Y1, Y2, Y3, Y4, Y5, Y6, and Y7”.
  • Y1 denotes amino acid residue “R”
  • Y2 denotes amino acid residue “G”
  • Y3 denotes amino acid residue “D”, in X1X2X3RGDX7X8X9X10 described in the present disclosure.
  • Y4 is denoted as “X 7 ” of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10
  • Y5 is denoted as “X 8 ” of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10
  • Y6 is denoted as X 9 ” of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10
  • Y7 is denoted as “X10” of X1X2X3RGDX7X8X9X10.
  • FIG.35 shows a density plot of the density of vector DNA at a specific enrichment score ((Log2(Fold)) for AAV9 capsids having specific amino acids in position 4 (“Y4”, or “X7” in X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 ), position 5 (“Y5”, or “X8” in SEQ ID NO: 44915), position 6 (“Y6”, or “X9” in X1X2X3RGDX7X8X9X10) or position 7 (“Y7”, or “X10” in X1X2X3RGDX7X8X9X10) within the targeting peptide sequence.
  • FIG.36A shows the muscle-tropic sequence motif identified from the experiments described in Example 11.
  • FIG.36B is a scatter plot showing muscle enrichment through distribution of scaled enrichment scores of variants tested in Example 11 (an inverse coefficient of variation (ICV)).
  • the red dots on the scatter plot represent variants with a muscle-tropic sequence motif.
  • the mean-ICV plot shows the relationship between mean enrichment score and inverse CV (ICV) of the enrichment score for each variant in the library.
  • the ICV plot is useful for selecting variants with high average tissue enrichment score and low variability (high ICV).
  • FIG.36C compares distribution scaled enrichment scores of variants with and without the muscle-tropic motif.
  • FIG.37A shows a sequence motif identified from top 10 myotropic variants as described in Table 27 that was shown to have significant sequence similarity.
  • FIG.37B is a scatter plot showing muscle enrichment scores of variants (an inverse coefficient of variation (ICV)) where the top muscle targeting peptides are in red.
  • the mean-ICV plot shows the relationship between mean enrichment score and inverse CV (ICV) of the enrichment score for each variant in the library.
  • the ICV plot is useful for selecting variants with high average tissue enrichment score and low variability (high ICV).
  • FIG.37C is a network plot where the lines connect dots representing variants with similar sequences.8 out of 10 top variants were connected in a sequence similarity network.
  • FIG.37C shows the sequence similarity network of SEQ ID NO: 238 (“ATLVT013XX38181”), the top targeting peptide for muscle tropism.
  • FIG.37D shows a sequence motif enriched in muscle tropic capsids.
  • FIG.37E shows a network comparison of the targeting peptides described herein to literature myopeptides.
  • FIG.38A provides a network plot and FIG.38B provides a scatter plot showing tissue enrichment scores for each tissue region assessed.
  • the network analysis and scatter Atty Docket No.: 38053-57988/WO (112WO) plot show that SEQ ID NO: 238 (“ATLVT013XX38181”) outperformed wild-type AAV9, AAV9 deco1 (AAV9 capsid with the deco1 peptide), and AAV9 myo3E (AAV9 capsid with a myo3E peptide) capsids for muscle tropism, and reduces liver toxicity due to the liver- detargeting phenotype Mut1 as compared to wild-type AAV9.
  • the targeting peptide SEQ ID NO: 238 (“ATLVT013XX38181”) consistently ranked at the top for target enrichment in all tissues except for the triceps.
  • FIG.38C shows a plot of tissue enrichment scores (log10 scale of expression) for liver and the indicated muscle tissues.
  • Capsids tested included AAV9 and AAV9 comprising an M3 (MYODV6 (SEQ ID NO: 44864)) targeting peptide located in VR VIII or an M2 (RGDRSVV (SEQ ID NO: 239)) targeting peptide located in VR VIII between amino acids at positions 588 and 589.
  • Muscle tissues analyzed included ventricle wall of the heart, biceps femoris, diaphragm, tibialis anterior, and triceps brachii. Higher tissue enrichment score values represent greater tropism.
  • FIG.39 shows the probability of the top 100 targeting peptides at positions Y4, Y5, Y6 and Y7, where amino acid “R” at position 4 (Y4) is enriched, and amino acids “V, I, and L” are enriched at position 7 (Y7).
  • FIG.40 shows a comparison of Anc80 variants with and without: a deco1 (SEQ ID NO: 1) targeting peptide inserted into the VR VIII region of the Anc80 variant capsid.6 out of 7 Anc80 variants with a deco1 insertion at VR VIII enhanced muscle delivery of Anc80 variants.
  • the present inventors created additional targeting peptide variants for insertion, substitution, or modification within the VR8 region of an AAV capsid by modifying the 3 amino acids preceding amino acids “RGD” in the targeting peptide having an amino acid sequence of SEQ ID NO: 44910 (termed “triplet”) and the 4 amino acids after “RGD” in the targeting peptide having an amino acid sequence of SEQ ID NO: 44910 peptide (termed “quad” or “quadruplet”).
  • FIG.41 shows a list of 20 targeting peptide variants (myoDV1-myoDV10 and myoCD5-myoCD14).
  • FIG.41 shows tissue enrichment scores for each of the 20 targeting peptide variants in muscle tissue (diaphragm, flexor digitorum profundus, heart left ventricle wall, tibialis anterior, and triceps brachii). Higher tissue enrichment score values represent greater muscle tropism, with MYODV6 and MYODV8 having the highest averages tissue enrichment score for all muscle tissue regions.
  • FIG.42 shows a network plot showing log2 fold change (tissue enrichment score) for each of the muscle tissues analyzed for SEQ ID NO: 44880, MYODV6 (SEQ ID NO: 44864), MYODV8 (SEQ ID NO: 44866), MYOCD10 (SEQ ID NO: 44874), and MYOCD12 (SEQ ID NO: 44876) targeting peptides.
  • MYODV6 and MYODV8 each have the highest overall log2 fold change of tissue enrichment in each muscle tissue compared to MYOCD10 and MYCD12 (which contain amino acid residues “SNR” (triplet) preceding “RGD” within the targeting peptide.
  • FIG.43 shows a plot of tissue enrichment scores (log10 scale of expression) for liver and the indicated muscle tissues.
  • FIG.44 shows a plot of tissue enrichment scores (log10 scale of expression) for liver and the indicated muscle tissues.
  • Capsids tested included AAV9 and AAV9 comprising an S1 (RGDISRT (SEQ ID NO: 263)) targeting peptide located in VR VIII between amino acids at positions 588 and 589 or an S2 (RGDRSQT (SEQ ID NO: 251)) targeting peptide located in VR VIII between amino acids at positions 588 and 589.
  • FIG.45 shows a plot for median percent GFP + cells for liver and the indicated muscle tissues.
  • FIG.46 shows representative IHC images for anti-GFP staining for the indicated tissues (quads, heart, and liver) for various AAV capsid proteins (AAV9, AAV9 comprising SEQ ID NO: 44880, AAV9 comprising an M3 targeting peptide (SEQ ID NO: 48391), and an AAV9 comprising an M1 targeting peptide (SEQ ID NO: 44864). Images were taken at 10x magnification. The IHC images in FIG.46 served, in part, as the basis for the quantification in FIG.45. [0093] FIG.47 shows a plot for ddPCR data for cells for liver and the indicated muscle tissues.
  • FIG.48 provides a summary of eGFP mRNA expression in various tissues of C57BL/6 mice treated with different AAV vectors, as measured by RT-ddPCR and eGFP vector genomes copies per DPG (DNA) in various tissues of C57BL/6 mice treated with different AAV vectors, as measure by RT-ddPCR.
  • FIG.49A provides a peptide segment within variable region I (VRI) of the AAV9 capsid protein, various amino acid positions (P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12) within the peptide segment and modifications made to the various positions for capsids present in the AAV -Lib1 capsid library.
  • FIG.49B provides a sequence alignment of variable region I in certain AAV variants using AAV2 variable region I as a reference for numbering.
  • FIG.50 shows vector genome (DNA) in liver of C57BL/6 mice treated with the AAV -Lib1 Library.
  • FIG.51A is a schematic showing the amino acid residues in VRI with a box highlighting position P6, including the two amino acid residues at P6 for the AAV -Lib1 library: alanine (A) and glycine (G).
  • FIG.51B shows a density plot of the amount of vector DNA in the liver (Log2(Fold)) for the sum of all capsids in AAV -Lib1 that include an alanine (A) at P6 (amino acid position 266) and a density plot of the amount of vector DNA in the liver (Log2(Fold)) for the sum of all capsids in AAV -Lib1 that include a glycine (G) at P 6 (amino acid position 266).
  • A alanine
  • G glycine
  • FIG.51C is an empirical cumulative distribution function (eCDF) plot showing the amount of vector DNA in the liver (Log2(Fold)) for the sum of all capsids in AAV -Lib1 that include an alanine (A) at P 6 (amino acid position 266) and the amount of vector DNA in the liver (Log2(Fold)) for the sum of all capsids in AAV -Lib1 that include a glycine (G) at P 6 (amino acid position 266).
  • FIG.52A is a schematic showing the amino acid residues in VRI with boxes highlighting positions of interest: P1, P3, P5, P6, P8, and P12.
  • FIG.52A provides an example formula for calculating an enrichment score for the modified capsid proteins in the AAV -Lib1 library.
  • FIG.52B is an empirical cumulative distribution function (eCDF) plot showing the amount of vector DNA in the liver (Log2(Fold)) for the sum of all capsids in the AAV -Lib1 library that include either an alanine (A), a glutamate (E), a glutamine (Q), and a threonine (T) at P 3 (amino acid position 263).
  • eCDF empirical cumulative distribution function
  • FIG.52C is an empirical cumulative distribution function (eCDF) plot showing the amount of vector DNA in the liver (Log2(Fold)) for the sum of all capsids in the AAV -Lib1 library that include either an alanine (A) or a glycine (G) at P 5 (amino acid position 265).
  • eCDF empirical cumulative distribution function
  • FIG.52D is an empirical cumulative distribution function (eCDF) plot showing the amount of vector DNA in the liver (Log2(Fold)) for the sum of all capsids in the AAV -Lib1 library that include either an alanine (A) or a glycine (G) at P 6 (amino acid position 266).
  • FIG.53A is an enrichment plot showing the amount of vector DNA in the liver for the sum of all the capsids the AAV -Lib1 library that include either an alanine (A) or a glycine (G) at P6 (amino acid position 266).
  • FIG.53B is an enrichment plot showing the amount of vector DNA in the liver for the sum of all the capsids the AAV -Lib1 library having a P 5 P 6 combination of AA, AG, GA, or GG.
  • FIG.53C is an enrichment plot showing the amount of vector DNA in the liver for the sum of all the capsids in the AAV -Lib1 library having a P 3 , P 5 , and P 6 combination of AAA, AAG, AGA, AGG, EAA, EAG, EGA, EGG, QAA, QAG, QGA, QGG, TAA, TAG, TGA, and TGG (appearing from left to right on the x-axis).
  • FIG.54 shows output of a network analysis of data from the AAV -Lib1 library. Each circle indicates a capsid variant and a line connecting the capsids indicates sequence similarity.
  • FIG.55 is an illustration of the structure of an AAV VP1 protein with positions P 3 , P 5 , P 6 , and P 8 in VRI highlighted.
  • FIG.56A is an empirical cumulative distribution function (eCDF) plot showing RNA expression in the liver (Log2(Fold)) for the sum of all capsids in the AAV -Lib1 library that include an alanine (A), a glutamate (E), a glutamine (Q), or a threonine (T) at P 3 (amino acid position 263).
  • eCDF empirical cumulative distribution function
  • FIG.56B is an empirical cumulative distribution function (eCDF) plot showing RNA expression in the heart (Log2(Fold)) for the sum of all capsids in the AAV -Lib1 library that include an alanine (A), a glutamate (E), a glutamine (Q), or a threonine (T) at P 3 (amino acid position 263).
  • eCDF empirical cumulative distribution function
  • FIG.56C is an empirical cumulative distribution function (eCDF) plot showing RNA expression in the liver (Log2(Fold)) for the sum of all capsids in the AAV -Lib1 library that include either an alanine (A) or a glycine (G) at P 5 (amino acid position 265).
  • eCDF empirical cumulative distribution function
  • FIG.56D is an empirical cumulative distribution function (eCDF) plot showing the RNA expression in the heart (Log2(Fold)) for the sum of all capsids in the AAV- Lib1 library that include either an alanine (A) or a glycine (G) at P 5 (amino acid position 265).
  • FIG.56E is an empirical cumulative distribution function (eCDF) plot showing RNA expression in the liver (Log2(Fold)) for the sum of all capsids in the AAV -Lib1 library that include either an alanine (A) or a glycine (G) at P 6 (amino acid position 266).
  • FIG.56F is an empirical cumulative distribution function (eCDF) plot showing RNA expression in the heart (Log2(Fold)) for the sum of all capsids in the AAV -Lib1 library that include either an alanine (A) or a glycine (G) at P6 (amino acid position 266).
  • FIG.57A shows enrichment in liver DNA (x-axis) plotted against enrichment in liver RNA (y-axis) for capsids comprising various combination of amino acid modifications at P3, P5, and P6. Positions of AAV mut1 and AAV9 controls are indicated by arrows and corresponding text.
  • FIG.57B shows enrichment in liver DNA (x-axis) plotted against enrichment in heart RNA (y-axis) for capsids comprising various combination of amino acid modification at P 3 , P 5 , and P 6 . Positions of AAV mut1 and AAV9 controls are indicated by arrows and corresponding text.
  • FIG.58A shows enrichment in liver DNA (x-axis) plotted against enrichment in liver RNA (y-axis) for capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid modifications at P 3 , P 5 , and P 6 , respectively.
  • T threonine
  • G glycine
  • G glycine
  • FIG.58B shows enrichment in liver DNA (x-axis) plotted against enrichment in heart RNA (y-axis) for capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid modifications at P3, P5, and P6, respectively.
  • Positions of AAV mut1 and AAV9 controls are indicated by arrows and corresponding text.
  • FIG.58C shows enrichment in liver DNA (x-axis) plotted against enrichment in heart RNA (y-axis) for capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid modifications at P3, P5, and P6, respectively.
  • Sub-populations of Atty Docket No.: 38053-57988/WO (112WO) the capsids having a threonine (T) (yellow dots) or an asparagine (N)/a serine (S) (blue dots) at position P1 are color-coded.
  • Positions of AAV mut1 and AAV9 controls are indicated by arrows and corresponding text.
  • FIG.58D shows enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid residues at P 3 , P 5 , and P 6 , respectively.
  • T threonine
  • G glycine
  • G glycine
  • Capsids having a particular amino acid residue (or combinations of amino acid residues) at positions P1, P2, P4, and P12 e.g., an asparagine (N), a glycine (G), a threonine (T), a serine (S), and/or an alanine (A)
  • N asparagine
  • G glycine
  • T threonine
  • S serine
  • A alanine
  • Particular subpopulations having NGTT, NSTT, SGAA, and SGAT at positions P1, P2, P4, and P12 are identified.
  • Positions of AAV mut1 and AAV9 controls are indicated by arrows and corresponding text.
  • FIG.58E shows enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for capsids having a glutamine (Q), a glycine (G), and a glycine (G) combination of amino acid residues at P3, P5, and P6, respectively.
  • Each dot represent a capsid having TGG at P 3 , P 5 , and P 6 .
  • a subpopulation having a serine (S), a glycine (G), a threonine, and a histidine (H) at positions at positions P 1 , P 2 , P 4 , and P 12 is identified.
  • FIG.58F shows average enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for all AAV -Lib1 capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid residues at P3, P5, and P6, respectively.
  • Positions of AAV mut1 and AAV9 controls are indicated by corresponding text.
  • FIG.58G shows enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for 24 selected capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid residues at P3, P5, and P6, respectively.
  • T threonine
  • G glycine
  • G glycine
  • the 24 selected capsids correspond to groups: 1 NSTSGGP 7 P 8 NDNH (e.g., SEQ ID NO: 44927); group 2: NSTTGGP7P8NDNH (SEQ ID NO: 44928); group 4: SGTAGGP7P8NDNT (SEQ ID NO: 44930); group 5: SGTSGGP7P8NDNA (SEQ ID NO: 44931); group 6: SGTTGGP 7 P 8 NDNT (SEQ ID NO: 44932); and group 7: SSTAGGP 7 P 8 NDNA (SEQ ID NO: 44933). Positions of AAV mut1 and AAV9 controls are indicated by corresponding text.
  • FIGs.58H-58I shows enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for 6 groups where each group has a threonine (T), a Atty Docket No.: 38053-57988/WO (112WO) glycine (G), and a glycine (G) combination of amino acid residues at P3, P5, and P6, respectively.
  • FIG.58H includes dark circles that identify the VR1 mini-Lib peptide segments in the key.
  • FIG.58I includes dark circles that identify the VR1 mini-Lib peptide segments in the key.
  • FIG.59 shows vector genome (DNA) in liver of NHP treated with the AAV- Lib1 Library.
  • FIG.60 is a box plot showing the distribution of liver RNA log2FC enrichment score from capsids in all sequence groups defined by P3, P5, P6, P8 positions (see also positions P3, P5, P6, P8 in VR I).
  • FIG.61 is a plot showing liver enrichment for rAAV from AAV- Lib1 in mice (y-axis) versus liver enrichment for rAAV from AAV- Lib1 in NHP (x-axis).
  • FIGs.62A-62E shows non-limiting examples of combination of targeting peptides, comprising: variable triplets, a constant RGD, a variable quad, and, optionally, peptide segments in the AAV-Lib3 library in Example 27.
  • FIG.62A shows possible amino acid residues in the variable triplet corresponding X 1 , X 2 , and X 3 where the color indicates the type of amino acid.
  • FIG.62B shows the constant RGD quad that is in each targeting peptide.
  • FIG.62C shows the amino acid residue category (genus) and specific quads.
  • FIG. 62D shows non-limiting examples of peptide segments present in VR I.
  • FIG.62E shows amino acids at positions X 7 , X 8 , X 9 , and X 10 , where the color indicates the type of amino acid residue.
  • FIGs.63A-63B show an enrichment plots (Avg_logFC versus Inverse CV (Coefficient of Variation)) for capsids in the AAV -Lib2 library.
  • FIG.63A is an enrichment plot showing capsid performance in muscle (i.e., all muscle tissue) for all 50,500 capsids in the AAV -Lib2 library.
  • FIG.63B shows enrichment plots for capsids from the AAV -Lib2 library having a quad selected from: FNNL, FNNT, and RGQI.
  • FIG.64 shows an enrichment plot of the amount of vector RNA in muscle tissue for the sum of all the capsids in the AAV -Lib2 library having the indicated quad sequences. Atty Docket No.: 38053-57988/WO (112WO)
  • FIG.65 shows a heatmap of tissue enrichment scores in muscle tissues for each of the capsids in the AAV -Lib2 library.
  • AAV -Lib2 library includes about 50,500 capsids having one of the 20 quad and one of XX triplet sequences.
  • FIG.66A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV -Lib2 library having a quad selected from: RGQI, RSVV, and RGVV (Group 1). The top 100 capsids are identified by the larger of the two dots on the plot.
  • FIG.66B shows a motif plot of the targeting peptides for the top 100 capsids in the AAV -Lib2 library having a quad selected from: RGQI, RSVV, and RGVV (Group 1). Data used to generate the motif plot is averaged across all muscle tissue.
  • FIG.66C shows a plot of unique variant count for the indicated triplets for the top 100 capsids in the AAV -Lib2 library having a quad selected from: RGQI, RSVV, and RGVV (Group 1).
  • FIG.67A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV -Lib2 library having a quad selected from: HGVL, YSSV, YSTM, and YTSV (Group 2).
  • the top 100 capsids are identified by the larger of the two dots on the plot.
  • FIG.67B shows a motif plot of the targeting peptides for the top 100 capsids in the AAV -Lib2 library having a quad selected from: HGVL, YSSV, YSTM, and YTSV (Group 2). Data used to generate the motif plot is averaged across all muscle tissue.
  • FIG.67C shows a plot of unique variant count for the indicated triplets for the top 100 capsids in the AAV -Lib2 library having a quad selected from: HGVL, YSSV, YSTM, and YTSV (Group 2).
  • FIG.68A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV -Lib2 library having a quad selected from: FNNT, FNNL, FQNT, and YNSL (Group 3).
  • the top 100 capsids are identified by the larger of the two dots on the plot.
  • FIG.68B shows a motif plot of the targeting peptides for the top 100 capsids in the AAV -Lib2 library having a quad selected from: FNNT, FNNL, FQNT, and YNSL (Group 3). Data used to generate the motif plot is averaged across all muscle tissue. Atty Docket No.: 38053-57988/WO (112WO) [00141]
  • FIG.68C shows a plot of unique variant count for the indicated triplets for the top 100 capsids in the AAV -Lib2 library having a quad selected from: FNNT, FNNL, FQNT, and YNSL (Group 3).
  • FIG.69 shows an enrichment plot of the amount of vector RNA in liver tissue for the sum of all the capsids in the AAV -LIB2 library having the indicated quad sequences.
  • FIG.70A shows enrichment scores for liver tissue for each of the capsids in the AAV -Lib2 library having a quad selected from: RGQI, RSVV, RGVV, RQGI, and RSQT. The top 100 capsids are identified by the larger of the two dots on the plot.
  • FIG.70B shows a motif plot of the targeting peptides for the top 100 capsids in the AAV -Lib2 library (from FIG.70A) having a quad selected from: RGQI, RSVV, RGVV, RQGI, and RSQT. Data used to generate the motif plot is averaged across the liver tissue.
  • FIG.71A shows an enrichment score plots (inverse VC versus AverageMN_FC) for biceps femoris, quadriceps, diaphragm, heart – atria, heart – ventricle, and liver for for each of the capsids in the AAV -Lib2 library.
  • the top 10 performing capsids are identified by the darker shaded circles.
  • FIG.71B shows a table of enrichment scores for the top 10 capsids, where the top 10 capsids include the indicated targeting peptide.
  • FIG.71C shows a table of the amino acid residues (at each position for the targeting peptides described in FIG.71B and shown also in FIG.71A.
  • FIG.72A shows an enrichment score plot (LogFC mean versus inverse CV (count of variants)) for capsids having the indicated quads. Targeting peptides with specific triplets are identified.
  • FIG.72B shows a table of the amino acid residues at each position or the targeting peptides described in FIG.72A.
  • FIG.73A shows enrichment score plot (LogFC mean versus inverse CV (count of variants)) for capsids having the indicated quads. Targeting peptides with specific triplets are identified.
  • FIG.73B shows a table of the amino acid residues (and properties for the respective amino acid residues (e.g., polar)) for the targeting peptides described in FIG.73A Atty Docket No.: 38053-57988/WO (112WO)
  • FIG.74A shows enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV -Lib2 library. The 10 capsids are highlighted.
  • FIG.74B shows enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV -Lib2 library. The 10 capsids are highlighted.
  • FIG.75A shows enrichment scores in skeletal muscle versus enrichment scores heart for each of the capsids in the AAV -Lib2 library.
  • FIG.75B shows enrichment scores in skeletal muscle versus enrichment scores in heart for each of the capsids in the AAV -Lib2 library having the quad “LIGR”.
  • FIG.75C shows enrichment scores in skeletal muscle versus enrichment scores in heart for each of the capsids in the AAV -Lib2 library having the quad (“QSTL”).
  • FIG.75D shows enrichment scores in skeletal muscle versus enrichment scores in heart for each of the capsids in the AAV -Lib2 library having the quad (“RGVV”).
  • FIG.75E shows enrichment scores for heart over enrichment scores in skeletal muscle for each of the capsids in the AAV -Lib2 library having the indicated quads.
  • FIGs.76A, 76B, and 76C show non-limiting examples of combination of targeting peptides, comprising: variable triplets, a constant RGD, a variable quad, and optionally, VR1 peptide segments in the AAV -Lib3 library in Example 27.
  • FIG.76A shows possible amino acid residues in the variable triplet corresponding X1, X2, and X3 where the color indicates the type of amino acid.
  • FIG.76B shows the functional category, VR1 peptide, VR8 insert name, conserved RGD, and specific quads.
  • FIG.76C shows non- limiting examples of peptide segments present in VR I.
  • FIG.77 shows the scaled enrichment scores of capsids having a targeting peptide with the indicated VR1 and VR8 modifications.
  • Mut1_2679 is an AAV9 capsid with a Mut1 modification in VR1 region and a targeting peptide with the sequence of SAQRGDARTL (SEQ ID NO: 98979) in VR8 region.
  • AAV9_DV6 is an AAV9 capsid with a wild type VR1 region and a targeting peptide with the sequence of ENRRGDFNNL (SEQ ID NO: 44864) in VR8 region.
  • 47_DV6 is an AAV9 capsid with a 47VR1 modification and a targeting peptide with the sequence of ENRRGDFNNL (SEQ ID NO: 44864) in VR8 region.
  • 49_DV6 is an AAV9 capsid with a 49VR1 modification and a targeting peptide with the sequence of ENRRGDFNNL (SEQ ID NO: 44864) in VR8 region.
  • 47_1000 is an AAV9 capsid with a 47VR1 modification and a Atty Docket No.: 38053-57988/WO (112WO) targeting peptide with the sequence of ENRRGDFNNT (SEQ ID NO: 44880) in VR8 region.
  • AAV9_1000 is an AAV9 capsid with a wild type VR1 region and a targeting peptide with the sequence of ENRRGDFNNT (SEQ ID NO: 44880) in VR8 region.
  • 50_1000 is an AAV9 capsid with a 50VR1 modification and a targeting peptide with the sequence of ENRRGDFNNT (SEQ ID NO: 44880) in VR8 region.
  • AAV9_DV8 is an AAV9 capsid with a wild type VR1 region and a targeting peptide with the sequence of ENRRGDFQNT (SEQ ID NO: 44866) in VR8 region.
  • 47_DV8 is an AAV9 capsid with a 47VR1 modification and a targeting peptide with the sequence of ENRRGDFQNT (SEQ ID NO: 44866) in VR8 region.
  • Mut1_20169 is an AAV9 capsid with a Mut1 modification in VR1 region and a targeting peptide with the sequence of SAQRGDHVNL (SEQ ID NO: 104033) in VR8 region.
  • Mut1_38181 is an AAV9 capsid with a Mut1 modification in VR1 region and a targeting peptide with the sequence of SAQRGDRGQI (SEQ ID NO: 48391) in VR8 region.
  • AAV9_38181 is an AAV9 capsid with a wild type VR1 region and a targeting peptide with the sequence of SAQRGDRGQI (SEQ ID NO: 48391) in VR8 region.
  • Mut1_38249 is an AAV9 capsid with a Mut1 modification in VR1 region and a targeting peptide with the sequence of SAQRGDRGVV (SEQ ID NO: 48394) in VR8 region.
  • Mut1_39374 is an AAV9 capsid with a Mut1 modification in VR1 region and a targeting peptide with the sequence of SAQRGDRNVV (SEQ ID NO: 129300) in VR8 region.
  • Mut1_39441 is an AAV9 capsid with a Mut1 modification in VR1 region and a targeting peptide with the sequence of SAQRGDRQGI (SEQ ID NO: 131827) in VR8 region.
  • Mut1_40049 is an AAV9 capsid with a Mut1 modification in VR1 region and a targeting peptide with the sequence of SAQRGDRSVV (SEQ ID NO: 136881) in VR8 region.
  • FIGs.78A, 78B, and 78C show the enrichment scores in muscle versus liver for the capsids in the AAV -Lib3 library.
  • FIG.78A shows the overall enrichment scores in muscle versus liver for the all capsids in the AAV -Lib3 library.
  • ATLVT022XXAAV9_1000 is an AAV9 capsid with a wild type VR1 region and a targeting peptide with the sequence of ENRRGDFNNT (SEQ ID NO: 44880) in VR8 region.
  • ATLVT022XXAAV9_DV6 is an AAV9 capsid with a wild type VR1 region and a targeting peptide with the sequence of ENRRGDFNNL (SEQ ID NO: 44864) in VR8 region.
  • ATLVT022XXAAV9_38181 is an AAV9 capsid with a wild type VR1 region and a targeting peptide with the sequence of SAQRGDRGQI (SEQ ID NO: 48391) in VR8 region.
  • ATLVT022XXAAV9 is the wild type AAV9 capsid.
  • FIG.78B shows a zoomed in view of Atty Docket No.: 38053-57988/WO (112WO) FIG.78A with the muscle enrichment score between 0.83 and 0.95.
  • FIG.78C shows the enrichment scores in muscle versus liver in capsids having different VR1 variants.
  • FIG.79 shows the box plot of enrichment scores in muscle tissue for the capsids in the AAV -Lib3 library having the indicated quad sequences and the indicated VR1 sequences.
  • FIG.80 shows that the performance of selected quads with different VR1 backgrounds, measured in the AAV -Lib2 library study (x-axis) or AAV -Lib3 library study (y- axis).
  • FIG.81 shows a heatmap of tissue enrichment scores in muscle tissues for each of the capsids in the AAV -Lib3 library.
  • Data for each capsid appears as a cell in the heatmap where the row indicates the quad and VR1 modification and the column indicates the triplet.
  • Data is grouped using hierarchical clustering. Clustering identified Quads cluster1, Quads cluster2, Quads cluster3, Triplets cluster1, Triplets cluster2, Triplets cluster3, and Triplets cluster4.
  • FIGs.82A, 82B, 82C, and 82D show motif plots that identify the enriched motifs of Quads cluster 1, with FIG.82A showing the motif plot of Quads cluster 1 combined with Triplets cluster1, FIG.82B showing the motif plot of Quads cluster 1 combined with Triplets cluster2, FIG.82C showing the motif plot of Quads cluster 1 combined with Triplets cluster3, and FIG.82D showing the motif plot of Quads cluster 1 combined with Triplets cluster4.
  • FIGs.83A, 83B, and 83C show motif plots that identify the enriched motifs of Quads cluster 2, with FIG.83A showing the motif plot of Quads cluster 2 combined with Triplets cluster1, FIG.83B showing the motif plot of Quads cluster 2 combined with Triplets cluster2, and FIG.83C showing the motif plot of Quads cluster 2 combined with Triplets cluster3.
  • FIG 84 show motif plots that identify the enriched motifs of Quads cluster 3 combined with Triplets cluster1.
  • FIGs.85A and 85B show the effect of liver de-targeting by VR1 sequences and by quad sequences, with FIG.85A showing the effect of liver de-targeting by VR1 sequences and FIG.85B showing the effect of liver de-targeting by quad sequences.
  • FIG.86 shows a heatmap of tissue enrichment scores in liver tissues for each of the capsids in the AAV -Lib3 library. Data for each capsid appears as a cell in the heatmap where the row indicates the quad and the VR1 modification and the column indicates the triplet. Data is grouped using hierarchical clustering. Clustering identified liver Cluster 1, liver Cluster 2, and liver Cluster 3.
  • FIGs.87A, 87B, and 87C show motif plots that identify the enriched triplet motifs of liver Cluster 1, liver Cluster 2, and liver Cluster 3, with FIG.87A showing the triplet motif plot of liver Cluster 1, FIG.87B showing the triplet motif plot of liver Cluster 2, and FIG.87C showing the triplet motif plot of liver Cluster 3.
  • FIGs.88A and 88B show the heart versus skeletal muscle enrichment scores of the capsids in the AAV -Lib3 library.
  • FIG.88A shows the overall enrichment scores in skeletal muscle versus heart muscle for the all capsids in the AAV -Lib3 library.
  • FIG.88B shows the enrichment scores for heart muscle over skeletal muscle for the capsids in the AAV -Lib3 library having the indicated quads.
  • FIG.89 shows the percent GFP+ cells in cervical DRG, thoracic DRG, and lumbar DRG for wild type AAV9 and AAV9 comprising an M3 targeting peptide, an M1 targeting peptide with a wild type VRI, or an M1 targeting peptide with mut1 VRI substitution.
  • FIGs.90A and 90B show the levels of liver enzymes for wild type AAV9 and AAV9 comprising an M3 targeting peptide, an M1 targeting peptide with wild type VRI, or an M1 targeting peptide with mut1 VRI substitution, with FIG.90A showing the level of alanine transaminase (ALT) and FIG.90B showing the level of aspartate transaminase (AST).
  • ALT alanine transaminase
  • AST aspartate transaminase
  • FIGs.91A and 91B show the eGFP expression in heart tissues of mice received M1 (AAV9-38181-mut1) or AAV9 expressing eGFP at multiple dose levels, with FIG.91A showing the immunohistochemistry (IHC) staining in heart tissue sections of mice received M1 or AAV9 and FIG.91B showing the RT-ddPCR results of heart tissues of mice received M1 or AAV9.
  • FIG.92 shows eGFP mRNA expression in the heart tissues of mice received M1 or M3 at post-injection day 28, day 60, and day 90.
  • FIGs.93A, 93B, and 93C show the transcription efficiency of eGFP in heart tissues of mice received M1, M3, or AAV9 at multiple dose levels, with FIG.93A showing Atty Docket No.: 38053-57988/WO (112WO) the GFP mRNA level in heart tissues of mice, FIG.93B showing the GFP DNA level in heart tissues of mice, and FIG.93C showing the mRNA/DNA ratio of M1 and M3 over AAV9.
  • FIG.94 shows the immunohistochemistry (IHC) staining in heart, liver, and DRG tissue sections of NHPs received M1, M3 or AAV9 expressing eGFP at high dose level.
  • IHC immunohistochemistry
  • FIG.95 shows the immunohistochemistry (IHC) staining in heart tissue sections of NHPs received M1, M3 or AAV9 expressing eGFP at low dose level and high dose level.
  • FIGs.96A and 96B show the percentage of GFP+ cells in different heart regions of NHPs received M1, M3 or AAV9 expressing eGFP, with FIG.96A showing the percentage of GFP+ cells with low dose of M1, M3 or AAV9 and FIG.96B showing the percentage of GFP+ cells with high dose of M1, M3 or AAV9.
  • FIGs.97A and 97B show the GFP mRNA level in different heart regions of NHPs received M1, M3 or AAV9 expressing eGFP, with FIG.97A showing the GFP mRNA level with low dose of M1, M3 or AAV9 and FIG.97B showing the GFP mRNA level with high dose of M1, M3 or AAV9.
  • FIG.98 shows the immunohistochemistry (IHC) staining in different heart region sections of NHPs received M1 or AAV9 expressing eGFP at low dose level.
  • FIG.99 shows the GFP mRNA level in skeletal muscle of NHPs received M1, M3 or AAV9 expressing eGFP at low dose level and high dose level.
  • FIG.100 shows the immunohistochemistry (IHC) staining in different skeletal muscle tissue sections of NHPs received M1, M3 or AAV9 expressing eGFP at high dose level.
  • FIGs.101A and 101B show the level of AAV9 and the modified AAV9 comprising the indicated targeting peptide in the blood circulation of NHPs at various time points, with FIG.101A showing the data calculated as VGC/ng of whole blood and FIG. 101B showing the data calculated as VGC/mL of whole blood.
  • FIGs.102A and 102B show the levels of liver enzymes in cynomolgus macaques injected with wild type AAV9 or M1, with FIG.102A showing the level of alanine transaminase (ALT) and FIG.102B showing the level of aspartate transaminase (AST). Dotted line denotes maximum levels reported in healthy cynomolgus macaques. Atty Docket No.: 38053-57988/WO (112WO) 6. DETAILED DESCRIPTION 6.1. Definitions [00186] “AAV” is adeno-associated virus and may be used to refer to the virus itself or derivatives thereof.
  • AAV capsid protein or simply “capsid protein” refers to a VP1, VP2, or VP3 capsid protein.
  • the AAV capsid protein is a wild type or modified capsid protein of AAV9; AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56- B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh.19-B; rh.49-B; rh.52-B
  • modified AAV capsid protein or simply “modified capsid protein” refers to a capsid protein that is modified as compared to such naturally occurring or synthetic / artificial capsid protein, which is referred to as the “reference AAV capsid protein” or “reference capsid protein.”
  • the reference AAV capsid protein as used herein may be a VP1, VP2, or VP3 capsid protein of a naturally occurring AAV variant or a non- naturally occurring VP1, VP2, or VP3 capsid protein that is known in the art.
  • targeting peptide refers to a 10 amino acid sequence (X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 ) within the variable region VIII (VRVIII) of a modified AAV capsid protein introduced by one or more modifications described herein.
  • AAVs comprising a modified capsid protein with a targeting peptide can have localization and distribution in a target cell, tissue or organ different from the AAV with a capsid protein without the target peptide.
  • peptide segment refers to a part of variable region I (VR I) of an AAV capsid protein comprising 10, 11, or 12 amino acids. In preferred embodiments, the peptide segment is positioned between amino acid 250 and 280 of the AAV capsid protein.
  • a modified AAV capsid protein provided herein includes a peptide segment having a sequence different from the corresponding sequence of a reference AAV capsid protein by having one or more modifications.
  • the peptide segment can be referred to as P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 10 P 11 P 12 within VR I where “Pn” refers to a position in the peptide segment.
  • amino acid position within an AAV capsid protein as here herein refers to a position of an amino acid residue in an AAV VP1 protein sequence, counted from the first amino acid in the N terminal.
  • amino acid comprises naturally occurring L- and D- amino acids and artificial, i.e. non-naturally occurring, ⁇ -amino acids.
  • the amino acid is a naturally occurring amino acid.
  • the amino acid is a naturally occurring L- ⁇ -amino acid.
  • the indication that an insertion site is at amino acid position X means that the targeting peptide is inserted between amino acids X and X+l, i.e., the targeting peptide is inserted after the indicated amino acid.
  • the term “liver off” is used herein to describe an AAV having a lower tropism to liver or less biodistribution in liver when administered to a mammalian subject compared to other AAV variants.
  • liver off is also used to describe a modification in the AAV capsid protein that reduces the tropism to liver or biodistribution in liver when administered to a mammalian subject.
  • liver off is also used to describe a species of an AAV capsid with a variable region 1 (VRI) that reduces the tropism to liver or biodistribution in liver when administered to a mammalian subject.
  • VRI variable region 1
  • liver on is used herein to describe an AAV having a higher tropism to liver or more biodistribution in liver when administered to a mammalian subject compared to other AAV variants.
  • liver on is also used to describe a modification in the AAV capsid protein that increases the tropism to liver or biodistribution in liver when administered to a mammalian subject.
  • CAG when used in relation to a promoter or ERE refers to a promoter or ERE with chicken beta actin promoter and CMV enhancer sequences.
  • ERE constitutive promoter or ERE as used herein refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • expression regulatory element or “ERE” as used herein in the context of the rAAV of the disclosure refers to a nucleic acid sequence which is required for expression of the MTM1 coding sequence operably linked to the ERE.
  • an ERE sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product, for example exon sequences.
  • the term “functional fragment” in the context of the myotubularin or MTM1 refers to a biologically functional fragment of myotubularin or MTM1. As would be understood in the art, a biologically functional fragment is a portion or portions of a full length sequence that retain a biological function of the full length sequence. An exemplary functional fragment corresponds to amino acids 29-486 of SEQ ID NO:165 (and is disclosed herein as SEQ ID NO:164).
  • Biological functions of MTM1 include the ability cleave or hydrolyze an endogenous phosphoinositide substrate known in the art, or an artificial phosphoinositide substrate for in vitro assays (i.e., a phosphoinositide phosphatase activity), to recruit and/or associate with other proteins such as, for example, the GTPase Rab5, the PI 3-kinase Vps34 or Vps15 (i.e., proper localization), or treat myotubular myopathy.
  • a phosphoinositide phosphatase activity i.e., a phosphoinositide phosphatase activity
  • the term “functional variant” in the context of the myotubularin or MTM1 refers to various splicing isoforms, variants, fusion proteins, and modified forms of the wildtype MTM1 polypeptide or a functional fragment thereof. Such isoforms, bioactive fragments or variants, fusion proteins, and modified forms of the MTM1 polypeptides retain at least one biological function of the full length MTM1 protein (e.g., a protein of SEQ ID NO:165).
  • inducible promoter or ERE refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • internalizing moiety refers to a moiety capable of interacting with a target tissue or a cell type to effect delivery of the attached molecule into Atty Docket No.: 38053-57988/WO (112WO) the cell (i.e., penetrate desired cell; transport across a cellular membrane).
  • an MTM1 polypeptide encoded by the rAAV of the disclosure can be a fusion protein comprising an internalizing moiety.
  • the internalizing moiety selectively, although not necessarily exclusively, targets and penetrates muscle cells.
  • the internalizing moiety has limited cross-reactivity, and thus preferentially targets a particular cell or tissue type.
  • suitable internalizing moieties include, for example, antibodies, monoclonal antibodies, or derivatives or analogs thereof.
  • Other internalizing moieties include for example, homing peptides, receptors, and ligands.
  • the internalizing moiety mediates transit across cellular membranes via an ENT2 transporter.
  • ITR inverted terminal repeat
  • liver-toggle mutant refers to a capsid protein comprising a sequence different from a reference AAV capsid protein by having one or more mutations (e.g., amino acid substitutions) that alter tropism, specificity or distribution in a liver as compared to the reference AAV capsid protein when administered to a mammalian subject (such a sequence difference referred to herein as a “liver toggle mutation”).
  • mutations e.g., amino acid substitutions
  • the mammalian subject can be a human, non-human primate (NHP), mice, rats, birds, rabbits, guinea pigs, hamsters, farm animals (including pigs and sheep), dogs, or cats.
  • Exemplary liver toggle mutations are disclosed in WO2019/217911 and WO2021/050614, incorporated by reference in their entireties herein.
  • the liver toggle mutations comprise (i) an alanine (A) or guanine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1 and/or b) a lysine (K) or arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1.
  • a liver-toggle mutant of a reference AAV capsid protein is a capsid protein comprising a sequence different from the reference AAV capsid protein by having an alanine (A) amino acid residue at an amino acid position corresponding to position 267 in AAV9 VP1 protein and a threonine (T) Atty Docket No.: 38053-57988/WO (112WO) amino acid residue at an amino acid position corresponding to position 269 in AAV9 VP1.
  • A alanine
  • T threonine
  • the liver toggle mutations comprise a sequence different from the reference AAV capsid protein by having any combination of (i) an arginine (R) instead of serine (S) at position 446; (ii) an alanine (A) instead of an arginine (R) at position 471; and (iii) a threonine (T) or alanine (A) instead of a valine (V) at position 708, in each case numbered according to an AAV2 reference capsid protein (SEQ ID NO:1 of WO2021/050614, which is incorporated by reference herein).
  • MTM1 coding sequence is used herein to refer to a specific sequence of nucleotides in a polynucleotide, such as an rAAV genome or mRNA produced thereby, that encodes an MTM1 polypeptide.
  • MTM1 polypeptide refers to a polypeptide comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to human MTM1 (SEQ ID NO:165) or a functional fragment (e.g., SEQ ID NO:164) or functional variant thereof.
  • operably linked refers to the functional relationship of the nucleic acid sequences with regulatory sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences and indicates that two or more DNA segments are joined together such that they function in concert for their intended purposes.
  • operative linkage of nucleic acid sequences, typically DNA, to a regulatory sequence or promoter region refers to the physical and functional relationship between the DNA and the regulatory sequence or promoter such that the transcription of such DNA is initiated from the regulatory sequence or promoter, by an RNA polymerase that specifically recognizes, binds and transcribes the DNA.
  • parenteral administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
  • peptide”, “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • rAAV refers to a recombinant adeno-associated viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide, sometimes referred to herein as a “genome”.
  • rAAV can include a genome that comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome), such as a heterologous polynucleotide encoding a gene delivered to a mammalian cell such as the MTM1 gene.
  • the heterologous nucleotide is sometimes referred to as a transgene.
  • self-complementary” rAAV vector or genome as used herein means a fully or partially self-complementary rAAV vector or genome, respectively.
  • a “fully self-complementary” rAAV vector refers to a vector containing a genome generated by the absence of a terminal resolution site (TR) from one of the ITRs of the rAAV. The absence of a TR prevents the initiation of replication at the vector terminus where the TR is not present.
  • fully self-complementary rAAV vectors generate single-stranded, inverted repeat genomes, with a wild-type (wt) AAV TR at each end and a mutated TR (mTR) in the middle.
  • a fully self-complementary rAAV genome is typically a single stranded polynucleotide having, in the 5′ to 3′ direction, a first ITR sequence, a heterologous sequence (e.g., MTM1 coding sequence and/or ERE), a second ITR sequence, a second heterologous sequence that is complementary to the first heterologous sequence, and a third ITR sequence.
  • a heterologous sequence e.g., MTM1 coding sequence and/or ERE
  • a “partially self-complementary” rAAV genome refers to a single stranded polynucleotide having, in the 5′ to 3′ direction or the 3′ to 5′ direction, a first ITR sequence, a heterologous sequence (e.g., MTM1 coding sequence and/or ERE), a second ITR sequence, and a self-complementary region that is complementary to a portion of the heterologous sequence and has a length that is less than the entire length the heterologous sequence.
  • a heterologous sequence e.g., MTM1 coding sequence and/or ERE
  • tissue-specific promoter or ERE refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • treatment refers to generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be Atty Docket No.: 38053-57988/WO (112WO) prophylactic in terms of completely or partially preventing a disease, condition, or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition.
  • Treatment covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition (e.g., arresting its development); or (c) relieving the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).
  • vector refers to an rAAV that comprises a heterologous polynucleotide, e.g., a transgene.
  • variable region refers to one or more of nine sequence variable regions (e.g., VRI to VRIX) in an AAV capsid protein previously defined by comparison and alignment of various AAV capsid proteins. See e.g., Govindasamy et al., Structurally mapping the diverse phenotype of adeno-associated virus serotype 4, J. Virol (2006); Meyer et al. Structure of the gene therapy vector, adeno- associated virus with its cell receptor, AAVR, eLife (2019).
  • the VRs are known to contain amino acids that contribute to slight differences in surface topologies and distinct functional phenotypes, such as in receptor binding, transduction efficiency, and antigenic re-activity.
  • the relative positions of the VR I, VR IV and VIIII are illustrated in FIG.1, but the specific positions of the variable regions within a capsid protein can vary depending on the capsid protein and/or the sequence alignment method.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below.
  • AAV capsid proteins [00218]
  • One aspect of the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising (i) a targeting peptide at a site within variable region VIII (VR VIII); or (ii) a peptide segment within variable region I (VR I), wherein the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X1, X2, X3, X7, X8, X9 and X 10 are independently selected from any amino acid residue, wherein the peptide segment has an amino acid sequence of P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 and P 1, P 2 , P 3 , P 4 , P 5 , P 6 , P 7 , P 8 , and P12 are independently selected from any amino acid residue.
  • the modified AAV capsid protein comprises both (i) the targeting peptide and (ii) the peptide segment. In some embodiments, the AAV capsid protein comprises the targeting peptide but not the peptide segment. In some embodiments, the AAV capsid protein comprises the peptide segment but not the targeting peptide.
  • AAV adeno-associated virus
  • a modified adeno-associated virus (AAV) capsid protein comprising: a sequence of a reference AAV capsid protein comprising: (i) a targeting peptide at a site within variable region VIII (VR VIII) of the reference AAV capsid protein; and (ii) one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X1, X2, X3, X7, X8, X9 and X 10 are independently selected from any amino acid residue, wherein the peptide segment has an amino acid sequence of P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 and P 1, P 2 , P 3 , P 4 , P 5 , P 6 , P 7 , P 8 , and P12 are independently selected from any amino acid residue.
  • the modified AAV capsid protein does not comprise RGDLLLS (SEQ ID NO: 1).
  • the modified AAV capsid protein has a peptide segment that does not comprise an alanine (A) at P6 and a threonine (T) at P8
  • the modified AAV capsid does not comprise RGDLLLS (SEQ ID NO: 1), and the peptide segment does not comprise an alanine (A) at P 6 and a threonine (T) at P8.
  • the modified AAV capsid protein has one or more amino acid insertions, deletions, substitutions, or combinations thereof as compared to a reference AAV capsid. Atty Docket No.: 38053-57988/WO (112WO) [00224]
  • the reference AAV capsid protein further comprises one or more modifications comprising an amino acid insertion, deletion, substitution, or a combination thereof to introduce the targeting peptide within VR VIII of the reference AAV capsid protein.
  • the capsid protein further comprises one or more modifications outside of the VR I and VR VIII of the reference AAV capsid protein.
  • the reference AAV capsid protein includes one or more modifications comprising an amino acid insertion, deletion, substitution, or a combination thereof to introduce the targeting peptide within VR VIII (e.g., wherein VR VIII corresponds to amino acids between positions 565 and 595 of the reference AAV capsid protein).
  • the reference AAV capsid protein includes one or more modifications outside of VR I (e.g., VR I corresponds to amino acids between position 259 to position 275 of the reference AAV capsid protein) of the reference capsid protein.
  • the reference AAV capsid protein includes one or more modifications outside of VR VIII (e.g., VR VIII corresponds to amino acids between positions 565 and 595 of the reference AAV capsid protein) of the reference AAV capsid protein.
  • the reference AAV capsid protein includes one or more modifications outside of VR I (e.g., VR I corresponds to amino acids between position 259 to position 275 of the reference AAV capsid protein) and VR VIII (e.g., VR VIII corresponds to amino acids between positions 565 and 595 of the reference AAV capsid protein) of the reference AAV capsid protein.
  • the one or more modifications is an amino acid insertion, deletion, substitution, or a combination thereof to introduce the targeting peptide within VR VIII of the reference AAV capsid protein.
  • the one or more modifications is an amino acid insertion, substitution, or a combination in a region outside of the VR VIII region of the reference AAV capsid protein.
  • the one or more modifications outside of the VR VIII is a modification in the VR I region of the reference AAV capsid protein.
  • the one or more modifications results in a liver-off phenotype.
  • the one or more modifications outside of the VR VIII is a liver toggle mutation. 6.2.1.
  • the targeting peptide has a sequence X1X2X3RGDX7X8X9X10 , wherein X1, X2, X3, X7, X8, X9 and X10 are independently selected Atty Docket No.: 38053-57988/WO (112WO) from any amino acid residue.
  • X7, X8, X9 and X10 are independently selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y.
  • X7, X 8 , X 9 and X 10 are independently selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, and V.
  • the modified AAV capsid protein has at least 90% sequence identity to the sequence of the reference AAV capsid protein. In some embodiments, the modified AAV capsid protein has at least 95% sequence identity to the sequence of the reference AAV capsid protein. In some embodiments, the modified AAV capsid protein has at least 96% sequence identity to the sequence of the reference AAV capsid protein. In some embodiments, the modified AAV capsid protein has at least 97% sequence identity to the sequence of the reference AAV capsid protein.
  • the modified AAV capsid protein has at least 98% sequence identity to the sequence of the reference AAV capsid protein. In some embodiments, the modified AAV capsid protein has at least 99% sequence identity to the sequence of the reference AAV capsid protein.
  • X 1 , X 2 , and X 3 are independently selected from any amino acid residue.
  • X1, X2, and X3 have not been modified from the amino acids at corresponding positions of the reference AAV capsid protein. In this case, X 1 , X 2 , and X 3 are identical to the amino acids at corresponding positions of the reference AAV capsid protein.
  • X 1 , and X 2 have not been modified from the amino acids at corresponding positions of the reference AAV capsid protein.
  • X1 has not been modified from the amino acids at corresponding position of the reference AAV capsid protein.
  • X2 has not been modified from the amino acids at corresponding position of the reference AAV capsid protein.
  • X1, X2, and/or X3 are the natural amino acid residues of the reference AAV capsid protein.
  • X 1 , X 2 , and X 3 can be introduced by amino acid substitutions, insertions, mutations, and/or deletions of the amino acids at the corresponding sites in the reference AAV capsid.
  • any one of the corresponding sites X1, X2, and X 3 in the reference AAV capsid can be deleted or substituted.
  • the Atty Docket No.: 38053-57988/WO (112WO) corresponding site X1 in the reference AAV capsid is deleted or substituted.
  • the corresponding site X2 in the reference AAV capsid is deleted or substituted.
  • the corresponding site X 3 in the reference AAV capsid is deleted or substituted. In some embodiments, the corresponding sites X1, and X2 in the reference AAV capsid are deleted or substituted. In some embodiments, the corresponding sites X2 and X3 in the reference AAV capsid are deleted or substituted. In some embodiments, the corresponding sites X 1 and X 3 in the reference AAV capsid are deleted or substituted. In some embodiments, the corresponding sites X1, X2, and X3 in the reference AAV capsid are deleted or substituted. [00235] In some embodiments, X 1 is selected from S, E, A, D, N, Q, or T. In some embodiments, X1 is D or E.
  • X1 is S, A or T. In some embodiments, X1 is S, A or E. In some embodiments, X1 is selected from S or E. In some embodiments, X1 is S. In some embodiments, X 1 is E. [00236] In some embodiments, X2 is selected from N, A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X2 is selected from N or A. In some embodiments, X 2 is selected from K, E, D, A, S, F, N, V or L. In some embodiments, X 2 is selected from K, E or D.
  • X 2 is selected from N, A or Y. In some embodiments, X2 is selected from N, Y or S. [00237] In some embodiments, X 3 is selected from R, Q, A, D, E, F, G, H, I, K, L, M, N, P, S, T, V, W, or Y. In some embodiments, X 2 is N. In some embodiments, X 2 is A. In some embodiments, X3 is selected from R or Q. In some embodiments, X3 is R. In some embodiments, X 3 is Q. In some embodiments, X 2 is N; and X 3 is R.
  • X 1 is E; X 2 is N; and X 3 is R.
  • X 1 is S; X 2 is N; and X 3 is R.
  • X3 is selected from Y, V or F.
  • X3 is selected from N, A, Y or S.
  • X3 is selected from I, Q, R, V, T or M.
  • X 2 X 3 is NR.
  • X 1 X 2 X 3 is ENR.
  • X1X2X3 is SNR.
  • X1X2X3 is selected from the groups consisting of: DII, DWM, EEI, DML, DWI, SLE, EIN, NHE, DFI, EEL, TEQ, TDA, EDT, NEV, TDW, QFE, EDY, DTT, EPL, SEN, SEQ, TAE, EVN, ELN, DVQ, ETI, EVI, ESV, ETW, SEW, DNW, EVF, EAW, EPF, EIY, EIF, EPY, DVI, DMM, DQI, DHL, DTL, DVL, NDL, DLL, DMQ, NEF, DFL, DIM, TEW, DYI, SDY, DYY, DHF, DKE, DTW, DTI, ELY, TEY, TEI, DAI, Atty Docket No.: 38053-57988/WO (112WO) DQY, DMY, EWG,
  • X 1 X 2 X 3 is selected from the group consisting of: DAV, DKW, EAY, AEY, DFV, DKF, DKI, DKL, DNV, DNY, DSL, DSV, EFI, SEF, SEY, SLY, ADF, ADY, ALY, AVF, DAF, DAL, DAM, DAT, DHV, DIV, DKA, DKM, DKT, DKV, DKY, DMI, DNF, DNI, DQT, DSI, DVY, DYN, DYV, EAT, EAW, EFV, EGL, EIY, EMF, EMY, ENF, EPF, EPY, EQY, ESY, ETF, EWI, EWT, EYI, EYV, NEM, QDF, QDY, QEY, QLY, QND, QVF, QVY, SDL, SDV, SEH, SII, SIY
  • X1X2X3 is selected from the group consisting of: APW, TEL, TDA, QPY, SPN, EHY, DWK, DLK, DFK, DVK, NSI, DIR, SPF, SEL, DRT, DRF, ADL, TDL, SDL, DNY, DKI, NDV, DKM, DNH, DNF, DSS, EST, EWT, DKN, DKS, SEH, ESQ, ESL, QND, EAH, AIF, AVF, QVF, TMY, ALY, NNG, NIF, NTF, NFF, AWF, NPY, SWF, AII, AYF, AQW, NFY, AGP, QQF, TKE, TNG, NSF, NAW, QAG, ERG, NKD, QSG, QNG, EAK, QWF, SWY, TFF, TYF, NYY, QFG, NWA,
  • X 1 X 2 X 3 is selected from the group consisting of: ANY, SNI, AAI, AAM, ANT, AST, AYQ, EHK, ENK, ENR, SFQ, SSI, TAY, TDK, TNT, AAF, AAL, AAY, ADK, AFA, ANF, ANI, ANQ, ANS, AQM, ARE, ASV, AYH, AYT, EMK, EWK, NNM, QAF, QAI, QAM, QAT, QAY, QFT, QGM, QHL, QNF, QNQ, QNS, QNT, QNV, QNY, SAH, SAI, SAL, SFT, SFV, SHI, SHV, SMM, SNF, SNM, SNN, SNQ, SNV, SNY, SQI, SQV, SSL, SWQ, SWS, SYI, SYM, SYN, SYQ, TAM,
  • X 1 X 2 X 3 is selected from the group consisting of: DMK, ATD, EEK, QMD, EFS, ERD, DDR, TDM, SAE, EHS, ENH, SWE, SNE, NNG, QAG, ERG, QSG, QNG, ASG, QFG, AMF, ELR, NFM, NNS, NNI, SDR, EQR, EHR, EWR, EQK, ESR, EKQ, EYR, ENR, EMK, EYK, EHK, EWK, QNI, TNI, TYI, SNY, DRQ, AWI, Atty Docket No.: 38053-57988/WO (112WO) QWI, DFR, EKG, QYG, QWQ, EKN, EKF, EKT, AFH, ENK, NYS, DKH, AAG, QMF, QFH, QKD, ARE, AHQ, ADK,
  • X 1 X 2 X 3 is selected from the group consisting of: ADR, ASI, EFK, EHK, EWK, SYQ, AAF, AAT, AAY, AFI, AFQ, AGI, AGT, AHI, ANH, ANM, ANN, AQI, ASA, ASH, AST, ASV, AWT, AYQ, AYT, DAR, DMK, DQR, DVR, EAR, EFR, EMK, EMR, EQK, ERA, ERS, ESR, NDA, NDR, NMI, NMV, NNM, NNN, NYL, NYM, NYN, NYQ, NYV, QAM, QFQ, QFV, QGV, QNH, QNI, QNM, QQV, QSF, QSY, SAI, SAM, SAS, SDR, SFQ, SGH, SGM, SGT, SGV, SHL, SHM, SHQ, SH
  • the targeting peptide comprises a 7-mer peptide (RGDX7X8X9X10) having an amino acid sequence selected from SEQ ID NOs.: 238-44858.
  • X 1 X 2 X 3 can be the amino acids at the corresponding positions of the reference AAV capsid.
  • X 1 X 2 X 3 can be the naturally occurring amino acids at the corresponding positions of the reference AAV capsid.
  • the targeting peptide comprises a 7-mer peptide (RGDX 7 X 8 X 9 X 10 ) having an amino acid sequence with at least 90%, 95%, 98%, 99% sequence identity to the amino acid sequence selected from SEQ ID NOs.: 238-44858.
  • the modified AAV capsid protein comprises targeting peptide including a 7-mer peptide (RGDX 7 X 8 X 9 X 10 ) having an amino acid sequence selected from SEQ ID NOs.: 238-247.
  • X1X2X3 can be the amino acids at the corresponding positions of the reference AAV capsid.
  • X1X2X3 has a sequence that is different from the sequence at the corresponding positions of the reference AAV capsid. In some embodiments, any one of the corresponding positions X1, X2, X3 of the reference AAV can be substituted with any amino acid.
  • the 7-mer has a sequence selected from SEQ ID NOs.: 238-337. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs.: 238-437. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs.: 238-537. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs.: 238-637.
  • the 7-mer has a sequence selected from SEQ ID NOs.: 238-737. In some embodiments, the 7-mer has a Atty Docket No.: 38053-57988/WO (112WO) sequence selected from SEQ ID NOs.: 238-837. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs.: 238-937. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs.: 238-1037.
  • the targeting peptide comprises a 7-mer peptide (RGD X7 X8 X9 X10) having an amino acid sequence selected from SEQ ID NOs.: 3881, 12092, 14601, 15342, 21498, and 31396.
  • the targeting peptide comprises a 7-mer peptide (RGD X7 X8 X9 X10) having an amino acid sequence of SEQ ID NO: 238.
  • the targeting peptide comprises an amino acid sequence selected from SEQ ID NOs.:.44859- 44883, 44911, 44912, 44913, and 44918- 44919.
  • the targeting peptide comprises an amino acid sequence selected from SEQ ID NOs.:.44859- 44878. In some embodiments, the targeting peptide comprises an amino acid sequence of 44859. In some embodiments, the targeting peptide comprises an amino acid sequence of 44860. In some embodiments, the targeting peptide comprises an amino acid sequence of 44861. In some embodiments, the targeting peptide comprises an amino acid sequence of 44862. In some embodiments, the targeting peptide comprises an amino acid sequence of 44863. In some embodiments, the targeting peptide comprises an amino acid sequence of 44864. In some embodiments, the targeting peptide comprises an amino acid sequence of 44865. In some embodiments, the targeting peptide comprises an amino acid sequence of 44866.
  • the targeting peptide comprises an amino acid sequence of 44867. In some embodiments, the targeting peptide comprises an amino acid sequence of 44868. In some embodiments, the targeting peptide comprises an amino acid sequence of 44869. In some embodiments, the targeting peptide comprises an amino acid sequence of 44870. In some embodiments, the targeting peptide comprises an amino acid sequence of 44871. In some embodiments, the targeting peptide comprises an amino acid sequence of 44872. In some embodiments, the targeting peptide comprises an amino acid sequence of 44873. In some embodiments, the targeting peptide comprises an amino acid sequence of 44874. In some embodiments, the targeting peptide comprises an amino acid sequence of 44875.
  • the targeting peptide comprises an amino acid sequence of 44876. In some embodiments, the targeting peptide comprises an amino acid sequence of 44877. In some embodiments, the targeting peptide comprises an amino acid sequence of 44878. In some embodiments, the targeting peptide comprises an amino acid sequence of 44879. In some embodiments, the targeting peptide Atty Docket No.: 38053-57988/WO (112WO) comprises an amino acid sequence of 44880. In some embodiments, the targeting peptide comprises an amino acid sequence of 44881. In some embodiments, the targeting peptide comprises an amino acid sequence of 44882. In some embodiments, the targeting peptide comprises an amino acid sequence of 44883.
  • the targeting peptide comprises an amino acid sequence of 44911. In some embodiments, the targeting peptide comprises an amino acid sequence of 44912. In some embodiments, the targeting peptide comprises an amino acid sequence of 44913. In some embodiments, the targeting peptide comprises an amino acid sequence of 44918. In some embodiments, the targeting peptide comprises an amino acid sequence of 44919. In some embodiments, the targeting peptide does not comprise a peptide selected from: SEQ ID NOs: 44855 and 3000. In some embodiments, the targeting peptide does not comprise a peptide selected from: SEQ ID NOs: 44880 and 44910.
  • the targeting peptide comprises an amino acid sequence having at least 90%, 95%, 98%, 99% sequence identity to the amino acid sequence selected from SEQ ID NOs.: 44859- 44883, 44911, 44912, 44913, and 44918-44919. [00250] In some embodiments, the targeting peptide comprises an amino acid sequence selected from SEQ ID NOs.: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919.
  • the targeting peptide comprises an amino acid sequence having at least 90%, 95%, 98%, 99% sequence identity to the amino acid sequence selected from SEQ ID NOs.: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918- 44919.
  • the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44864.
  • the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44865.
  • the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44866.
  • the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44867.
  • the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44879. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44880. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44881. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44882. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44883. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44911. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44912.
  • the targeting peptide comprises an amino acid sequence of SEQ ID NO: Atty Docket No.: 38053-57988/WO (112WO) 44913. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44918. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44919. [00251] In some embodiments, the targeting peptide comprises a sequence X7 is selected from R, F, H, L, Q, R, and Y. In some embodiments, X7 is R. In some embodiments, X 7 is Y or H. In some embodiments, X 7 is F or Y.
  • X 8 is selected from S, G, D, I, L, N, Q, T, and V. In some embodiments, X8 is S or G. In some embodiments, X8 is S. In some embodiments, X8 is G. In some embodiments, X 8 is T, G or S. In some embodiments, X 8 is N or Q. In some embodiments, X 9 is selected from S or V. [00253] In some embodiments, X9 is selected from any of the amino acids. In some embodiments, X9 is selected from V, S, N, G, Q, L, T, and Y. In some embodiments, X9 is selected from V or Q.
  • X 9 is selected from N or S.
  • X10 is selected from I, V, S, L, M, R, T, and Q.
  • X10 is I or V.
  • X10 is I.
  • X10 is V.
  • X 10 is V, L or M.
  • X 10 is T or L.
  • the targeting peptide comprises a sequence of RGDRX8X9X10.
  • X8 is S or G.
  • X8 is S.
  • X 8 is G.
  • X 9 is selected from any amino acid residue.
  • X 9 is selected from V, S, N, G, and Q. In some embodiments, X 9 is V. In some embodiments, X9 is S. In some embodiments, X9 is N. In some embodiments, X9 is G. In some embodiments, X 9 is Q. In some embodiments, X 10 is selected from I, V, S, L and Q. In some embodiments, X 10 is I. In some embodiments, X 10 is V. In some embodiments, X 10 is S. In some embodiments, X10 is L. In some embodiments, X10 is Q. In some embodiments, X10 is I or V.
  • the targeting peptide has a sequence of X1X2X3RGDRGVV (SEQ ID NO: 98928), X1X2X3RGDRSVV (SEQ ID NO: 98931), X1X2X3RGDRGQI (SEQ ID NO: 98927), X1X2X3RGDRSQT (SEQ ID NO: 98930), X 1 X 2 X 3 RGDRQGI (SEQ ID NO: 98929), X 1 X 2 X 3 RGDFQNT (SEQ ID NO: 98934), X 1 X 2 X 3 RGDHGVL (SEQ ID NO: 98938), X 1 X 2 X 3 RGDYTSV (SEQ ID NO: 98941), X1X2X3RGDYTSM (SEQ ID NO: 98942), X1X2X3RGDLTVT (SEQ ID NO: 98935), X 1 X 2 X 3 RGD
  • the targeting peptide has a sequence of X 1 X 2 X 3 RGDRGVV (SEQ ID NO: 98928), X 1 X 2 X 3 RGDRSVV (SEQ ID NO: 98931) or X 1 X 2 X 3 RGDRGQI (SEQ ID NO: 98927).
  • the targeting peptide has a sequence of X 1 X 2 X 3 RGDYTSV (SEQ ID NO: 98941), X 1 X 2 X 3 RGDYTSM (SEQ ID NO: 98942), X 1 X 2 X 3 RGDRGVV (SEQ ID NO: 98928), X 1 X 2 X 3 RGDRSVV (SEQ ID NO: 98931), X1X2X3RGDYSSV (SEQ ID NO: 98937), or X1X2X3RGDHGVL (SEQ ID NO: 98938).
  • the targeting peptide has a sequence of X 1 X 2 X 3 RGDFQNT (SEQ ID NO: 98934), X 1 X 2 X 3 RGDHGVL (SEQ ID NO: 98938), X1X2X3RGDLIGR (SEQ ID NO: 98925), X1X2X3RGDRGQI (SEQ ID NO: 98927), X1X2X3RGDRGVV (SEQ ID NO: 98928), X1X2X3RGDYTSM (SEQ ID NO: 98942) or X 1 X 2 X 3 RGDYTSV (SEQ ID NO: 98941).
  • X 1 X 2 X 3 is selected from the group consisting of EFK, AAY, DQK, QVY, DKL, DNV, ENF, EWK, QNV, and TFM.
  • the modified sequence comprises a 7-mer peptide selected from: RGDRSX 9 I, RGDRGX 9 I, RGDRSX 9 V, or RGDRGX 9 V.
  • the targeting peptide has an amino acid sequence selected from X 1 X 2 X 3 RGDRGQI (SEQ ID NO: 98927), X 1 X 2 X 3 RGDRSVV (SEQ ID NO: 98931) or X 1 X 2 X 3 RGDRGVV (SEQ ID NO: 98928).
  • X1X2X3 is selected from the group consisting of DAV, DKW, EAY, AEY, DFV, DKF, DKI, DKL, DNV, DNY, DSL, DSV, EFI, SEF, SEY, SLY, ADF, ADY, ALY, AVF, DAF, DAL, DAM, DAT, DHV, DIV, DKA, DKM, DKT, DKV, DKY, DMI, DNF, DNI, DQT, DSI, DVY, DYN, DYV, EAT, EAW, EFV, EGL, EIY, EMF, EMY, ENF, EPF, EPY, EQY, ESY, ETF, EWI, EWT, EYI, EYV, NEM, QDF, QDY, QEY, QLY, QND, QVF, QVY, SDL, SDV, SEH, SII, SIY, SSL
  • X1X2X3 is selected from the group consisting of: ETI, DQN, DLL, EKW, DNN, EYS, or TVF.
  • the targeting peptide has an amino acid sequence selected from: X 1 X 2 X 3 RGDHGVL (SEQ ID NO: 98938), X 1 X 2 X 3 RGDYSSV (SEQ ID NO: 98937), X 1 X 2 X 3 RGDYTSM (SEQ ID NO: 98942) or X 1 X 2 X 3 RGDYTSV (SEQ ID NO: 98941).
  • X 1 X 2 X 3 is selected from the group consisting of: ANY, SNI, AAI, AAM, ANT, AST, AYQ, EHK, ENK, ENR, SFQ, SSI, TAY, TDK, TNT, AAF, AAL, AAY, ADK, AFA, ANF, ANI, ANQ, ANS, AQM, ARE, ASV, AYH, AYT, EMK, EWK, NNM, QAF, QAI, QAM, QAT, QAY, QFT, QGM, QHL, QNF, QNQ, QNS, QNT, QNV, QNY, SAH, SAI, SAL, SFT, SFV, SHI, SHV, SMM, SNF, SNM, SNN, SNQ, SNV, SNY, SQI, SQV, SSL, SWQ, SWS, SYI, SYM, SYN, SYQ, TAM,
  • X 1 X 2 X 3 is selected from TVF, APM, AQI, TTS, NQF, NFL or SMN.
  • X7 is selected from F and Y;
  • X8 is selected from N and Q;
  • X 9 is selected from N and S;
  • X 10 is selected from T and L.
  • the targeting peptide has an amino acid sequence selected from: X1X2X3RGDFNNT (SEQ ID NO: 98943), X1X2X3RGDFNNL (SEQ ID NO: 98933), X 1 X 2 X 3 RGDFQNT (SEQ ID NO: 98934) or X 1 X 2 X 3 RGDYNSL (SEQ ID NO: 98940).
  • X1X2X3 is selected from the group consisting of: ADR, ASI, EFK, EHK, EWK, SYQ, AAF, AAT, AAY, AFI, AFQ, AGI, AGT, AHI, ANH, ANM, ANN, AQI, ASA, ASH, AST, ASV, AWT, AYQ, AYT, DAR, DMK, DQR, DVR, EAR, EFR, EMK, EMR, EQK, ERA, ERS, ESR, NDA, NDR, NMI, NMV, NNM, NNN, NYL, NYM, NYN, NYQ, NYV, QAM, QFQ, QFV, QGV, QNH, QNI, QNM, QQV, QSF, QSY, SAI, SAM, SAS, SDR, SFQ, SGH, SGM, SGT, SGV, SHL, SHM, SHQ, SHV,
  • X1X2X3 is selected from the group consisting of: TVF, APM, AQI, TTS, NQF, NFL or SMN.
  • the modified sequence comprises a 7-mer peptide selected from: RGDRGVX10(SEQ ID NO: 157058), RGDRGSX10(SEQ ID NO: 157059), RGDRGNX10(SEQ ID NO: 157060), RGDRGGX10(SEQ ID NO: 157061), RGDRGQX 10 (SEQ ID NO: 157062), RGDRGX 9 V(SEQ ID NO: 157063), RGDRGX 9 I(SEQ ID NO: 157064), RGDRGX 9 S(SEQ ID NO: 157065), RGDRGX 9 L(SEQ ID NO: 157066), RGDRGX9Q(SEQ ID NO: 157067), RGDHX8X9L(SEQ ID NO: 157068), RGDRX8X9I(SEQ ID NO: 157069), RGDRX 8 X 9 V(SEQ ID NO: 157070), RGDRX 8 X 9 L(SEQ ID NO:
  • the targeting peptide comprises a sequence of H1 (RGDLIGR (SEQ ID NO: 1422)) or a sequence of H2 (RGDQSTL (SEQ ID NO: 3052)).
  • the targeting peptide comprises a sequence of H1 (RGDLIGR (SEQ ID NO: 1422)) targeting peptide located in VR VIII between amino acids at positions 588 and 589 or a sequence of H2 (RGDQSTL (SEQ ID NO: 3052)) targeting peptide located in VR VIII between amino acids at positions 588 and 589.
  • a modified AAV capsid protein comprising an H1 or H2 targeting peptide have increased tissue enrichment (tropism) in cardiac muscle tissue as compared to skeletal muscle tissue.
  • the targeting peptide comprises a sequence of S1 (RGDISRT (SEQ ID NO: 263)) targeting peptide located in VR VIII or a sequence of S2 (RGDRSQT (SEQ ID NO: 251)).
  • the targeting peptide comprises a sequence of S1 (RGDISRT (SEQ ID NO: 263)) targeting peptide located in VR VIII between amino acids 588 and 589 or a sequence of S2 (RGDRSQT (SEQ ID NO: 251)) targeting peptide located in VR VIII between amino acids at positions 588 and 589.
  • a modified AAV capsid protein comprising an S1 or S2 targeting peptide have increased tissue enrichment (tropism) in skeletal tissue as compared to cardiac muscle tissue.
  • the modified sequence does not comprise a 7-mer peptide selected from: RGDRMVF, RGDRTVI, SRGDRPM, and ISLRGDR.
  • the modified sequence does not comprise a 7-mer peptide having an amino acid sequence of SEQ ID NO: 1 (RGDLLLS). In certain embodiments, the modified sequence does not comprise a 7-mer peptide of RGDRMVF. In certain embodiments, the Atty Docket No.: 38053-57988/WO (112WO) modified sequence does not comprise a 7-mer peptide of RGDRTVI. In certain embodiments, the modified sequence does not comprise a 7-mer peptide selected from SRGDRPM, or ISLRGDR. In some embodiments, the modified sequence does not comprise a 7-mer peptide selected from: RGDRMVF, RGDRTVI, SRGDRPM, and ISLRGDR.
  • the modified sequence does not comprise a 7-mer peptide of X1SLRGDR, where X1 is any amino acid residue. In some embodiments, the modified sequence does not comprise a 7-mer peptide of X 1 X 2 LRGDR, where X 1 and X 2 are independently any amino acid residue.
  • the targeting peptide has a sequence selected from: X 1 X 2 X 3 RGDHVNL (SEQ ID NO: 98924); X 1 X 2 X 3 RGDLIGR (SEQ ID NO: 98925); X 1 X 2 X 3 RGDQSTL (SEQ ID NO: 98926); X 1 X 2 X 3 RGDRGQI (SEQ ID NO: 98927); X1X2X3RGDRGVV (SEQ ID NO: 98928); X1X2X3RGDRQGI (SEQ ID NO: 98929); X1X2X3RGDRSQT (SEQ ID NO: 98930); X1X2X3RGDRSVV (SEQ ID NO: 98931); X 1 X 2 X 3 RGDLLLS (SEQ ID NO: 98932); X 1 X 2 X 3 RGDFNNL (SEQ ID NO: 98933); X1X2X3
  • X 1 is selected from S, E, A, D, N, Q, or T. In some embodiments, X 1 is selected from S or E. In some embodiments, X 1 is S. In some embodiments, X 1 is E. In some embodiments, X2 is selected from N, A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X 2 is selected from N or A. In some embodiments, X 2 is N. In some embodiments, X 2 is A.
  • X 3 is selected from R, Q, A, D, E, F, G, H, I, K, L, M, N, P, S, T, V, W, or Y.
  • X3 is selected from R or Q.
  • X3 is R.
  • X3 is Q.
  • X2 is N; and X 3 is R.
  • X 1 is E; X 2 is N; and X 3 is R.
  • X 1 is S; X2 is N; and X3 is R.
  • the modified AAV capsid protein further includes one or more mutations outside of the VRVIII.
  • the mutant outside of VRVIII can comprise (1) an alanine (A) or glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1; and/or (2) a lysine (K) or arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1.
  • the modified AAV Atty Docket No.: 38053-57988/WO (112WO) capsid protein can comprise an alanine (A) amino acid residue at an amino acid position 267 and a threonine (T) amino acid residue at an amino acid position 269 in AAV9 VP1.
  • the modified AAV capsid protein further includes one or more deletions or substitutions within the VRVIII. In some embodiments, the modified AAV capsid protein further includes one or more deletions or substitutions between positions 565 and 595 of the reference capsid protein. In some embodiments, the modified AAV capsid protein further includes one or more deletions or substitutions within the VRVIII, followed by an insertion of the targeting peptide. In some embodiments, the modified AAV capsid protein further includes one or more substitutions within the VRVIII, followed by an insertion of the targeting peptide. In some embodiments, the modified AAV capsid protein further includes one or more substitutions within the VRVIII.
  • the modified AAV capsid protein further includes one or more substitutions between positions 565 and 595 of the reference capsid protein. In some embodiments, the modified AAV capsid protein further includes one or more substitutions within the VRVIII, followed by an insertion of the targeting peptide.
  • the targeting peptide can enhance targeting of an AAV to a brain, muscle, spinal cord, eye, liver, heart, muscle, or other organ. In some embodiments, the targeting peptide can decrease targeting of an AAV to a brain, muscle, spinal cord, eye, liver, heart, muscle, or other organ.
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDYNSL (SEQ ID NO: 44859).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDYNNL (SEQ ID NO: 44860). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDYNST (SEQ ID NO: 44861). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence Atty Docket No.: 38053-57988/WO (112WO) identity to the amino acid sequence of ENRRGDYNNT (SEQ ID NO: 44862).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDFNSL (SEQ ID NO: 44863). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDFNNL (SEQ ID NO: 44864). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDFNST (SEQ ID NO: 44865).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDFQNT (SEQ ID NO: 44866). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDEQNT (SEQ ID NO: 44867). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDQQNT (SEQ ID NO: 44868).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDYNSL (SEQ ID NO: 44869). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDYNNL (SEQ ID NO: 44870). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDYNST (SEQ ID NO: 44871).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDYNNT (SEQ ID NO: 44872). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDFNSL (SEQ ID NO: 44873). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDFNNL (SEQ ID NO: 44874).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDFNST (SEQ ID NO: 44875). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of Atty Docket No.: 38053-57988/WO (112WO) SNRRGDFQNT (SEQ ID NO: 44876).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDEQNT (SEQ ID NO: 44877). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDQQNT (SEQ ID NO: 44878). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDFNGL (SEQ ID NO: 44879).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of ENRRGDFNNT (SEQ ID NO: 44880). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SAQRGDFNNT (SEQ ID NO: 44881). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SAQRGDLLLS (SEQ ID NO: 44882).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of SNRRGDFNNT (SEQ ID NO: 44883). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of (SEQ ID NO: 44911). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of (SEQ ID NO: 44912).
  • the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of (SEQ ID NO: 44913). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of (SEQ ID NO: 44918). In some embodiments, the targeting peptide comprises at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence of (SEQ ID NO: 44919). In some embodiments, the targeting peptide does not have the amino acid sequence of SEQ ID NO: 44910.
  • the targeting peptide does not have the amino acid sequence of SEQ ID NO: 44880. In some embodiments, the targeting peptide does not have the amino acid sequence selected from SEQ ID NOs: 44855-44858. Atty Docket No.: 38053-57988/WO (112WO) [00283]
  • X7, X8, and X9 are independently selected from L, G, V, and A; and X7 is S, V, A, G, or L. In some embodiments, X 7 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y.
  • X8 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y.
  • X9 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y.
  • X 10 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y.
  • X 7 is selected from L, Q, D, H, M, P, and K. In some embodiments, X7 is L.
  • X8 is selected from G, V, S, D, M, and N. In some embodiments, X 8 is G. In some embodiments, X 9 is selected from V, M, P, S, and D. In some embodiments, X 9 is V. In some embodiments, X 10 is selected from S, N, L, H, and M. In some embodiments, X10 is S. [00285] In some embodiments, X7 is L. In further embodiments, X8 is G. In further embodiments, X 9 is L. In further embodiments, X 10 is S. [00286] In some embodiments, X7 is A. In further embodiments, X8 is V. In further embodiments, X9 is G.
  • X10 is V.
  • X 7 is L.
  • X 8 is L.
  • X 9 is L.
  • X 10 is S.
  • X 7 is L, X 8 is G or L, and/or X 10 is S.
  • X 8 and X 9 is G or L.
  • X7, X8, and X9 are independently selected from L, V, and A; at least two of X 7 , X 8 , and X 9 are independently L. In some embodiments, X 7 , X 8 , and X 9 are L. In certain embodiments, X 8 is L.
  • the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X7X8X9X10 is a quad selected for enhanced targeting to muscle.
  • the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X7X8X9X10 is a quad selected for enhanced targeting to a specific muscle (e.g., biceps, quadriceps, diaphragm, heart). [00291] In some embodiments, the targeting peptide has a sequence of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 , wherein X 7 X 8 X 9 X 10 is a quad selected for enhanced targeting to Atty Docket No.: 38053-57988/WO (112WO) skeletal muscle.
  • the quad is FNNL, YNSL, RQGI, FQNT, YVGL, YSSV, YTSM, RSVV, YTSV, RDYL, or FNNT.
  • the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X7X8X9X10 is a quad selected for enhanced targeting to cardiac muscle.
  • the quad is LIGR, QSTL, or RGVV.
  • the targeting peptide has a sequence of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 , wherein X 7 X 8 X 9 X 10 is a quad selected for enhanced targeting to liver.
  • the quad is RGVV, RSVV, RGQI, RSQT, RQGI, FQNT, HGVL, YTSV, YTSM, LTVT, FNNT, YSSV, or HVNL
  • the targeting peptide comprises the amino acid sequence selected from RGDLRVS (SEQ ID NO: 153), RGDAVGV (SEQ ID NO: 154), RGDFTPTS (SEQ ID NO: 155), RGDLGLS (SEQ ID NO: 156), and RGDMSRE (SEQ ID NO: 157), and/or a sequence comprising at most two, preferably at most one, amino acid substitution compared to one of the aforesaid specific sequences.
  • the targeting peptide does not comprise an amino acid sequence selected from RGDLRVS (SEQ ID NO: 153), RGDAVGV (SEQ ID NO: 154), RGDFTPTS (SEQ ID NO: 155), RGDLGLS (SEQ ID NO: 156), and RGDMSRE (SEQ ID NO: 157).
  • the targeting peptide comprises a sequence of RGDLLLS (SEQ ID NO: 1).
  • the targeting peptide comprises a sequence selected from: SEQ ID NOs.: 238-44858.
  • the modified AAV capsid protein comprises a targeting peptide comprising an amino acid sequence selected from SEQ ID NOs.: 238-247.
  • the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-337.
  • the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-437.
  • the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-537.
  • the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-637.
  • the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238- 737.
  • the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-837. In some embodiments, the targeting peptide comprises a sequence selected Atty Docket No.: 38053-57988/WO (112WO) from SEQ ID NOs.: 238-937. In some embodiments, the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-1037. [00298] In some embodiments, the targeting peptide is the targeting peptide disclosed in US2017/0166926, incorporated by reference in its entirety herein. [00299] The targeting peptide can have any of the sequences selected from SEQ ID NOs: 2-51 and 53 provided herein.
  • the targeting peptide is the 7-mer peptide TLAVPFK (SEQ ID NO: 53). [00300] In some embodiments, the targeting peptide is not a targeting peptide disclosed in US2017/0166926, WO2019/028306, WO2020/072683, WO2021/042909, WO2021/050974, WO2021/077000, or WO2021/222831, incorporated by reference in their entireties herein. 6.2.2. Targeting peptide site [00301] A modified AAV capsid protein of the present disclosure comprises a targeting peptide within VR VIII of the reference AAV capsid protein (FIG.1).
  • the targeting peptide is at a site exposed to the exterior of the capsid, preferably based on structure predictions and/or experimental data. More preferably, the targeting peptide is at a site exposed to the exterior of the AAV capsid in a manner that does not interfere with the activity of said protein in capsid assembly.
  • the position of the targeting peptide in an AAV capsid protein that “corresponds to” the position in the AAV9 capsid protein can be established by the skilled person by known methods, preferably by aligning the amino acids of the capsid proteins.
  • the site of the targeting peptide corresponds to amino acid position 588 of the AAV9 VP1 capsid protein.
  • X 1 of the targeting peptide is at a site corresponding to amino acid position 580 of the AAV9 VP1 capsid protein.
  • X1 of the targeting peptide is at a site corresponding to amino acid position 581 of the AAV9 VP1 capsid protein.
  • X 1 of the targeting peptide is at a site corresponding to amino acid position 582 of the AAV9 VP1 capsid protein.
  • X 1 of the targeting peptide is at a site corresponding to amino acid position 583 of the AAV9 VP1 capsid protein.
  • X 1 of the targeting peptide is at a site corresponding to amino acid position 584 of the AAV9 VP1 capsid protein. In other words, X 1 of the targeting peptide is at a site corresponding to amino acid position 588 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is at a site corresponding to amino acid position 585 of the Atty Docket No.: 38053-57988/WO (112WO) AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is at a site corresponding to amino acid position 586 of the AAV9 VP1 capsid protein.
  • X 1 of the targeting peptide is at a site corresponding to amino acid position 587 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is at a site corresponding to amino acid position 588 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is at a site corresponding to amino acid position 587 of the AAV9 VP1 capsid protein. In other words, X 1 of the targeting peptide is at a site corresponding to amino acid position 586 of the AAV9 VP1 capsid protein.
  • X1 of the targeting peptide is at a site corresponding to amino acid position 585 of the AAV9 VP1 capsid protein. In other words, X 1 of the targeting peptide is at a site corresponding to amino acid position 589 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is at a site corresponding to amino acid position 590 of the AAV9 VP1 capsid protein. In other words, X 1 of the targeting peptide is at a site corresponding to amino acid position 591 of the AAV9 VP1 capsid protein.
  • X1 of the targeting peptide is at a site corresponding to amino acid position 592 of the AAV9 VP1 capsid protein.
  • the position of the targeting peptide can be any one of those described in WO2019/207132, incorporated by reference in its entirety herein. Some of the sites are provided below in Table 1 and highlighted in FIGs.2A-2C and FIGs 3A-3D. In Table 1, the preferred sites are indicated by a “-” relative to wild type VP1 capsid polypeptide.
  • the targeting peptide is inserted between 2 amino acid positions, where the insertion site is denoted as “-”.
  • the targeting peptide is inserted between positions D590 and P591.
  • the AAV capsid protein is modified by way of mutation or substitution of one or more amino acids, followed by insertion of the targeting peptide.
  • insertion site 3 of Table 1 3 amino acids positioned between the insertion sites are substituted or mutated prior to insertion of the targeting peptide.
  • the targeting peptide is inserted between position Q585 and T589 after the amino acids “SSS” positioned between Q585 and T589 (S586, S587, and S588) are deleted, denoted as “Q-SSS-T”.
  • the targeting peptide is at between 560 and 600 within the VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is at between 565 and 595 within the VR VIII of the modified AAV capsid protein. [00307] In some embodiments, the targeting peptide is at between 570 and 610 within the VR VIII of the modified AAV capsid protein.
  • the targeting peptide Atty Docket No.: 38053-57988/WO (112WO) is at between 580 and 610 within the VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is at between 580 and 595 within the VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is at between 582 and 600 within the VR VIII of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV1 or a modification thereof and the targeting peptide is between D590 and P591 or between S588 and T589 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV1 or a modification thereof and the targeting peptide is between positions 587 and 594 or between positions 585 and 592 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAV1 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. [00309] In some embodiments, the reference AAV capsid protein is a capsid protein of AAV2 or a modification thereof and the targeting peptide is between R585 and Q589 or between N587 and R588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV2 or a modification thereof and the targeting peptide is between positions 582 and 592 or between positions 585 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAV2 or a modification thereof and the targeting peptide is between Q584 and R588. [00310] In some embodiments, the reference AAV capsid protein is a capsid protein of AAV3 or a modification thereof and the targeting peptide is between S586 and S587 or between N588 and T589 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV3 or a modification thereof and the targeting peptide is between positions 583 and 590 or between positions 585 and 592 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAV3 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. [00311] In some embodiments, the reference AAV capsid protein is a capsid protein of AAV4 and the targeting peptide is between S584 and N585 or between S586 and N587 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV4 or a modification thereof and the targeting peptide is between Atty Docket No.: 38053-57988/WO (112WO) positions 581 and 586 or between positions 583 and 590 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV4 or a modification thereof and the targeting peptide is between G581 and N585 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV5 or a modification thereof and the targeting peptide is between S575 and S576 or between T577 and T578 of the capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAV5 or a modification thereof and the targeting peptide is between positions 572 and 579 or between positions 574 and 581 of the capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAV5 and the targeting peptide is between Q574 and T589 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV6 or a modification thereof and the targeting peptide is between D590 and P591 or S588 and T589 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV6 or a modification thereof and the targeting peptide is between positions 587 and 594 or positions 585 and 592 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV6 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV7 or a modification thereof and the targeting peptide is between N589 and T590 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAV7 or a modification thereof and the targeting peptide is between positions 586 and 593 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAV7 or a modification thereof and the targeting peptide is between Q586 and T590 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV8 and the targeting peptide is between N590 and T591 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV8 or a modification thereof and the targeting peptide is between positions 587 and 594 of the modified AAV capsid protein.
  • the reference AAV capsid Atty Docket No.: 38053-57988/WO (112WO) protein is a capsid protein of AAV8 or a modification thereof and the targeting peptide is between Q587 and T591 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV9 and the targeting peptide is between Q588 and A589 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV9 or a modification thereof and the targeting peptide is between positions 585 and 592 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV9 or a modification thereof and the targeting peptide is between Q585 and A589 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAVrh10 or a modification thereof and the targeting peptide is between N590 and A591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAVrh10 or a modification thereof and the targeting peptide is between positions 587 and 594 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of AAVrh10 or a modification thereof and the targeting peptide is between Q587 and A591 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAVpo.1 or a modification thereof and the targeting peptide is between N567 and S568 or between N569 and T570 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAVpo.1 or a modification thereof and the targeting peptide is between positions 570 and 571 or between positions 566 and 573 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAVpo.1 or a modification thereof and the targeting peptide is between N564 and S568 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV12 or a modification thereof and the targeting peptide is between N592 and A593 or between T594 and T595 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV12 or a modification thereof and the targeting peptide is between positions 589 and 596 or between positions 591 and 598 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of AAV12 or a modification thereof and the targeting peptide is between N589 and A593 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80 or a modification thereof and the targeting peptide is between T589 and A590 or between N587 and T588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80 or a modification thereof and the targeting peptide is between positions 586 and 593 or between positions 584 and 591 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80L65 or a modification thereof and the targeting peptide is between T589 and A590 or between N587 and T588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80L65 or a modification thereof and the targeting peptide is between positions 586 and 593 or between positions 584 and 591 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80L65 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-55 or a modification thereof and the targeting peptide is between T589 and A590 or between N587 and T588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-55 or a modification thereof and the targeting peptide is between positions 586 and 593 or between positions 584 and 591 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-55 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-129 or a modification thereof and the targeting peptide is between T589 and A590 or between N587 and T588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-129 or a modification thereof and the targeting peptide is between positions 586 and 593 or between positions 584 and 591 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-129 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein.
  • Atty Docket No.: 38053-57988/WO (112WO) [00324]
  • the reference AAV capsid protein is a capsid protein of Anc80-156 or a modification thereof and the targeting peptide is between T589 and A590 or between N587 and T588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-156 or a modification thereof and the targeting peptide is between positions 586 and 593 or between positions 584 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of Anc80-156 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. [00325] In some embodiments, the reference AAV capsid protein is a capsid protein of Anc80-751 or a modification thereof and the targeting peptide is between T589 and A590 or between N587 and T588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-751 or a modification thereof and the targeting peptide is between positions 586 and 593 or between positions 584 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of Anc80-751 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. [00326] In some embodiments, the reference AAV capsid protein is a capsid protein of Anc80-1029 or a modification thereof and the targeting peptide is between T589 and A590 or between N587 and T588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-1029 or a modification thereof and the targeting peptide is between positions 586 and 593 or between positions 584 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of Anc80-1029 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. [00327] In some embodiments, the reference AAV capsid protein is a capsid protein of Anc80-1712 or a modification thereof and the targeting peptide is between T589 and A590 or between N587 and T588 of the modified AAV capsid protein.
  • the reference AAV capsid protein is a capsid protein of Anc80-1712 or a modification thereof and the targeting peptide is between positions 586 and 593 or between positions 584 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is a capsid protein of Anc80-1712 or a modification thereof and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. Atty Docket No.: 38053-57988/WO (112WO) 6.2.3.
  • modified AAV capsid proteins comprising a reference AAV capsid protein with one or more modifications to comprise a targeting peptide at a site within VR VIII of the reference AAV capsid protein, wherein the targeting peptide has a sequence X1X2X3RGDX7X8X9X10.
  • the modified AAV capsid protein comprises a targeting peptide selected from SEQ ID NOs.: 238-44858 introduced into the VR VIII.
  • the modified AAV capsid protein comprises a targeting peptide having at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence selected from SEQ ID NOs.: 238-44858 introduced into the VRVIII.
  • the modified AAV capsid protein is an AAV9 capsid protein containing a targeting peptide, RGDLLLS (SEQ ID NO: 1), inserted into the VR VIII.
  • the modified AAV capsid protein has a sequence of SEQ ID NO: 158.
  • the modified AAV capsid protein has the amino acids 138 to 736 of SEQ ID NO: 158.
  • the modified AAV capsid protein has the amino acids 203 to 736 of SEQ ID NO: 158. In some embodiments, the modified AAV capsid protein has the amino acid sequence selected from SEQ ID NOs.: 44900-44909. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44900. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44901. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44902. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44903.
  • the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44904. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44905. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44906. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44907. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44908. In some embodiments, the modified AAV capsid protein has the amino acid sequence of SEQ ID NO.: 44909.
  • the modified AAV capsid protein has a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from: SEQ ID NOs.: 44900-44909. Atty Docket No.: 38053-57988/WO (112WO) [00330] In some embodiments, the modified AAV capsid protein has a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 158.
  • the present disclosure provides a modified AAV capsid protein comprising (i) a) an alanine (A) amino acid residue at an amino acid position corresponding to position 266 in Anc80; or b) a lysine (K) amino acid residue at an amino acid position corresponding to position 168 in Anc80; and (ii) a targeting peptide introduced into a site within VR VIII of the liver-toggle mutant.
  • the modified AAV capsid protein is an AAV9 capsid protein containing a targeting peptide, RGDLLLS (SEQ ID NO: 1), inserted into the VR VIII.
  • the modified AAV capsid protein can comprise one or more additional modifications to comprise (i) a) an alanine (A) amino acid residue at an amino acid position corresponding to position 266 in Anc80; or b) a lysine (K) amino acid residue at an amino acid position corresponding to position 168 in Anc80.
  • the modified AAV capsid protein has a sequence of SEQ ID NO: 159.
  • the modified AAV capsid protein has the amino acids 138 to 736 of SEQ ID NO: 159.
  • the modified AAV capsid protein has the amino acids 203 to 736 of SEQ ID NO: 159.
  • the modified AAV capsid protein has a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 159.
  • a modified AAV capsid protein of the present disclosure can change the tropism, specificity and/or bio-distribution of an AAV comprising the modified AAV capsid protein.
  • an AAV comprising the modified AAV capsid protein has increased targeting to a target cell, tissue or organ when administered to a subject.
  • an AAV comprising the modified AAV capsid protein has decreased distribution outside of a target cell, tissue or organ when administered to a subject.
  • tropism of a modified AAV capsid protein can be measured using an enrichment score.
  • the modified AAV capsid protein comprises (i) a reference AAV capsid protein, and (ii) a 7-mer peptide
  • the reference AAV capsid protein used in various embodiments of the present disclosure is a VP1, VP2 or VP3 capsid protein of an AAV known in the art. It can be a VP1, VP2 or VP3 capsid protein of a naturally occurring or non-naturally occurring AAV variant.
  • the non-naturally occurring VP1, VP2, or VP3 capsid protein includes a capsid protein generated by biological or chemical alteration or in silico design, or variation of a naturally occurring AAV capsid protein.
  • the reference AAV capsid protein includes, but is not limited to, a capsid protein of various AAV serotypes (e.g., AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9) or a variant thereof.
  • a non-naturally occurring VP1, VP2, or VP3 capsid protein further includes an artificial capsid protein created by in silico design or synthesis.
  • An artificial capsid protein includes, but is not limited to, AAV capsid proteins disclosed in PCT/US2014/060163, USP9695220, PCT/US2016/044819, PCT/US2018/032166, PCT/US2019/031851, and PCT/US2019/047546, which are incorporated herein by reference in their entireties.
  • the reference AAV capsid protein is the capsid protein of AAV9 (Genbank Ace. No: AAS99264.1), AAV1 (Genbank Ace. No: AAD27757.1), AAV2 (Genbank Ace. No: AAC03780.1), AAV3 (Genbank Ace. No: AAC55049.1), AAV3b (Genbank Ace.
  • AAV4 Genebank Ace. No: AAC58045.1
  • AAV5 Genebank Ace. No: AAD13756.1
  • AAV6 Genebank Ace. No: AF028704.1
  • AAV7 Genebank Ace. No: AAN03855.1
  • AAV 8 Genebank Ace. No: AAN03857.1
  • AAV10 Genebank Ace. No: AAT46337.1
  • AAVrh10 Genebank Ace. No: AY243015.1
  • AAV11 Genebank Ace. No: AAT46339.1
  • AAV12 Genebank Ace. No: ABI16639.1
  • AAV13 Genebank Ace. No: ABZ10812.1
  • AAVpol Genebank Ace.
  • the AAV capsid protein is the capsid protein of AAV9 (Genbank Ace. No: AAS99264.1). Atty Docket No.: 38053-57988/WO (112WO) [00342]
  • the reference AAV capsid protein can be VP1 capsid protein having a sequence selected from: SEQ ID NO: 54 (AAV1 (AAD27757)), SEQ ID NO: 55 (AAV2 (AAC03780)), SEQ ID NO: 56 (AAV3 (AAC55049)), SEQ ID NO: 57 (AAV5 (AAD13756)), SEQ ID NO: 58 (AAV6 (AAB95450)), SEQ ID NO: 59 (AAV7 (AF513851_2)), SEQ ID NO: 60 (AAV8 (AF513852_2)), SEQ ID NO: 61 (AAV9 (AAS99264)), SEQ ID NO: 62 (AAV10 (AAT46337)), SEQ ID NO: 54 (AAV1 (A
  • the reference AAV capsid protein can be a VP2 or VP3 protein having a part of one of the sequences.
  • VP2 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1
  • VP3 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 protein.
  • the reference AAV capsid protein can be VP1 capsid protein having any member sequence of the ancestral AAV library selected from SEQ ID NO: 132 (Anc80), SEQ ID NO: 133 (Anc81 (AKU89596)), SEQ ID NO: 134 (Anc82 (AKU89597)), SEQ ID NO: 135 (Anc83 (AKU89598)), SEQ ID NO: 136 (Anc84 (AKU89599)), SEQ ID NO: 137 (Anc94) SEQ ID NO: 138 (Anc110 (AKU89600)), SEQ ID NO: 139 (Anc113 (AKU89601)), SEQ ID NO: 140 (Anc126 (AKU89602)), SEQ ID NO: 141 Anc127 (AKU89603), and SEQ ID NO: 142 (Anc80L65 (AKU89595)).
  • the reference AAV capsid protein can be a VP2 or VP3 protein having a part of one of the sequences.
  • VP2 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1
  • VP3 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 protein.
  • SEQ ID NO for a library sequence refers to a sequence of any one member of the library.
  • the reference AAV capsid protein is a liver-toggle mutant described in WO2019/217911, which is incorporated by reference in its entirety herein.
  • the reference AAV capsid protein is a capsid protein (VP1, VP2 or VP3) of an AAV variant selected from the group consisting of: AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; AAV hu.68; Atty Docket No.: 38053-57988/WO (112WO) AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6- A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45-B; rh.57-B; rh.35-B; rh
  • the reference AAV capsid protein is a capsid protein of any member protein of an ancestral AAV library selected from: Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; Anc113; Anc126; and Anc127. [00346] In some embodiments, the reference AAV capsid protein is a protein having a sequence selected from SEQ ID Nos: 54-131 and 143-152.
  • the reference AAV capsid protein is a protein having a VP2 (corresponding to amino acids 138 to 736 of AAV9 VP1) or VP3 portion (corresponding to amino acids 138 to 736 of AAV9 VP1) of the protein having a sequence selected from SEQ ID NOs: 54-131 and 143-152. [00347] In some embodiments, the reference AAV capsid protein is a capsid protein of the AAV variant modified to include one or more liver-toggle mutations described in WO2019/217911.
  • the reference AAV capsid protein comprises (1) an alanine (A) or glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1 and/or (2) a lysine (K) or arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1.
  • the reference AAV capsid protein comprises (i) an alanine (A) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1 and/or b) a lysine (K) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1.
  • the reference AAV capsid protein comprises (ii) an alanine (A) amino acid residue at an amino acid position corresponding to position 267 in AAV9 VP1 protein and/or a threonine (T) amino acid residue at an amino acid position corresponding to position 269 in AAV9 VP1.
  • the reference AAV capsid protein is a liver toggle mutant of a capsid protein of Anc80L65.
  • the reference AAV capsid protein is a capsid protein having a sequence selected from SEQ ID NOs.: 44885-44898, 44916-44917, or a fragment thereof. In some embodiments, the reference AAV capsid protein is a capsid protein having a sequence with at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to the amino acid sequence selected from SEQ ID NOs.: 44885-44898, 44916-44917, or a fragment thereof.
  • the reference AAV capsid protein comprises one or more modifications as described in WO2019/217911 or WO 2021/050614, which are both incorporated by reference herein in their entireties. In some embodiments, the reference AAV capsid protein comprises one or more modifications as described in PCT Application No. PCT/US2022/015842, which is herein incorporated by reference in its entirety. 6.2.5. Peptide Segment [00351] In some embodiments, the modified AAV capsid protein comprises a peptide segment within variable region I (VR I). In some embodiments, the modified AAV capsid protein comprises a tropism-altering peptide segment within VR I.
  • the modified AAV capsid protein comprises a peptide segment of 12 amino acids (P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 10 P 11 P 12 ) within VR I which is different from the reference AAV capsid protein.
  • 12 amino acids among the 12 amino acids, only one amino acid is different from the amino acids in the corresponding positions within the reference AAV capsid protein.
  • more than one amino acid residues in the peptide segment are different from the amino acid residues in the corresponding positions within the reference AAV capsid protein.
  • the peptide segment comprises 12 amino acids positioned between about amino acid 259 to about amino acid 275 in a reference AAV capsid protein (e.g., any of the reference AAV capsid proteins described herein). In some embodiments, the peptide segment comprises 12 amino acids (P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 10 P 11 P 12 ) located between position 261 and position 274 in a reference AAV capsid protein or between position 260 and position 273 in a reference AAV capsid protein.
  • the peptide segment comprises 11 amino acids (P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 10 P 11 P 12 ) between about amino acid 259 to about amino acid 275 in a reference AAV capsid protein (e.g., any of the reference AAV capsid proteins described herein). In some embodiments, the peptide segment comprises 10 amino acids between about amino acid 259 to about amino acid 275 in Atty Docket No.: 38053-57988/WO (112WO) a reference AAV capsid protein (e.g., any of the reference AAV capsid proteins described herein).
  • one or more amino acids in P1P2P3P4P5P6P7P8P9P10P11P12 are same as the one or more amino acids at corresponding positions within the reference AAV capsid protein.
  • the peptide segment has a sequence selected from SEQ ID NOs: 44935-47387. In some embodiments, the peptide segment has a sequence having at least 80% or at least 90% sequence identity to a sequence selected from SEQ ID NOs: 44935- 47387.
  • a modified adeno-associated virus (AAV) capsid protein includes a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 and P1, P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue.
  • AAV adeno-associated virus
  • a modified adeno-associated virus (AAV) capsid protein includes a peptide segment within variable region I (VR I), wherein the peptide segment has an amino acid sequence of P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 and P 1, P 2 , P 3 , P 4 , P 5 , P 6 , P 7 , P8, and P12 are independently selected from any amino acid residue.
  • VR I variable region I
  • the modified AAV capsid protein can include a peptide segment having the amino acid sequence of P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 NDNP 12 where: (i) P1 is independently selected from an asparagine (N), a serine (S), or a threonine (T), (ii) P 2 is independently selected from a serine (S) or a glycine (G), (iii) P3 is independently selected from a threonine (T), a glutamine (Q), an alanine (A), or glutamate (E), (iv) P 4 is independently selected from a serine (S), a threonine (T), or an alanine (A), (v) P5 is independently selected from a glycine (G) or an alanine (A), (vi) P6 is independently selected from a glycine (G) or an alanine (A), (vii) P 7 is
  • a modified adeno-associated virus (AAV) capsid protein comprises a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 10 P 11 P 12 , wherein positions P 9 , P 10 , and P 11 , or a combination thereof, are not modified compared to a reference AAV capsid protein.
  • AAV adeno-associated virus
  • a modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), wherein the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8 P9P10P11P12, wherein positions P9, P10, and P11, or a combination thereof, are not modified compared to a reference AAV capsid protein.
  • AAV a modified adeno-associated virus
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, where the peptide segment has an amino acid sequence P1P2P3P4GGP7P8NDNP12 (SEQ ID NO: 44921), wherein P1, P2, P3, P4, P7, P8, and P12 are independently selected from any amino acid residue.
  • AAV adeno-associated virus
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a peptide segment within variable region I (VR I), where the peptide segment has an amino acid sequence P 1 P 2 P 3 P 4 GGP 7 P 8 NDNP 12 (SEQ ID NO: 44921), wherein P 1 , P 2 , P 3 , P 4 , P 7 , P 8 , and P 12 are independently selected from any amino acid residue.
  • AAV adeno-associated virus
  • the modified AAV capsid protein can comprise a peptide segment having the amino acid sequence of P 1 P 2 P 3 P 4 GGP 7 P 8 NDNP 12 (SEQ ID NO: 44921) where: (i) P1 is independently selected from an asparagine (N) or a serine (S) ; (ii) P 2 is independently selected from a serine (S) or a glycine (G); (iii) P 3 is independently selected from a threonine (T) or a glutamine (Q); (iv) P4 is independently selected from a serine (S), a threonine (T), or an alanine (A); Atty Docket No.: 38053-57988/WO (112WO) (v) P7 is independently selected from an alanine (A) or a serine (S); (vi) P8 is independently selected from a serine (S) or a threonine (T); and (vii)
  • the modified AAV capsid protein can comprise a peptide segment having a sequence of P 1 P 2 TP 4 GGP 7 P 8 NDNP 12 (SEQ ID NO: 44922), wherein P 1 , P 2 , P 4 , P 7 , P 8 , and P 12 are independently selected from any amino acid residue.
  • the modified AAV capsid protein can comprise a peptide segment has a sequence of P 1 P 2 QP 4 GGP 7 P 8 NDNP 12 (SEQ ID NO: 44923), wherein P 1 , P 2 , P 4 , P 7 , P 8 , and P 12 are independently selected from any amino acid residue.
  • the modified AAV capsid protein can comprise a peptide segment having a sequence of P1P2TP4GGP7TNDNP12 (SEQ ID NO: 44924), wherein P 1 , P 2 , P 4 , P 7 , and P 12 are independently selected from any amino acid residue.
  • the modified AAV capsid protein can comprise a peptide segment has a sequence of NP2TP4GGP7P8NDNP12 (SEQ ID NO: 44925), wherein P 2 , P 4 , P 7 , P 8 , and P 12 are independently selected from any amino acid residue.
  • the modified AAV capsid protein can comprise a peptide segment has a sequence of SP2TP4GGP7P8NDNP12 (SEQ ID NO: 44926), wherein P2, P 4 , P 7 , P 8 , and P 12 are independently selected from any amino acid residue.
  • a modified adeno-associated virus (AAV) capsid protein includes a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, where the peptide segment has an amino acid sequence of P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 10 P 11 P 12 , wherein positions P5, P6, or a combination thereof, are not modified compared to a reference AAV capsid protein.
  • a modified adeno-associated virus (AAV) capsid protein includes a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, where the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8 P9P10P11P12, wherein positions P 5 , P 6 , P 9 , P 10 , and P 11 , or a combination thereof, are not modified compared to a reference AAV capsid protein.
  • AAV capsid protein includes a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, where the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8 P9P10P11P12, wherein positions P 5 , P 6 , P 9 , P 10 , and P 11 , or
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of NSTSGGP7P8NDNH (SEQ ID NO: 44927), wherein P7 and P8 are independently selected from any amino acid residue.
  • AAV adeno-associated virus
  • P 7 is independently selected from an alanine (A) or a serine (S) and P 8 is independently selected from a serine (S) or a threonine (T).
  • the modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), wherein the peptide segment has an amino acid sequence of NSTSGGP 7 P 8 NDNH (SEQ ID NO: 44927), wherein P 7 and P 8 are independently selected from any amino acid residue.
  • P7 is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T).
  • the peptide segment includes (i) NSTSGGASNDNH (SEQ ID NO: 46026), (ii) NSTSGGATNDNH (SEQ ID NO: 46029), (iii) NSTSGGSSNDNH (SEQ ID NO: 46031), (iv) NSTSGGSTNDNH (SEQ ID NO: 46034), or a corresponding sequence having one or more modifications (e.g., insertion, deletion, mutation, and/or substitution).
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of NSTTGGP 7 P 8 NDNH (SEQ ID NO: 44928), wherein P 7 and P 8 are independently selected from any amino acid residue.
  • P 7 is independently selected from an alanine (A) or a serine (S)
  • P8 is independently selected from a serine (S) or a threonine (T).
  • the modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), wherein the peptide segment has an amino acid sequence of NSTTGGP7P8NDNH (SEQ ID NO: 44928), wherein P7 and P8 are independently selected from any amino acid residue.
  • P7 is independently selected from an alanine (A) or a serine (S) and P 8 is independently selected from a serine (S) or a threonine (T).
  • the peptide segment includes: (i) NSTTGGASNDNH (SEQ ID NO: 46073), (ii) NSTTGGATNDNH (SEQ ID NO: 46076), (iii) NSTTGGSSNDNH (SEQ ID NO: 46079), (iv) NSTTGGSTNDNH (SEQ ID NO: Atty Docket No.: 38053-57988/WO (112WO) 46082), or a corresponding sequence having one or more modifications (e.g., insertion, deletion, mutation, and/or substitution).
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of SGQTGGP 7 P 8 NDNH (SEQ ID NO: 44929), wherein P 7 and P 8 are independently selected from any amino acid residue.
  • P7 is independently selected from an alanine (A) or a serine (S) and P 8 is independently selected from a serine (S) or a threonine (T).
  • the modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), wherein the peptide segment has an amino acid sequence of SGQTGGP7P8NDNH (SEQ ID NO: 44929), wherein P7 and P 8 are independently selected from any amino acid residue.
  • P 7 is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T).
  • the peptide segment includes: (i) SGQTGGASNDNH (SEQ ID NO: 46505), (ii) SGQTGGATNDNH (SEQ ID NO: 46508), (iii) SGQTGGSSNDNH (SEQ ID NO: 46511), (iv) SGQTGGSTNDNH (SEQ ID NO: 46514), or a corresponding sequence having one or more modifications (e.g., insertion, deletion, and/or substitution).
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of SGTAGGP7P8NDNT (SEQ ID NO: 44930), wherein P7 and P8 are independently selected from any amino acid residue.
  • P7 is independently selected from an alanine (A) or a serine (S)
  • P 8 is independently selected from a serine (S) or a threonine (T).
  • the modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), wherein the peptide segment has an amino acid sequence of SGTAGGP 7 P 8 NDNT (SEQ ID NO: 44930), wherein P 7 and P 8 are independently selected from any amino acid residue.
  • P 7 is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T).
  • the peptide segment has a sequence of: (i) SGTAGGASNDNT (SEQ ID NO: 46554), (ii) SGTAGGSSNDNT (SEQ ID Atty Docket No.: 38053-57988/WO (112WO) NO: 46560), or a corresponding sequence having one or more modifications (e.g., insertion, deletion, and/or substitution).
  • the peptide segment does not comprise SGTAGGATNDNT (SEQ ID NO: 46557) or SGTAGGSTNDNT (SEQ ID NO: 46563).
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of SGTSGGP7P8NDNA (SEQ ID NO: 44931), wherein P7 and P8 are independently selected from any amino acid residue.
  • P 7 is independently selected from an alanine (A) or a serine (S)
  • P 8 is independently selected from a serine (S) or a threonine (T).
  • the modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), wherein the peptide segment has an amino acid sequence of SGTSGGP 7 P 8 NDNA (SEQ ID NO: 44931), wherein P 7 and P 8 are independently selected from any amino acid residue.
  • P7 is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T).
  • the peptide segment includes: (i) SGTSGGASNDNA (SEQ ID NO: 46600), (ii) SGTSGGATNDNA (SEQ ID NO: 46600), (iii) SGTSGGSSNDNA (SEQ ID NO: 46603), (iv) SGTSGGSTNDNA (SEQ ID NO: 46609), or a corresponding sequence having one or more modifications (e.g., insertion, deletion, and/or substitution).
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of SGTTGGP7P8NDNT (SEQ ID NO: 44932), wherein P7 and P8 are independently selected from any amino acid residue.
  • P 7 is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T).
  • the modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of SGTTGGP 7 P 8 NDNT (SEQ ID NO: 44932), wherein P7 and P8 are independently selected from any amino acid residue.
  • P 7 is independently selected from an alanine (A) or a serine (S)
  • P 8 is independently selected from a serine (S) or a threonine (T).
  • the peptide segment includes: (i) SGTTGGASNDNT (SEQ ID NO: 46650), (ii) SGTTGGATNDNT (SEQ ID NO: 46653), (iii) SGTTGGSSNDNT (SEQ ID NO: 46656), (iv) SGTTGGSTNDNT (SEQ ID NO: 46659), or a corresponding sequence having one or more modifications (e.g., insertion, deletion, and/or substitution).
  • the peptide segment is SGTTGGSSNDNT (SEQ ID NO: 46656).
  • the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has an amino acid sequence of SSTAGGP 7 P 8 NDNA (SEQ ID NO: 44933), wherein P 7 and P 8 are independently selected from any amino acid residue.
  • P7 is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T).
  • themodified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), wherein the peptide segment has an amino acid sequence of SSTAGGP7P8NDNA (SEQ ID NO: 44933), wherein P7 and P8 are independently selected from any amino acid residue.
  • P 7 is independently selected from an alanine (A) or a serine (S) and P 8 is independently selected from a serine (S) or a threonine (T).
  • the peptide segment includes: (i) SSTAGGASNDNA (SEQ ID NO: 47128), (ii) SSTAGGATNDNA (SEQ ID NO: 47131), (iii) SSTAGGSSNDNA (SEQ ID NO: 47134), (iv) SSTAGGSTNDNA (SEQ ID NO: 47137), or a corresponding sequence having one or more modifications (e.g., insertion, deletion, and/or substitution).
  • the peptide segment is SSTAGGASNDNA (SEQ ID NO: 47128).
  • the peptide segment is SSTAGGATNDNA (SEQ ID NO: 47131).
  • the peptide segment is NSTSGASTNDNA (SEQ ID NO: 48390). [00377] In some embodiments, the peptide segment is selected a peptide segment as shown in Table 20. [00378] In some embodiments, a modified AAV capsid protein of the present disclosure can change the tropism, specificity and/or bio-distribution of an AAV comprising the modified AAV capsid protein. In some embodiments, an AAV comprising the modified AAV capsid protein has increased targeting to a target cell, tissue or organ when Atty Docket No.: 38053-57988/WO (112WO) administered to a subject.
  • an AAV comprising the modified AAV capsid protein has decreased distribution outside of a target cell, tissue or organ when administered to a subject.
  • tropism of a modified AAV capsid protein can be measured using an enrichment score.
  • An enrichment score is based on the combination of amino acid residues present in the modified sequence within VR I, where the modified sequence includes P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 10 P 11 P 12 .
  • FIG.52A provides an exemplary enrichment score formula, which is reproduced below.
  • X1 refers to, for example, amino acid position 1 (i.e., P1)
  • a refers to the metric calculated for the assigned amino acid residue at the amino acid position
  • T refers to the amino acid residue at the X amino acid position. 6.2.6.
  • variable region I (VR I) corresponds to sequences between about position 259 to about position 275 in the modified AAV capsid protein. In some embodiments, variable region I (VR I) corresponds to sequences between about position 259 to about position 275 in the reference AAV capsid protein (e.g., any of the reference AAV capsid proteins described herein (see, e.g., Section 4.2.4)).
  • a modified adeno-associated virus (AAV) capsid protein comprises a reference AAV capsid protein with one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein
  • the peptide segment is at a position between S261 and Y274 of an AAV9 capsid protein (SEQ ID NO: 61).
  • the peptide segment is at a position c between S260 and Y273 of an Anc80 capsid protein (SEQ ID NO: 132).
  • the peptide segment is at a position between S260 and Y273 of an Anc80L65 capsid protein (SEQ ID NO: 142). Atty Docket No.: 38053-57988/WO (112WO) [00385] In some embodiments, the peptide segment is at a position S260 and Y273 of an AAV2 capsid protein (SEQ ID NO: 55). 6.2.7. Other modifications [00386] In some embodiments, the modified AAV capsid protein comprises one or more additional modifications compared to a reference AAV capsid protein. The one or more additional modifications can be a insertion, deletion, substitution or a combination thereof, and can be located in the VRVIII or outside of the VRVIII.
  • the modified AAV capsid protein is different from the reference AAV capsid protein by having one or more amino acid substitutions at a variable region of the reference AAV capsid protein.
  • the one or more amino acid substitutions is at a variable region, VR I, of the reference AAV capsid protein (FIG.1).
  • the modified AAV capsid protein can be biologically or chemically produced.
  • the modified AAV capsid protein can have tropism, specificity or localization different from the reference AAV capsid protein, particularly in liver, when administered to a mammalian subject.
  • the mammalian subject can be a human, non-human primate (NHP), mice, rats, birds, rabbits, guinea pigs, hamsters, farm animals (including pigs and sheep), dogs, or cats.
  • the modified AAV capsid protein comprises a sequence different from a reference AAV capsid protein by having an amino acid substitution at an amino acid position corresponding to position 266 in Anc80 VP1 and/or at an amino acid position corresponding to position 168 in Anc80 VP1.
  • the modified AAV capsid protein comprises a sequence different from a reference AAV capsid protein by having (1) an alanine (A) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1 or (2) a lysine (K) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1.
  • the modified AAV capsid protein comprises a sequence different from a reference AAV capsid protein by having (1) an alanine (A) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1 and (2) a lysine (K) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1.
  • the modified AAV capsid protein comprises a sequence different from a reference AAV capsid protein by having a glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80; or an arginine (R) Atty Docket No.: 38053-57988/WO (112WO) amino acid residue at an amino acid position corresponding to position 168 in Anc80.
  • G glycine
  • R arginine
  • the modified AAV capsid protein comprises a sequence different from a reference AAV capsid protein by having a glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80; and an arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80.
  • G glycine
  • R arginine
  • An amino acid position corresponding to position 266 in Anc80 VP1 and an amino acid position corresponding to position 168 in Anc80 VP1 in various VP1 protein sequences are indicated with boxes in FIGs.3A-C and FIGs.4A-D.
  • the modified AAV capsid protein is different from a reference AAV capsid protein only at an amino acid position corresponding to position 266 in Anc80 VP1 or an amino acid position corresponding to position 168 in Anc80 VP1, other than the targeting peptide. In some embodiments, the modified AAV capsid protein is different from a reference AAV capsid protein only at two amino acid positions – an amino acid position corresponding to position 266 in Anc80 VP1 and an amino acid position corresponding to position 168 in Anc80 VP1, other than the targeting peptide. In some embodiments, the modified AAV capsid protein is different from a reference AAV capsid protein by more than the two amino acid substitutions.
  • the modified AAV capsid protein comprises a sequence different from a reference AAV capsid protein by having an alanine (A) amino acid residue at an amino acid position corresponding to position 267 in AAV9 VP1 protein or a threonine (T) amino acid residue at an amino acid position corresponding to position 269 in AAV9 VP1.
  • the modified AAV capsid protein comprises a sequence different from a reference AAV capsid protein by having an alanine (A) amino acid residue at an amino acid position corresponding to position 267 in AAV9 VP1 protein and a threonine (T) amino acid residue at an amino acid position corresponding to position 269 in AAV9 VP1.
  • the modified AAV capsid protein is different from a reference AAV capsid protein only at an amino acid position corresponding to position 267 in AAV9 VP1 protein or an amino acid position corresponding to position 269 in AAV9 VP1, other than the targeting peptide. In some embodiments, the modified AAV capsid protein is different from a reference AAV capsid protein only at two amino acid positions – an amino acid position corresponding to position 267 in AAV9 VP1 protein and an amino acid position corresponding to position 269 in AAV9 VP1, other than the targeting peptide.
  • a reference AAV capsid is an AAV capsid protein disclosed in WO2019/217911, which is incorporated by reference in its entirety herein.
  • an AAV capsid protein that is described therein to generate a “liver off” (“liver de-targeting”) AAV can be used in embodiments herein.
  • an AAV capsid protein that is described therein to generate a “liver on” (“liver targeting”) AAV can be used herein.
  • two amino acid positions corresponding to position 266 and position 168 of Anc80 VP1 protein are used as liver-toggle positions.
  • AAV with a capsid protein having an alanine (A) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1 or b) a lysine (K) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1 exhibits liver-off phenotypes.
  • AAV with a capsid protein having a glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80 VP1 or b) an arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80 VP1 exhibits liver-on phenotypes.
  • G glycine
  • R arginine
  • more than one toggle region residues are introduced to enhance the liver-off or the liver-on phenotypes.
  • a double mutant AAV9 G267A S269T is used.
  • a reference AAV capsid is Anc80L65 capsid protein with a G266A mutation.
  • a reference AAV capsid is Anc80-55 capsid protein with a G266A mutation. In some embodiments, a reference AAV capsid is Anc80- 129 capsid protein with a G266A mutation. In some embodiments, a reference AAV capsid is Anc80-156 capsid protein with a G266A mutation. In some embodiments, a reference AAV capsid is Anc80-751 capsid protein with a G266A mutation. In some embodiments, a reference AAV capsid is Anc80-1029 capsid protein with a G266A mutation.
  • a reference AAV capsid is Anc80-1712 capsid protein with a G266A mutation. In some embodiments, a reference AAV capsid is AAV9 capsid protein with a G267A mutation. In some embodiments, a reference AAV capsid is AAV9 capsid protein with G267A and S269T mutations. [00400] In some embodiments, a reference AAV capsid comprises (1) an alanine (A) amino acid residue at an amino acid position corresponding to position 504 in AAV9; and (2) Atty Docket No.: 38053-57988/WO (112WO) an alanine (A) amino acid residue at an amino acid position corresponding to position 505 in AAV9.
  • the modified AAV capsid protein does not include one or more modifications as described in WO2019/217911 or WO 2021/050614.
  • WO 2021/050614 showed that in a non-naturally occurring AAV capsid that A266 variants were less efficient in liver uptake compared to G266 variants.
  • Amino acid position 266 from Anc80 in WO 2021/050614 corresponds to position 267 in AAV9 VP1.
  • the reference AAV capsid protein does not include an alanine (A) at an amino acid position corresponding to position 267 in AAV9 VP1.
  • the one or more modifications to a reference AAV capsid protein to comprise a peptide segment within variable region I do not include modification to an alanine (A) at an amino acid position corresponding to position 267 in AAV9 VP1.
  • the reference AAV capsid protein comprises a glycine (G) at an amino acid position corresponding to position 267 in AAV9 VP1.
  • the modified AAV capsid protein does not include an alanine (A) at an amino acid position corresponding to position 267 in AAV9 VP1 protein and a threonine (T) at an amino acid position corresponding to position 269 in AAV9 VP1.
  • the modified AAV capsid protein does not include an alanine (A) at an amino acid position corresponding to position 267 in AAV9 VP1 protein and a threonine (T) at an amino acid position corresponding to position 269 in AAV9 VP1. 6.2.8.
  • A alanine
  • T threonine
  • a modified AAV capsid protein comprises: (i) a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 238-44858; and (ii) a peptide segment within variable region I (VR I), wherein the peptide segment as a sequence selected from SEQ ID NOs: 44935-47387.
  • a modified AAV capsid protein comprises: (i) a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391- 157057; and (ii) a peptide segment within variable region I (VR I), wherein the peptide segment has a sequence selected from SEQ ID NOs: 46026, 46029, 46031, 46035, 46073, Atty Docket No.: 38053-57988/WO (112WO) 46076, 46079, 46082, 46505, 46508, 46511, 46514, 46554, 46557, 46560, 46563, 46609, 46600, 46603, 46606, 46650, 46653, 46656, 46659, 47128, 47131, 47134
  • the modified AAV capsid protein comprises an (i) a targeting peptide having an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879- 44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) a peptide segment that has the amino acid of sequence SGTAGGASNDNT (SEQ ID NO: 46554).
  • the modified AAV capsid protein comprises an (i) a targeting peptide having an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and a peptide segment that has the amino acid sequence of SGTSGGSTNDNA (SEQ ID NO: 46609).
  • the modified AAV capsid protein comprises an (i a targeting peptide having an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) a peptide segment that has the amino acid sequence of SGTTGGSTNDNT (SEQ ID NO: 46659).
  • the modified AAV capsid protein comprises an (i) a targeting peptide having an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) a peptide segment that has the amino acid sequence of SGTTGGSTNDNT (SEQ ID NO: 47128).
  • the modified AAV capsid protein comprises an (i) a targeting peptide having an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) a peptide segment that has the amino acid sequence of SSTAGGATNDNA (SEQ ID NO: 47131).
  • the modified AAV capsid protein comprises an (i) a targeting peptide having an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) a peptide segment that has the amino acid sequence of NSTSGGSSNDNA (SEQ ID NO: 48388).
  • the modified AAV capsid protein comprises an (i) a targeting peptide having an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879-44883, 44911, 44912, 44913,44918-44919, and 48391-157057; and (ii) a peptide segment that has the amino acid sequence of NSTSGASTNDNA (SEQ ID NO: 48390).
  • a modified adeno-associated virus (AAV) capsid protein comprising: a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 48882, 44864, 44866, 44911, 44918, 98944-157057 and a peptide segment within VR I, wherein the peptide segment has a sequence selected from SEQ ID NOs: 48388, 48390, 46656, 47128, and 47131.
  • a modified adeno-associated virus (AAV) capsid protein comprising: a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 44882, 44911, 44918, 49394, and 98944- 144426 and a peptide segment within VR I, wherein the peptide segment has a sequence of NSTSGASTNDNA (SEQ ID NO: 48390).
  • a modified adeno-associated virus (AAV) capsid protein comprising: a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 44880, 44886, 44864, and 144427- 152004 and a peptide segment within VR I, wherein the peptide segment has a sequence of SGTTGGSSNDNT (SEQ ID NO: 46656).
  • a modified adeno-associated virus (AAV) capsid protein comprising: a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 44864 and 152005-154530 and a peptide segment within VR I, wherein the peptide segment has a sequence of SSTAGGASNDNA (SEQ ID NO: 47128).
  • a modified adeno-associated virus (AAV) capsid protein comprising: a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 44860 and 154531-157056 and a peptide segment within VR I, wherein the peptide segment has a sequence of SSTAGGATNDNA (SEQ ID NO: 47131).
  • a modified adeno-associated virus (AAV) capsid protein comprising: a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence of selected from: SAQRGDRGQI (SEQ ID NO: 44911), SAQRGDHASW (SEQ ID NO: 157057), ENRRGDFNNT (SEQ ID NO: 44880), ENRRGDFNNL (SEQ ID NO: 44864), ENRRGDFQNT (SEQ ID NO: 44866), and SAQRGDLLLS (SEQ ID NO: 44882)and a peptide segment within VR I, wherein the peptide segment has a sequence of NSTSGGSSNDNA (SEQ ID NO: 48388).
  • SAQRGDRGQI SEQ ID NO: 44911
  • SAQRGDHASW SEQ ID NO: 157057
  • ENRRGDFNNT SEQ ID NO: 44880
  • ENRRGDFNNL SEQ ID NO: 44864
  • ENRRGDFQNT
  • the present disclosure provides a polynucleotide encoding a modified AAV capsid protein described herein.
  • the polynucleotide is codon optimized for expression in a bacterial or mammalian cell.
  • the polynucleotide is inserted into an expression vector.
  • the polynucleotide is operably linked to a promoter or a sequence inducing expression of a protein from the polynucleotide.
  • the present disclosure provides a vector including the polynucleotide encoding a modified AAV capsid protein.
  • the vector can be used for generation of the modified AAV capsid protein.
  • the vector is used to generate an AAV virion comprising the modified AAV capsid protein.
  • the vector further comprises an AAV rep protein or a fragment thereof.
  • the reference capsid protein for the modified AAV capsid protein and the rep protein are originated from an AAV of the same clade.
  • the reference capsid protein for the modified AAV capsid protein and the rep protein are originated from an AAV of different clades.
  • the polynucleotide is transfected to a host cell.
  • the present disclosure provides a host cell comprising the polynucleotide encoding a modified AAV capsid protein.
  • the host cell can be a prokaryotic cell or eukaryotic cell.
  • the host cell is a mammalian cell or a yeast cell.
  • the host cell further comprises another polynucleotide encoding an AAV protein.
  • the host cell comprises a functional rep gene; a recombinant nucleic acid vector comprising AAV inverted terminal repeats (ITRs) and an expressible polynucleotide; and sufficient helper functions to permit packaging of the recombinant nucleic acid vector into the modified AAV capsid protein.
  • ITRs AAV inverted terminal repeats
  • the components required for the host cell to package a recombinant nucleic acid vector in a modified AAV capsid protein are provided to the host cell in trans.
  • any one or more of the required components are provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art.
  • a stable host cell contains the required component(s) under the control of an inducible Atty Docket No.: 38053-57988/WO (112WO) promoter.
  • the required component(s) is under the control of a constitutive promoter.
  • Recombinant nucleic acid vector containing an expressible polynucleotide provides a recombinant nucleic acid vector containing an expressible polynucleotide.
  • the recombinant nucleic acid vector is encapsulated in the modified AAV capsid proteins disclosed herein.
  • the expressible polynucleotide comprises a transgene (in cis or trans configuration with other viral sequences).
  • the transgene can be, for example, a reporter gene (e.g., beta-lactamase, beta- galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent polypeptide (GFP), chloramphenicol acetyltransferase (CAT), or luciferase, or fusion polypeptides that include an antigen tag domain such as hemagglutinin or Myc), or a therapeutic gene (e.g., genes encoding hormones or receptors thereof, growth factors or receptors thereof, differentiation factors or receptors thereof, immune system regulators (e.g., cytokines and interleukins) or receptors thereof, enzymes, RNAs (e.g., inhibitory RNAs or catalytic RNAs), or target antigens (e.g., oncogenic antigens, autoimmune antigens).
  • a reporter gene e.g., beta-lactamase, beta-
  • the modified rAAV comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA.
  • the transgene can be selected depending, at least in part, on the particular disease or deficiency being treated.
  • gene transfer or gene therapy can be applied to the treatment of hemophilia, retinitis pigmentosa, cystic fibrosis, leber congenital amaurosis, lysosomal storage disorders, inborn errors of metabolism (e.g., inborn errors of amino acid metabolism including phenylketonuria, inborn errors of organic acid metabolism including propionic acidemia, inborn errors of fatty acid metabolism including medium-chain acyl-CoA dehydrogenase deficiency (MCAD)), cancer, achromatopsia, cone- rod dystrophies, macular degenerations (e.g., age-related macular degeneration), lipopolypeptide lipase deficiency, familial hypercholesterolemia, spinal muscular atrophy, Duchenne’s muscular dystrophy, Alzheimer’s disease, Parkinson’s disease, obesity, inflammatory bowel disorder, diabetes, congestive heart failure, hypercholesterolemia, hearing loss, coronary heart disease, familial renal amyloidos
  • a transgene also can be, for example, an immunogen that is useful for immunizing a subject (e.g., a human, an animal (e.g., a companion animal, a farm animal, an endangered animal).
  • immunogens can be obtained from an organism (e.g., a pathogenic organism) or an immunogenic portion or component thereof (e.g., a toxin polypeptide or a by-product thereof).
  • pathogenic organisms from which immunogenic polypeptides can be obtained include viruses (e.g., picornavirus, enteroviruses, orthomyxovirus, reovirus, retrovirus), prokaryotes (e.g., Pneumococci, Staphylococci, Listeria, Pseudomonas), and eukaryotes (e.g., amebiasis, malaria, leishmaniasis, nematodes). It would be understood that the methods described herein and compositions produced by such methods are not to be limited by any particular transgene. 6.4.1.
  • viruses e.g., picornavirus, enteroviruses, orthomyxovirus, reovirus, retrovirus
  • prokaryotes e.g., Pneumococci, Staphylococci, Listeria, Pseudomonas
  • eukaryotes e.g., amebiasis, malaria, leishmaniasis, nematodes
  • the transgene is the MTM1 transgene for treatment of subjects (preferably human subjects) suffering from XLMTM and/or carrying mutations in the MTM1 gene as described in PCT Application No. PCT/US2022/015842, which is herein incorporated by reference in its entirety.
  • a modified rAAV of the present disclosure can be administered to a subject in a suitable pharmaceutical carrier, e.g., as described herein, and as described in PCT Application No.
  • Exemplary functional fragments of an MTM1 polypeptide include fragments comprising amino acids 29-486 of SEQ ID NO:165 (i.e., the amino acid sequence of SEQ ID NO:164).
  • the MTM1 polypeptides comprise amino acid residues 29-486 of SEQ ID NO:165 or the amino acid sequence of SEQ ID NO:164.
  • the MTM1 polypeptide comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to a functional fragment of human MTM1 having the amino acid sequence of SEQ ID NO:164.
  • the MTM1 polypeptide is a full length MTM1 polypeptide (e.g., a polypeptide of SEQ ID NO:165). Atty Docket No.: 38053-57988/WO (112WO) [00430]
  • the MTM1 polypeptide is a fusion polypeptide comprising an amino acid sequence having at least at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO:164 fused to another polypeptide portion, e.g., one or more polypeptide portions that enhance one or more of in vivo stability, in vivo half-life, uptake/administration, and/or purification.
  • the polypeptide portion is an internalizing moiety.
  • the MTM1 coding sequence comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO:166, which is of the native MTM1 coding sequence, or a portion thereof encoding a functional fragment of wild type MTM1, e.g., the functional fragment corresponding to amino acids 29-486 of MTM1 (SEQ ID NO:164).
  • the MTM1 coding sequence comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:166 or a portion thereof encoding a functional fragment of wild type MTM1, e.g., the functional fragment corresponding to amino acids 29- 486 of MTM1 (SEQ ID NO:164).
  • the MTM1 coding sequence comprises a nucleotide sequence having at least 80% sequence identity to any of SEQ ID NOs:167, 168 and 169, which are codon-optimized for expression in human cells, or to a portion of any of SEQ ID NOs:167, 168 and 169 encoding a functional fragment of wild type MTM1, e.g., the functional fragment corresponding to amino acids 29-486 of MTM1 (SEQ ID NO:164).
  • the MTM1 coding sequence comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs:167, 168 and 169, or to or to a portion of any of SEQ ID NOs:167, 168 and 169 encoding a functional fragment of wild type MTM1, e.g., the functional fragment corresponding to amino acids 29-486 of MTM1 (SEQ ID NO:164).
  • the MTM1 coding sequence may further comprise a nucleotide sequence that encodes a linker and/or an internalizing moiety.
  • the internalizing moiety is an antibody or an antigen-binding fragment thereof. 6.4.2. Regulatory Sequences [00434]
  • the recombinant nucleic acid vector of the disclosure typically comprise regulatory sequences operably linked to expressible polynucleotide (e.g., the MTM1 coding sequence), for example, as described in PCT Application No. PCT/US2022/015842, which is Atty Docket No.: 38053-57988/WO (112WO) herein incorporated by reference in its entirety.
  • the regulatory sequence can comprise a promoter, an enhancer, and/or a repressor. In some embodiments, the regulatory sequence is tissue specific.
  • the regulatory sequence induces expression of a transgene in a specific target such as heart, liver, skeletal muscle, cardiac muscle, or other muscle.
  • ITR sequences e.g., wild type ITRs or a combination of wild type ITR sequences and an ITR sequence lacking a functional terminal resolution site, for example as set forth in SEQ ID NO:178 and SEQ ID NO:179
  • a intron e.g., a chimeric intron comprising human herpesvirus beta and human globin 3 intronic sequences, for example as set forth in SEQ ID NO:176
  • a splice acceptor sequence 5′ of the MTM1 coding sequence e.g., a human globin 3 splice acceptor sequence, for example as set forth in SEQ ID NO:180
  • a polyadenylation sequence e.g., a rabbit globin polyadenylation sequence, for example as set forth in SEQ ID NO:177).
  • CAG Promoters Certain embodiments of the present disclosure are based in part on the discovery that an ERE comprising a CAG promoter can drive far greater expression levels of the expressible polynucleotide (e.g., MTM1 coding sequence) than the desmin promoter in clinical development, for example, as described in PCT Application No. PCT/US2022/015842, which is herein incorporated by reference in its entirety.
  • the expressible polynucleotide e.g., MTM1 coding sequence
  • the rAAV with the MTM1 coding sequence under the control of the CAG promoter can be therapeutically effective at lower doses than corresponding vectors in which the MTM1 coding sequence is under the control of the desmin promoter, and thus such vectors are believed to have improved therapeutic indexes as compared to corresponding vectors in which the MTM1 coding sequence is under the control of the desmin promoter.
  • the CMV enhancer component of the CAG promoter or ERE comprises a sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:171.
  • the chicken beta actin promoter component of the CAG promoter or ERE comprises a nucleotide sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:172.
  • Atty Docket No.: 38053-57988/WO (112WO) [00439]
  • the CAG promoter or ERE comprises a nucleotide sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:173.
  • An exemplary CAG ERE is used in the rAVE expression cassette (GeneDetect.com).
  • the CAG ERE further comprises a chimeric intron, for example a chimeric intron formed from introns from the human betaherpes virus and rabbit beta globin.
  • the chimeric intron comprises a nucleotide sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:174.
  • Further modifications of the CAG promoter can be used in the rAAV of the disclosure.
  • the intron in the 5′ untranslated region (UTR) of the CAG promoter can be truncated to accommodate larger inserts (Richardson et al., 2009, PLoS One, 4(4), e5308. doi: 10.1371/joumal.pone.0005308). Deletions in intron A of the hCMV promoter can also result in enhanced expression (Quilici et al., 2013, Biotechnol Lett.35(1), 21-27. doi: 10.1007/s10529-012-1043-z).
  • a person skilled in the art could modify the CAG ERE or promoter sequences without compromising the high MTM1 expression levels observed with the constructs disclosed in Example 7.
  • the rAAV of the disclosure may comprise, in lieu of a CAG ERE, an ERE comprising another constitutive promoter or a tissue specific or inducible promoter. Promoters that drive lower expression levels than a CAG promoter may be combined with other features that increase transgene expression (e.g., using codon optimized coding sequences) and/or reduce off target tropism of the virus (e.g., using muscle targeting and/or liver toggle capsid proteins). [00444] In various embodiments the promoter is a constitutive, tissue-specific (e.g., muscle-specific) or inducible promoter.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter. [00445] In some embodiments, the promoter is a skeletal-muscle specific promoter. In some embodiments, the promoter is a cardiac-specific promoter.
  • constitutive promoters include, without limitation, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, a phosphoglycerol kinase (PGK) promoter, and a EF1 ⁇ promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • SV40 promoter a SV40 promoter
  • dihydrofolate reductase promoter a ⁇ -actin promoter
  • PGK phosphoglycerol kinase
  • tissue-specific promoters include, without limitation a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an ⁇ -myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.
  • inducible promoters include a zinc-inducible metallothionine (MT) promoter, a dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, a tetracycline-inducible promoter, or a rapamycin-inducible promoter.
  • the present disclosure further provides a modified recombinant AAV (rAAV) virion comprising a modified AAV capsid protein described herein.
  • the modified rAAV comprises a modified AAV capsid protein and a recombinant nucleic acid vector.
  • the modified rAAV comprising a modified AAV capsid protein achieves higher infection of a target following administration to a mammalian subject as compared to an rAAV comprising a corresponding reference AAV capsid protein.
  • the modified rAAV achieves higher expression in a target of an expressible polynucleotide within the recombinant nucleic acid vector following administration to a mammalian subject when compared to expression of the expressible polynucleotide administered in an rAAV comprising a corresponding reference AAV capsid protein.
  • the modified rAAV comprising a modified AAV capsid protein achieves lower infection of an off-target following administration to a mammalian subject as compared to an rAAV comprising a corresponding reference AAV capsid protein.
  • the modified rAAV achieves lower expression in an off-target of an expressible polynucleotide within the recombinant nucleic acid vector following administration to a mammalian subject as compared to expression of the expressible polynucleotide administered in an rAAV comprising a corresponding reference AAV capsid protein.
  • the corresponding reference AAV capsid Atty Docket No.: 38053-57988/WO (112WO) protein is a capsid protein identical to the modified AAV capsid protein except that it does not include a targeting peptide and/or a liver-toggle mutation described above.
  • the target is brain, muscle, spinal cord, eye, liver, muscle, or other organ.
  • the off-target tissue is brain, muscle, spinal cord, eye, liver, muscle, or other organ.
  • the target is muscle.
  • the modified rAAV has less liver toxicity than an rAAV comprising a corresponding reference AAV capsid protein administered by the same route of administration and in the same dose. In some embodiments, the less liver toxicity is because of de-targeting of the modified rAAV to a liver. 6.6.
  • the rAAV of the disclosure comprise a recombinant nucleic acid vector containing an expressible polynucleotide.
  • the expressible polynucleotide is operably linked to an ERE.
  • the expressible polynucleotide and ERE optionally replace the AAV genomic coding region (e.g., replace the AAV rep and cap genes).
  • the expressible polynucleotide and ERE are generally flanked on either side by AAV inverted terminal repeat (ITR) regions, although a single ITR may be sufficient to carry out the functions normally associated with configurations comprising two ITRs (see, for example, WO 94/13788), and vector constructs with only one ITR can thus be employed in conjunction with the rAAV of the present disclosure.
  • the rAAV of the disclosure comprise an MTM1 coding sequence operably linked to an ERE.
  • the MTM1 coding sequence and ERE optionally replace the AAV genomic coding region (e.g., replace the AAV rep and cap genes).
  • the missing functions are complemented with a packaging gene, or a plurality thereof, which together encode the necessary functions for the various missing rep and/or cap gene products.
  • the packaging genes or gene cassettes are in one embodiment not flanked by AAV ITRs and in one embodiment do not share any substantial homology with the rAAV genome.
  • the rAAV vector construct, and the complementary packaging gene constructs can be implemented in a number of different forms. Viral particles, plasmids, and stably transformed host cells can all be used to introduce such constructs into the packaging cell, either transiently or stably.
  • the AAV vector and complementary packaging gene(s), if any, are provided in the form of bacterial plasmids, AAV particles, or any combination thereof.
  • either the AAV vector sequence, the packaging gene(s), or both are provided in the form of genetically altered (preferably inheritably altered) eukaryotic cells.
  • the development of host cells inheritably altered to express the AAV vector sequence, AAV packaging genes, or both provides an established source of the material that is expressed at a reliable level.
  • a variety of different genetically altered cells can thus be used in the context of this invention.
  • a mammalian host cell may be used with at least one intact copy of a stably integrated rAAV vector.
  • An AAV packaging plasmid comprising at least an AAV rep gene operably linked to a promoter can be used to supply replication functions (as described in U.S. Pat. No.5,658,776).
  • a stable mammalian cell line with an AAV rep gene operably linked to a promoter can be used to supply replication functions (see, e.g., WO 95/13392; WO 98/23018; and U.S. Patent No.5,656,785).
  • the AAV cap gene providing the encapsidation proteins as described above, can be provided together with an AAV rep gene or separately (see, e.g., the above-referenced patent documents as well as WO 98/27204.
  • the rAAV of the disclosure can be assembled by, for example, expression of its components in a packaging host cell.
  • the components of a virus particle e.g., rep sequences, cap sequences, inverted terminal repeat (ITR) sequences
  • ITR inverted terminal repeat
  • purified virus particles refer to virus particles that are removed from components in the mixture in which they were made such as, but not limited to, viral components (e.g., rep sequences, cap sequences), packaging host cells, and partially- or incompletely- assembled virus particles.
  • pharmaceutical composition comprising a modified AAV capsid protein or a modified rAAV of the present disclosure and a pharmaceutically acceptable carrier.
  • the modified rAAV can comprise a modified AAV Atty Docket No.: 38053-57988/WO (112WO) capsid protein as described herein and a recombinant nucleic acid vector containing an expressible polynucleotide.
  • the present disclosure provides a pharmaceutical composition
  • an rAAV whose genome comprising an MTM1 coding sequence operably linked to an expression regulatory element (ERE); and one, two or all three of the following features: (a) the ERE is a hybrid expression regulatory element (ERE) comprising a CMV enhancer and a chicken beta actin promoter operably linked to the MTM1 coding sequence; and/or (b) the rAAV comprises a modified AAV capsid protein comprising at least one liver-toggle mutation and/or one muscle-targeting element; and/or (c) the MTM1 coding sequence is codon optimized for expression in human cells, optionally wherein the coding sequence has at least 90%, at least 95%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOS:167 to 170.
  • ERE is a hybrid expression regulatory element (ERE) comprising a CMV enhancer and a chicken beta actin promoter operably linked to the MTM1 coding sequence
  • the pharmaceutical composition can be used to deliver the recombinant nucleic acid vector to a target within a mammalian subject.
  • the modified rAAV can achieve a higher infection of target cells following administration to a mammalian subject as compared to an rAAV comprising a corresponding reference AAV capsid protein administered by the same route of administration and in the same dose.
  • the modified rAAV achieves higher expression in target cells of an expressible polynucleotide within the recombinant nucleic acid genome following administration to a mammalian subject as compared to the expressible polynucleotide administered in an rAAV comprising a corresponding reference AAV capsid protein administered by the same route of administration and in the same dose.
  • the pharmaceutical composition can be formulated using one or more carriers, excipients, stabilizers and adjuvants to, for example: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; (4) alter the biodistribution (e.g., target the rAAV particle to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo.
  • Formulations of the pharmaceutical compositions provided herein can include, without limitation, saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, Atty Docket No.: 38053-57988/WO (112WO) pectin, water, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, nanoparticle mimics and combinations thereof.
  • Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology.
  • Such preparatory methods include the step of associating the active ingredient with a carrier and/or one or more other accessory ingredients (e.g., excipients, stabilizers and adjuvants).
  • a pharmaceutical composition in accordance with the present disclosure can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a unit dose refers to a discrete amount of the pharmaceutical composition including a predetermined amount of the active ingredient.
  • the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
  • Relative amounts of the active ingredient (e.g., rAAV), the pharmaceutically acceptable carrier, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure can vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered.
  • Various carriers, excipients, stabilizers and adjuvants for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 22nd Revised Ed., Pharmaceutical Press, 2012; incorporated herein by reference in its entirety). The use of suitable conventional carriers, excipients, stabilizers and adjuvants is contemplated within the scope of the present disclosure.
  • the pharmaceutical composition is in the form of a solution containing concentrations of from about 1 x 101 to about 1 x 1016 genome copies (GCs)/ml of rAAV (e.g., a solution containing concentrations of from about 1 x 103 to about 1 x 1014 GCs/ml). 6.7.1. Routes of Administration [00472] A modified rAAV of the present disclosure can be administered to a subject (e.g., a human or non-human mammal) in a suitable carrier.
  • a subject e.g., a human or non-human mammal
  • Suitable carriers include saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered Atty Docket No.: 38053-57988/WO (112WO) saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, and water.
  • buffering solutions e.g., phosphate buffered Atty Docket No.: 38053-57988/WO (112WO) saline
  • lactose lactose
  • sucrose sucrose
  • calcium phosphate gelatin
  • dextran agar
  • pectin agar
  • pectin pectin
  • routes of administration include, but are not limited to, direct delivery to an organ such as, for example, the muscle, liver or lung, orally, intranasally, intratracheally, intrathecally, intravenously, intramuscularly, intraocularly, subcutaneously, intradermally, or by other routes of administration. Routes of administration can be combined, if desired. 6.7.2. Dosages [00473] The dose of a viral vector administered to a subject will depend primarily on factors such as the condition being treated, and the age, weight, and health of the subject.
  • a therapeutically effective dosage of a viral vector to be administered to a human subject generally is in the range of from about 0.1 ml to about 10 ml of a solution containing concentrations of from about 1 x 10 1 to about 1 x 10 16 genome copies (GCs)/ml of viruses (e.g., a solution containing concentrations of from about 1 x 10 3 to about 1 x 10 14 GCs/ml).
  • a solution containing concentrations of from about 1 x 10 3 to about 1 x 10 14 GCs/ml e.g., a solution containing concentrations of from about 1 x 10 3 to about 1 x 10 14 GCs/ml.
  • the total dose of the rAAV administered to a subject is less than 3 x 10 14 GCs, e.g., 1 x 10 14 GCs or less, 5 x 10 13 GCs or less, 1 x 10 13 GCs or less, 5 x 10 12 GCs or less, or 1 x 10 12 GCs or less.
  • a therapeutically effective dosage of a viral vector to be administered to a human subject generally is in the range of from about 0.1 ml to about 10 ml of a solution containing concentrations of from about 1 x 10 1 to 1 x 10 12 genome copies (GCs) of viruses (e.g., about 1 x 10 3 to 1 x 10 9 GCs).
  • a therapeutically effective dosage of a viral vector to be administered to a human subject is about 1e12 vg/kg to 1e14 vg/kg. In some embodiments, a therapeutically effective dosage of a viral vector to be administered to a human subject is less than 1e14 vg/kg. In some embodiments, a therapeutically effective dosage of a viral vector to be administered to a human subject is 1.5e12 vg/kg to 1.5e13 vg/kg. In some embodiments, a therapeutically effective dosage of a viral vector to be administered to a human subject is about 1e13 vg/kg.
  • Transduction and/or expression of a transgene can be monitored at various time points following administration by DNA, RNA, or protein assays. In some instances, the levels of expression of the transgene can be monitored to determine the frequency and/or Atty Docket No.: 38053-57988/WO (112WO) amount of dosage. Dosage regimens similar to those described for therapeutic purposes also may be utilized for immunization. 6.7.3. Targeting [00477] Targeting of modified rAAVs can be tested in an experimental animal by measuring rAAV infection or expression of an expressible polynucleotide.
  • targeting is measured in a non-human primate (NHP), mice, rats, birds, rabbits, guinea pigs, hamsters, farm animals (including pigs and sheep), dogs, or cats.
  • NEP non-human primate
  • Targeting of modified rAAVs can be measured after systemic or local administration of rAAVs.
  • targeting of modified rAAVs is measured after intravenous infusion of rAAVs. 6.7.3.1 RNA data - Muscle:Liver Infection Ratio
  • targeting of modified rAAVs is measured by measuring the ratio between the copy numbers of the transgene transcripts and housekeeping gene (e.g., RPP30) transcripts.
  • the transcripts are measured by RT-ddPCR.
  • the ratio is measured after a first administration into a mammal, e.g., a mouse, or a non-human primate such as a marmoset or rhesus macaque.
  • a muscle:liver infection ratio is measured by comparing the ratios between the copy numbers of the transgene transcripts and housekeeping gene (e.g., RPP30) transcripts in the two different organs (e.g., muscle v. liver).
  • modified rAAV of the present disclosure provides a (transgene transcripts/housekeeping transcripts) ratio in liver of less than 1000, less than 900, less than 800, less than 700, less than 600, less than 500, less than 400, less than 300, less than 200, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10.
  • the muscle:liver infection ratio is reported as >10,000 by convention.
  • the modified rAAV of the present disclosure provides a muscle:liver infection ratio (RNA) of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 150, at least 200, at least 500, at least 1000.
  • the muscle is triceps surae, biceps, heart or quadricep.
  • modified rAAV of the present disclosure provides a muscle:liver infection ratio (RNA) of 1 to 10, 1 to 100, 10 to 20, 10 to 50, 10 to 80, 10 to 100, 20 to 100, 100 to 500, 100 to 1000, or 500 to 1000.
  • the muscle is triceps surae, bicep, heart or quadricep.
  • 6.7.3.2 DNA data - Muscle:Liver Infection Ratio targeting of modified rAAVs is measured by measuring the ratio between the copy numbers of the transgene DNA genomes to copy numbers of host genes or genetic loci (e.g., RPP30). In a particular embodiment, the genomes are measured by RT-ddPCR.
  • the ratio is measured after a first administration into a mammal, e.g., a mouse, or a non-human primate such as a marmoset or rhesus macaque.
  • a muscle:liver infection ratio is measured by comparing the ratios between the copy numbers of the transgene DNA genomes and housekeeping gene (e.g., RPP30) genomes in the two different organs (e.g., muscle v. liver).
  • modified rAAV of the present disclosure provides a (transgene genomes/housekeeping genomes) ratio
  • the modified rAAV of the present disclosure provides a muscle:liver infection ratio (DNA) of at least 1, at least 1.5, at least 2, at least 2.5, at least 3, Atty Docket No.: 38053-57988/WO (112WO) at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 500, at least 1,000 or at least 10,000
  • the muscle is triceps surae, biceps, heart or quadricep.
  • modified rAAV of the present disclosure provides a muscle:liver infection ratio (DNA) in the range of 0.5 to 1, 0.5 to 5, 0.5 to 10, 1 to 10, 1 to 100, 2 to 8, 5 to 10, 10 to 20, 20 to 80, 10 to 50, 10 to 100, 50 to 80, 100 to 500, 100 to 1000, or 500 to 1000.
  • the muscle is triceps surae, biceps, heart, or quadricep.
  • the modified rAAV achieves a muscle:liver infection ratio (DNA) of at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 500, at least 1000. In some embodiments, the modified rAAV achieves a muscle:liver infection ratio of 0.1 to 1, 1 to 5, 1 to 10, 1 to 20, 1 to 50, 1 to 100, 1 to 200, 1 to 300, 100 to 500, 250 to 750, or 500 to 1000.
  • targeting of modified rAAVs is calculated using the % of cells that have been successfully transduced and express a transgene in a tissue (e.g., eGFP).
  • a tissue e.g., eGFP
  • the transgene expression is measured by immunohistochemistry.
  • the ratio is measured after a first administration into a mammal, e.g., a mouse, or a non-human primate such as a marmoset or rhesus macaque.
  • a muscle:liver infection ratio is measured by comparing the ratios between the transgene %GFP + cells and housekeeping gene (e.g., RPP30) %GFP + cells in the two different organs (e.g., muscle v. liver).
  • housekeeping gene e.g., RPP30
  • modified rAAV of the present disclosure provides a (transgene %GFP/housekeeping %GFP) ratio in liver of less than 1, less than 5, less than 10, Atty Docket No.: 38053-57988/WO (112WO) or in a range from 1 to 10, 1 to 5, 1 to 2, 0.1 to 1, 0 to 1, 0.01 to 0.1, 0.01 to 0.5, or 0.01 to 0.05.
  • the modified rAAV of the present disclosure provides a muscle:liver infection ratio (IHC) of at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 500, at least 1000.
  • IHC muscle:liver infection ratio
  • modified rAAV of the present disclosure provides a muscle:liver infection ratio (IHC) of 1 to 5, 1 to 10, 1 to 100, 2 to 8, 10 to 20, 20 to 30, 10 to 50, 10 to 100, 20 to 80, 50 to 80, 100 to 500, 100 to 1000, or 500 to 1000.
  • IHC muscle:liver infection ratio
  • the muscle is triceps surae, bicep, heart or quadricep. 6.8.
  • a modified rAAV is for genetically modifying a cell in vitro or in vivo.
  • a modified rAAV is used for gene therapy or for vaccination in a human or animal. More specifically, a modified rAAV can be used for gene addition, gene augmentation, genetic delivery of a polypeptide therapeutic, genetic vaccination, gene silencing, genome editing, gene therapy, RNAi delivery, cDNA delivery, mRNA delivery, miRNA delivery, miRNA sponging, genetic immunization, optogenetic gene therapy, transgenesis, DNA vaccination, or DNA immunization of liver cells or non-liver cells.
  • a modified rAAV of the present disclosure is used for treatment of a muscle disease.
  • the disease is a muscular disease and/or the condition is muscle degeneration.
  • said muscular disease is a muscular dystrophy, a cardiomyopathy, a myotonia, a muscular atrophy, a myoclonus dystonia, a mitochondrial myopathy, a rhabdomyolysis, a fibromyalgia, and/or a myofascial Atty Docket No.: 38053-57988/WO (112WO) pain syndrome.
  • the modified rAAV is used to deliver the rAAV to a striated muscle, preferably heart or a skeletal muscle or diaphragm.
  • the rAAVs or pharmaceutical compositions described are useful in the treatment of subjects (preferably human subjects) suffering from XLMTM and/or carrying mutations in the MTM1 gene. “Treatment” of MTM encompasses a complete reversal or cure of the disease, or any range of improvement in conditions and/or adverse effects attributable to MTM.
  • treatment of MTM includes an improvement in any of the following effects associated with MTM or combination thereof: short life expectancy, respiratory insufficiency (partially or completely), poor muscle tone, drooping eyelids, poor strength in proximal muscles, poor strength in distal muscles, facial weakness with or without eye muscle weakness, abnormal curvature of the spine, joint deformities, and weakness in the muscles that control eye movement (ophthalmoplegia). Improvements in any of these conditions can be readily assessed according to standard methods and techniques known in the art. [00500] A modified rAAV of the present disclosure can be administered to a subject in a suitable pharmaceutical carrier, e.g., as described in Section 4.7.
  • the rAAV of the disclosure are typically administered in sufficient amounts to transduce or infect the desired cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit to subjects suffering from a disease.
  • the rAAV is administered in sufficient amounts to provide a therapeutic benefit to subjects suffering from XLMTM or carrying a mutation in the MTM1 gene, without undue adverse effects.
  • Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to an organ such as, for example, the muscle, liver or lung, orally, intranasally, intratracheally, intrathecally, intravenously, intramuscularly, intraocularly, subcutaneously, intradermally, or by other routes of administration.
  • a therapeutically effective dosage of a viral vector to be administered to a human subject generally is in the range of from about 0.1 ml to about 10 ml of a solution containing concentrations of from about 1 x 10 1 to about 1 x 10 16 genome copies (GCs)/ml of viruses Atty Docket No.: 38053-57988/WO (112WO) (e.g., a solution containing concentrations of from about 1 x 10 3 to about 1 x 10 14 GCs/ml).
  • the total dose of the rAAV administered to a subject is less than 3 x 10 14 GCs, e.g., 1 x 10 14 GCs or less, 5 x 10 13 GCs or less, 1 x 10 13 GCs or less, 5 x 10 12 GCs or less, or 1 x 10 12 GCs or less.
  • Transduction and/or expression of the transgene can be monitored at various time points following administration by DNA, RNA, or protein assays.
  • the present disclosure provides a method of treating and/or preventing a muscular disease and/or muscle degeneration by administering a modified rAAV described herein.
  • the modified rAAV capsid of the present disclosure is used to deliver a transgene to a target tissue.
  • the target tissue is cardiac muscle, diaphragm, tibialis anterior, quadriceps, biceps femoris, tibia gastrocnemius, tibialis anterior, triceps brachii, heart - ventricle wall, heart - atria, other skeletal muscle, or any muscle tissue described herein.
  • the target tissue is heart.
  • the target tissue is liver. 7. SPECIFIC EMBODIMENTS Embodiment 1.
  • a modified adeno-associated virus (AAV) capsid protein comprising: (i) a targeting peptide within VR VIII; or (ii) a peptide segment within VR I, wherein the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X1, X2, X3, X7, X8, X9 and X10 are independently selected from any amino acid residue, and wherein the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8NDNP12 and P1, P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue.
  • Embodiment 2 Embodiment 2.
  • the modified AAV capsid protein of embodiment 1, comprising: (i) the targeting peptide within VR VIII; and (ii) the peptide segment within VR I.
  • Embodiment 3. The modified AAV capsid protein of embodiment 1 or 2, wherein targeting peptide does not comprise RGDLLLS (SEQ ID NO: 1). Atty Docket No.: 38053-57988/WO (112WO) Embodiment 4.
  • the modified AAV capsid protein of embodiment 1 or 2 wherein the peptide segment does not comprise an alanine (A) at P6 and a threonine (T) at P8.
  • Embodiment 6 The modified AAV capsid protein of any one of embodiments 1-5, wherein the modified AAV capsid protein has one or more modifications comprising amino acid insertions, deletions, substitutions, or combinations thereof as compared to a reference AAV capsid protein.
  • Embodiment 8 The modified AAV capsid protein of any one of embodiments 6-7, further comprising one or more modifications outside of the VR I and VR VIII of the reference AAV capsid protein.
  • Embodiment 9 The modified AAV capsid protein of any one of embodiments 6-8, having at least 90%, 95%, 98%, 99% or 99.5% sequence identity to the sequence of the reference AAV capsid protein.
  • Embodiment 11 The modified AAV capsid protein of any one of embodiments 6-10, wherein the reference AAV capsid protein is a capsid protein of an AAV variant selected from the group consisting of: AAV9; Anc8065; Anc80-55; Anc80-129; Anc80-156; Anc80- 751; Anc80-1029; Anc80-1712; AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46- A; hu.48-A; hu.44-A; hu.43-A; AAV6-A;
  • Embodiment 12 The modified AAV capsid protein of any one of embodiments 6-11, wherein the reference AAV capsid protein is a capsid protein having a sequence selected from SEQ ID Nos: 54-152, 44885-44898, 44916-44917, or a fragment thereof.
  • Embodiment 13 The modified AAV capsid protein of any one of embodiments 6-12, wherein the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 61 or a fragment thereof.
  • Embodiment 14 The modified AAV capsid protein of any one of embodiments 6-12, wherein the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 132 or a fragment thereof.
  • Embodiment 15 The modified AAV capsid protein of any one of embodiments 6-12, the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 142 or a fragment thereof.
  • Embodiment 16 The modified AAV capsid protein of any one of embodiments 6-12, wherein the reference AAV capsid protein is a capsid protein selected from: Anc80-55, Anc80-129, Anc80-156, Anc80-751, Anc80-1029, and Anc80-1712.
  • Embodiment 17 The modified AAV capsid protein of embodiment 16, wherein the reference AAV capsid protein is a capsid protein having a sequence selected from SEQ ID NOs: 44885-44898, and 44916-44917.
  • Embodiment 18 The modified AAV capsid protein of any one of embodiments 1-10, having the sequence selected from SEQ ID NOs.: 44900-44909. Atty Docket No.: 38053-57988/WO (112WO) Embodiment 19.
  • Embodiment 20 The modified AAV capsid protein of any one of embodiments 1-19, wherein X1, X2, and X3 are independently selected from any amino acid residue.
  • Embodiment 22. The modified AAV capsid protein of embodiment 20, wherein X 1 is an amino acid that is identical to the amino acid at a corresponding position of the reference AAV capsid protein.
  • Embodiment 23. The modified AAV capsid protein of embodiment 20, wherein X2 is an amino acid that is identical to the amino acid at a corresponding position of the reference AAV capsid protein.
  • Embodiment 25. The modified AAV capsid protein of embodiment 20, wherein X1 and X3 are amino acids that are identical to the amino acids at corresponding positions of the reference AAV capsid protein.
  • Embodiment 26. The modified AAV capsid protein of embodiment 20, wherein X1 and X2 are amino acids that are identical to the amino acids at corresponding positions of the reference AAV capsid protein.
  • Embodiment 29. The modified AAV capsid protein of embodiment 20, wherein X1 is an amino acid that is not identical to the amino acid at a corresponding position of the reference AAV capsid protein.
  • Embodiment 31. The modified AAV capsid protein of embodiment 20, wherein X 3 is an amino acid that is not identical to the amino acid at a corresponding position of the reference AAV capsid protein.
  • Embodiment 32. The modified AAV capsid protein of embodiment 20, wherein X 1 and X 3 are amino acids that are not identical to the amino acids at corresponding positions of the reference AAV capsid protein.
  • Embodiment 34. The modified AAV capsid protein of embodiment 20, wherein X 2 and X 3 are amino acids that are not identical to the amino acids at corresponding positions of the reference AAV capsid protein.
  • Embodiment 35. The modified AAV capsid protein of any one of embodiments 1-34, wherein the modified AAV capsid protein comprises one or more substitutions, one or more insertions, one or more deletions, or a combination thereof into VR VIII of the reference AAV capsid protein.
  • the modified AAV capsid protein of embodiment 35 wherein one or more modifications comprises a substitution of one or more amino acids between amino acid positions 565 and 595 of the reference AAV capsid.
  • Embodiment 37 The modified AAV capsid protein of any one of embodiments 1-36, wherein X1 is selected from S, E, A, D, N, Q, or T.
  • Embodiment 38. The modified AAV capsid protein of embodiment 37, wherein X 1 is D or E.
  • Embodiment 39 The modified AAV capsid protein of embodiment 37, wherein X1 is S, A or T.
  • Embodiment 40 The modified AAV capsid protein of embodiment 37, wherein X1 is S, A or T.
  • Embodiment 41 The modified AAV capsid protein of any one of embodiments 1-36, wherein X 2 is selected from N, A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y.
  • Embodiment 42 The modified AAV capsid protein of embodiment 41, wherein X 2 is selected from K, E, D, A, S, F, N, V or L.
  • Embodiment 43 The modified AAV capsid protein of embodiment 41, wherein X2 is selected from K, E or D.
  • Embodiment 44 The modified AAV capsid protein of embodiment 41, wherein X2 is selected from K, E or D.
  • Embodiment 45 The modified AAV capsid protein of embodiment 41, wherein X 2 is selected from N, Y or S.
  • Embodiment 46 The modified AAV capsid protein of any one of embodiments 1-45, wherein X 3 is selected from R, Q, A, D, E, F, G, H, I, K, L, M, N, P, S, T, V, W, or Y.
  • Embodiment 47 The modified AAV capsid protein of embodiment 46, wherein X 3 is selected from Y, V or F.
  • Embodiment 48 The modified AAV capsid protein of embodiment 41, wherein X 3 is selected from Y, V or F.
  • Embodiment 53. The modified AAV capsid protein of any one of embodiments 1-36, wherein X1X2 X3 is selected from the group consisting of: DII, DWM, EEI, DML, DWI, SLE, EIN, NHE, DFI, EEL, TEQ, TDA, EDT, NEV, TDW, QFE, EDY, DTT, EPL, SEN, SEQ, TAE, EVN, ELN, DVQ, ETI, EVI, ESV, ETW, SEW, DNW, EVF, EAW, EPF, EIY, EIF, EPY, DVI, DMM, DQI, DHL, DTL, DVL, NDL, DLL, DMQ, NEF, DFL, DIM, TEW, DYI, SDY, DYY, DHF, DKE, DT
  • Embodiment 54 The modified AAV capsid protein of any one of embodiments 1-36, wherein X 1 X 2 X 3 is selected from the group consisting of: APW, TEL, TDA, QPY, SPN, EHY, DWK, DLK, DFK, DVK, NSI, DIR, SPF, SEL, DRT, DRF, ADL, TDL, SDL, DNY, Atty Docket No.: 38053-57988/WO (112WO) DKI, NDV, DKM, DNH, DNF, DSS, EST, EWT, DKN, DKS, SEH, ESQ, ESL, QND, EAH, AIF, AVF, QVF, TMY, ALY, NNG, NIF, NTF, NFF, AWF, NPY, SWF, AII, AYF, AQW, NFY, AGP, QQF, TKE, TNG, NSF, NAW, QAG, ERG
  • Embodiment 55 The modified AAV capsid protein of any one of embodiments 1-36, wherein X1X2X3 is selected from the group consisting of: DMK, ATD, EEK, QMD, EFS, ERD, DDR, TDM, SAE, EHS, ENH, SWE, SNE, NNG, QAG, ERG, QSG, QNG, ASG, QFG, AMF, ELR, NFM, NNS, NNI, SDR, EQR, EHR, EWR, EQK, ESR, EKQ, EYR, ENR, Atty Docket No.: 38053-57988/WO (112WO) EMK, EYK, EHK, EWK, QNI, TNI, TYI, SNY, DRQ, AWI, QWI, DFR, EKG, QYG, QWQ, EKN, EKF, EKT, AFH, ENK, NYS, DKH, AAG, QMF, QFH
  • Embodiment 56 The modified AAV capsid protein of any one of embodiments 1-55, wherein RGDX7X8X9X10 has an amino acid sequence selected from SEQ ID NOs.: 238- 44858.
  • Embodiment 57 The modified AAV capsid protein of any one of embodiments 1-55, wherein RGDX7X8X9X10 has an amino acid sequence selected from SEQ ID NOs.: 238-248.
  • Embodiment 58 The modified AAV capsid protein of any one of embodiments 1-55 wherein RGDX 7 X 8 X 9 X 10 has an amino acid sequence selected from SEQ ID NOs.: 238-338.
  • Embodiment 59 The modified AAV capsid protein of any one of embodiments 1-55, wherein RGDX 7 X 8 X 9 X 10 has an amino acid sequence selected from SEQ ID NOs.: 238-338.
  • Embodiment 64 The modified AAV capsid protein of any one of embodiments 1-55, wherein RGDX 7 X 8 X 9 X 10 has the amino acid sequence of SEQ ID NO: 238.
  • Embodiment 65 The modified AAV capsid protein of any one of embodiments 1-55, wherein X1X2X3RGDX7X8X9X10 has an amino acid sequence selected from SEQ ID NOs.: 44859- 44883, 44911, 44912, 44913, 44918-44919, and 48391-157057.
  • Embodiment 66 The modified AAV capsid protein of any one of embodiments 1-55, wherein X1X2X3RGDX7X8X9X10 has an amino acid sequence selected from SEQ ID NOs.: 44864-44867, and 44879-44883.
  • Embodiment 67 Embodiment 67.
  • Embodiment 68. The modified AAV capsid protein of any one of embodiments 1-55, wherein the X7 is selected from R, F, H, L, Q, R, and Y.
  • Embodiment 71 The modified AAV capsid protein of embodiment 68, wherein the X7 is F or Y.
  • Embodiment 72 The modified AAV capsid protein of any one of embodiments 1-71, wherein X8 is selected from S, G, D, I, L, N, Q, T, and V.
  • Embodiment 73 The modified AAV capsid protein of embodiment 72, wherein X 8 is T, G or S.
  • Embodiment 74 The modified AAV capsid protein of embodiment 72, wherein X8 is N or Q.
  • Embodiment 75 The modified AAV capsid protein of embodiment 72, wherein X8 is N or Q.
  • Embodiment 76 The modified AAV capsid protein of embodiments 1-75, wherein X9 is selected from V, S, N, G, Q, L, T, and Y.
  • Embodiment 77 The modified AAV capsid protein of embodiment 76, wherein X 9 is selected from V or Q.
  • Embodiment 79 The modified AAV capsid protein of embodiment 76, wherein X 9 is selected from N or S.
  • Embodiment 80 The modified AAV capsid protein of embodiment 76, wherein X 9 is selected from N or S.
  • Embodiment 80 The modified AAV capsid protein of embodiment 76, wherein X 9 is selected from N or S. Em
  • the modified AAV capsid protein of embodiment 80, wherein X 10 is T or L.
  • the modified AAV capsid protein of embodiment 1-55 wherein the targeting peptide has a sequence of X 1 X 2 X 3 RGDRGVV (SEQ ID NO: 98928), X1X2X3RGDRSVV (SEQ ID NO: 98931), X1X2X3RGDRGQI (SEQ ID NO: 98927), X1X2X3RGDRSQT (SEQ ID NO: 98930), X1X2X3RGDRQGI (SEQ ID NO: 98929), X 1 X 2 X 3 RGDFQNT (SEQ ID NO: 98934), X 1 X 2 X 3 RGDHGVL (SEQ ID NO: 98938), X 1 X 2 X 3 RGDYTSV (SEQ ID NO: 98941), X 1 X 2 X 3 RGDYTSM (SEQ ID NO: 98942), X1X2X3RGDLTVT (SEQ ID NO: 98935), X
  • Embodiment 85 The modified AAV capsid protein of embodiment 84, wherein the targeting peptide has a sequence of X1X2X3RGDRGVV (SEQ ID NO: 98928), X1X2X3RGDRSVV (SEQ ID NO: 98931) or X 1 X 2 X 3 RGDRGQI (SEQ ID NO: 98927).
  • Embodiment 86 The modified AAV capsid protein of embodiment 84, wherein the targeting peptide has a sequence of X1X2X3RGDRGVV (SEQ ID NO: 98928), X1X2X3RGDRSVV (SEQ ID NO: 98931) or X 1 X 2 X 3 RGDRGQI (SEQ ID NO: 98927).
  • the modified AAV capsid protein of embodiment 84 wherein the targeting peptide has a sequence of X1X2X3RGDYTSV (SEQ ID NO: 98941), X1X2X3RGDYTSM (SEQ ID NO: 98942), X 1 X 2 X 3 RGDRGVV (SEQ ID NO: 98928), X 1 X 2 X 3 RGDRSVV (SEQ Atty Docket No.: 38053-57988/WO (112WO) ID NO: 98931), X1X2X3RGDYSSV (SEQ ID NO: 98937), or X1X2X3RGDHGVL (SEQ ID NO: 98938).
  • Embodiment 87 Embodiment 87.
  • Embodiment 88 The modified AAV capsid protein of embodiment 87, wherein X1X2X3 is selected from the group consisting of EFK, AAY, DQK, QVY, DKL, DNV, ENF, EWK, QNV, and TFM.
  • Embodiment 89 The modified AAV capsid protein of any one of embodiments 1-55 wherein the targeting peptide has an amino acid sequence selected from: RGDRSX9I, RGDRGX 9 I, RGDRSX 9 V, or RGDRGX 9 V.
  • Embodiment 90 Embodiment 90.
  • the modified AAV capsid protein of embodiment 89 wherein the targeting peptide has an amino acid sequence selected from X1X2X3RGDRGQI (SEQ ID NO: 98927), X 1 X 2 X 3 RGDRSVV (SEQ ID NO: 98931) or X 1 X 2 X 3 RGDRGVV (SEQ ID NO: 98928).
  • Embodiment 91 The modified AAV capsid protein of any one of embodiments 89-90, wherein X1 is D or E, X2 is K, E, D, A, S, F or N, and X3 is Y, V, or F.
  • Embodiment 92 Embodiment 92.
  • X 1 X 2 X 3 is selected from the group consisting of: DII, DWM, EEI, DML, DWI, SLE, EIN, NHE, DFI, EEL, TEQ, TDA, EDT, NEV, TDW, QFE, EDY, DTT, EPL, SEN, SEQ, TAE, EVN, ELN, DVQ, ETI, EVI, ESV, ETW, SEW, DNW, EVF, EAW, EPF, EIY, EIF, EPY, DVI, DMM, DQI, DHL, DTL, DVL, NDL, DLL, DMQ, NEF, DFL, DIM, TEW, DYI, SDY, DYY, DHF, DKE, DTW, DTI, ELY, TEY, TEI, DAI, DQY, DMY, EWG, DMV, DMI, E
  • Embodiment 93 The modified AAV capsid protein of any one of embodiments 89-92, wherein X1X2X3 is selected from the group consisting of: DAV, DKW, EAY, AEY, DFV, DKF, DKI, DKL, DNV, DNY, DSL, DSV, EFI, SEF, SEY, SLY, ADF, ADY, ALY, AVF, DAF, DAL, DAM, DAT, DHV, DIV, DKA, DKM, DKT, DKV, DKY, DMI, DNF, DNI, DQT, DSI, DVY, DYN, DYV, EAT, EAW, EFV, EGL, EIY, EMF, EMY, ENF, EPF, EPY, EQY, ESY, ETF, EWI, EWT, EYI, EYV, NEM, QDF, QDY, QEY, QLY, QND, QVF, Q
  • Embodiment 94 The modified AAV capsid protein of any one of embodiments 1-55, wherein X 7 is selected from Y and H; X 8 is selected from T, G and S; X 9 is selected from S and V; X10 is selected from V, L and M.
  • Embodiment 95 The modified AAV capsid protein of any one of embodiments 1-55, wherein X 7 is selected from Y and H; X 8 is selected from T, G and S; X 9 is selected from S and V; X10 is selected from V, L and M.
  • the modified AAV capsid protein of embodiment 94 wherein the targeting peptide has an amino acid sequence selected from: X 1 X 2 X 3 RGDHGVL (SEQ ID NO: 98938), X1X2X3RGDYSSV (SEQ ID NO: 98937), X1X2X3RGDYTSM (SEQ ID NO: 98942) or X1X2X3RGDYTSV (SEQ ID NO: 98941).
  • Embodiment 96 The modified AAV capsid protein of embodiment 94 or 95, wherein X 1 is S, A or T, X2 is N, A or Y, and X3 is I, V, M. Q, T, Y or K.
  • Embodiment 97 Embodiment 97.
  • X 1 X 2 X 3 is selected from the group consisting of: APW, TEL, TDA, QPY, SPN, EHY, DWK, DLK, DFK, DVK, NSI, DIR, SPF, SEL, DRT, DRF, ADL, TDL, SDL, DNY, DKI, NDV, DKM, DNH, DNF, DSS, EST, EWT, DKN, DKS, SEH, ESQ, ESL, QND, EAH, Atty Docket No.: 38053-57988/WO (112WO) AIF, AVF, QVF, TMY, ALY, NNG, NIF, NTF, NFF, AWF, NPY, SWF, AII, AYF, AQW, NFY, AGP, QQF, TKE, TNG, NSF, NAW, QAG, ERG, NKD
  • Embodiment 98 The modified AAV capsid protein of any one of embodiments 94-97, wherein X1X2X3 is selected from ANY, SNI, AAI, AAM, ANT, AST, AYQ, EHK, ENK, ENR, SFQ, SSI, TAY, TDK, TNT, AAF, AAL, AAY, ADK, AFA, ANF, ANI, ANQ, ANS, AQM, ARE, ASV, AYH, AYT, EMK, EWK, NNM, QAF, QAI, QAM, QAT, QAY, QFT, Atty Docket No.: 38053-57988/WO (112WO) QGM, QHL, QNF, QNQ, QNS, QNT, QNV, QNY, SAH, SAI, SAL, SFT, SFV, SHI, SHV, SMM, SNF, SNM, SNN, SNQ, SNV, SNY, SQI, S
  • Embodiment 99 The modified AAV capsid protein of any one of embodiments 1-55, wherein X 7 is selected from F and Y; X 8 is selected from N and Q; X 9 is selected from N and S; X 10 is selected from T and L.
  • Embodiment 100 The modified AAV capsid protein of any one of embodiments 1-55, wherein X 7 is selected from F and Y; X 8 is selected from N and Q; X 9 is selected from N and S; X 10 is selected from T and L.
  • Embodiment 101 The modified AAV capsid protein of embodiment 99 or 100, wherein X1 is S, A or E, X 2 is N, Y or S, X 3 is I, Q, R, V, T, M or K.
  • X1X2X3 is selected from the group consisting of: DMK, ATD, EEK, QMD, EFS, ERD, DDR, TDM, SAE, EHS, ENH, SWE, SNE, NNG, QAG, ERG, QSG, QNG, ASG, QFG, AMF, ELR, NFM, NNS, NNI, SDR, EQR, EHR, EWR, EQK, ESR, EKQ, EYR, ENR, EMK, EYK, EHK, EWK, QNI, TNI, TYI, SNY, DRQ, AWI, QWI, DFR, EKG, QYG, QWQ, EKN, EKF, EKT, AFH, ENK, NYS, DKH, AAG, QMF, QFH, QKD, ARE, AHQ, ADK, ADR, AHN, QNS,
  • Embodiment 103 The modified AAV capsid protein of any one of embodiments 99-102, wherein X1X2X3 is selected from the group consisting of: ADR, ASI, EFK, EHK, EWK, SYQ, AAF, AAT, AAY, AFI, AFQ, AGI, AGT, AHI, ANH, ANM, ANN, AQI, ASA, ASH, AST, ASV, AWT, AYQ, AYT, DAR, DMK, DQR, DVR, EAR, EFR, EMK, EMR, EQK, ERA, ERS, ESR, NDA, NDR, NMI, NMV, NNM, NNN, NYL, NYM, NYN, NYQ, NYV, QAM, QFQ, QFV, QGV, QNH, QNI, QNM, QQV, QSF, QSY, SAI, SAM, SAS, SDR, SFQ, SGH, SGM, S
  • Embodiment 104 The modified AAV capsid protein of any one of embodiments 1-55, wherein RGDRX8X9X10 has an amino acid sequence selected from: RGDRGVX10 (SEQ ID NO: 157058), RGDRGSX10 (SEQ ID NO: 157059), RGDRGNX10 (SEQ ID NO: 157060), RGDRGGX 10 (SEQ ID NO: 157061), RGDRGQX 10 (SEQ ID NO: 157062), RGDRGX 9 V (SEQ ID NO: 157063), RGDRGX 9 I (SEQ ID NO: 157064), RGDRGX 9 S(SEQ ID NO: 157065), RGDRGX9L(SEQ ID NO: 157066), RGDRGX9Q (SEQ ID NO: 157067), RGDHX 8 X 9 L (SEQ ID NO: 157068), RGDRX 8 X 9 I (SEQ ID NO: 157069), RGDRX 8 X 9 V (SEQ ID NO
  • Embodiment 105 The modified AAV capsid protein of any one of embodiments 1-104, wherein the modified sequence does not comprise an amino acid sequence selected from: RGDRMVF (SEQ ID NO: 157080), RGDRTVI (SEQ ID NO: 157081), SRGDRPM (SEQ ID NO: 157082), ISLRGDR (SEQ ID NO: 157083), and RGDLLLS (SEQ ID NO: 1).
  • RGDRMVF SEQ ID NO: 157080
  • RGDRTVI SEQ ID NO: 157081
  • SRGDRPM SEQ ID NO: 157082
  • ISLRGDR SEQ ID NO: 157083
  • RGDLLLS SEQ ID NO: 1
  • Embodiment 107 The modified AAV capsid protein of any one of embodiments 1-106, wherein the targeting peptide is positioned between 565 and 595 within VR VIII of the modified AAV capsid protein. Atty Docket No.: 38053-57988/WO (112WO) Embodiment 108.
  • Embodiment 109 The modified AAV capsid protein of any one of embodiments 1-107, wherein: the reference AAV capsid protein is a capsid protein of AAV1 or a modification thereof and the targeting peptide is between D590 and P591 or between S588 and T589 of the reference AAV capsid protein; the reference AAV capsid protein is a capsid protein of AAV2 or a modification thereof and the targeting peptide is between R588 and Q589 or between N587 and R588 of the reference AAV capsid protein; the reference AAV capsid protein is a capsid protein of AAV3 or a modification thereof and the targeting peptide is between S586 and S587 or between N588 and T589 of the reference AAV capsid protein; the reference AAV capsid protein is a capsid protein of AAV4 or a modification thereof and the targeting peptide is between S584 and N585 or between S586 and N587 of the reference AAV caps
  • Embodiment 110 The modified AAV capsid protein of any one of embodiments 1-109, wherein (i) P1 is independently selected from an asparagine (N), a serine (S), or a threonine (T); (ii) P 2 is independently selected from a serine (S) or a glycine (G); (iii) P3 is independently selected from a threonine (T), a glutamine (Q), an alanine (A), or glutamate (E); (iv) P 4 is independently selected from a serine (S), a threonine (T), or an alanine (A); Atty Docket No.: 38053-57988/WO (112WO) (v) P5 is independently selected from a glycine (G) or an alanine (A); (vi) P6 is independently selected from a glycine (G) or an alanine (A); (vii) P 7 is independently selected from an alanine (
  • Embodiment 111 The modified AAV capsid protein of embodiment 110, wherein P 5 , P 6 or both P5 and P6 are not an alanine (A).
  • Embodiment 112. The modified AAV capsid protein of embodiment 110, wherein the peptide segment does not comprise an alanine (A) at P 6 and a threonine (T) at P 8 .
  • P 1 is independently selected from an asparagine (N) or a serine (S);
  • P 2 is independently selected from a serine (S) or a glycine (G);
  • P3 is independently selected from a threonine (T) or a glutamine (Q);
  • P 4 is independently selected from a serine (S), a threonine (T), or an alanine (A);
  • P7 is independently selected from an alanine (A) or a serine (S);
  • P 8 is independently selected from a serine (S) or a threonine (T);
  • P 12 is independently selected from a histidine (H), a threonine (T), or an alanine (A).
  • Embodiment 115 The modified AAV capsid protein of any embodiment one of embodiments 1-113, wherein the peptide segment has a sequence of P1P2TP4GGP7P8NDNP12 (SEQ ID NO: 44922), wherein P 1 , P 2 , P 4 , P 7 , P 8 , and P 12 are independently selected from any amino acid residue.
  • Embodiment 116 The modified AAV capsid protein of any embodiment one of embodiments 1-113, wherein the peptide segment has a sequence of P1P2TP4GGP7P8NDNP12 (SEQ ID NO: 44922), wherein P 1 , P 2 , P 4 , P 7 , P 8 , and P 12 are independently selected from any amino acid residue.
  • Embodiment 120 is independently selected from any amino acid residue.
  • Embodiment 121. The modified AAV capsid of protein of embodiment 120, wherein P 7 is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T).
  • Embodiment 123 Embodiment 123.
  • Embodiment 125 Embodiment 125.
  • Embodiment 126 The modified AAV capsid protein of any one of embodiments 1-113, wherein the peptide segment is SGQTGGP 7 P 8 NDNH (SEQ ID NO: 44929), wherein P 7 and P8 are independently selected from any amino acid residue.
  • the modified AAV capsid protein of embodiment 126 or 127 wherein the peptide segment comprises: (i) SGQTGGASNDNH (SEQ ID NO: 46505), (ii) SGQTGGATNDNH (SEQ ID NO: 46508), (iii) SGQTGGSSNDNH (SEQ ID NO: 46511), or Atty Docket No.: 38053-57988/WO (112WO) (iv) SGQTGGSTNDNH (SEQ ID NO: 46514).
  • Embodiment 129 Embodiment 129.
  • Embodiment 130. The modified AAV capsid protein of embodiment 129, wherein P7 is independently selected from an alanine (A) or a serine (S) and P 8 is independently selected from a serine (S) or a threonine (T).
  • the modified AAV capsid protein of embodiment 129 or 130 wherein the peptide segment comprises: (i) SGTAGGASNDNT (SEQ ID NO: 46554), or (ii) SGTAGGSSNDNT (SEQ ID NO: 46560).
  • Embodiment 132 The modified AAV capsid protein of embodiment 129 or 130, wherein the peptide segment does not comprise SGTAGGATNDNT (SEQ ID NO: 46557) or SGTAGGSTNDNT (SEQ ID NO: 46563).
  • Embodiment 133 Embodiment 133.
  • the modified AAV capsid protein of embodiment 133 or 134 wherein the peptide segment comprises: (i) SGTSGGASNDNA (SEQ ID NO: 46600), (ii) SGTSGGATNDNA (SEQ ID NO: 46603), (iii) SGTSGGSSNDNA (SEQ ID NO: 46606), or (iv) SGTSGGSTNDNA (SEQ ID NO: 46609).
  • Atty Docket No.: 38053-57988/WO (112WO) Embodiment 136 The modified AAV capsid protein of any one of embodiments 1-113, wherein the peptide segment is SGTTGGP7P8NDNT (SEQ ID NO: 44932), wherein P7 and P 8 are independently selected from any amino acid residue.
  • Embodiment 137 The modified AAV capsid protein of embodiment 136, wherein P7 is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T).
  • Embodiment 138 The modified AAV capsid protein of embodiment 136 or 137, wherein the peptide segment comprises: (i) SGTTGGASNDNT (SEQ ID NO: 46650), (ii) SGTTGGATNDNT (SEQ ID NO: 46653), (iii) SGTTGGSSNDNT (SEQ ID NO: 46656), or (iv) SGTTGGSTNDNT (SEQ ID NO: 46659).
  • Embodiment 139 Embodiment 139.
  • Embodiment 140. The modified AAV capsid protein of any one of embodiments 1-113, wherein the peptide segment is SSTAGGP 7 P 8 NDNA (SEQ ID NO: 44933), wherein P 7 and P 8 are independently selected from any amino acid residue.
  • Embodiment 141. The modified AAV capsid protein of embodiment 140, wherein P7 is independently selected from an alanine (A) or a serine (S) and P 8 is independently selected from a serine (S) or a threonine (T).
  • the modified AAV capsid protein of embodiment 140 or 141 wherein the peptide segment comprises: (i) SSTAGGASNDNA (SEQ ID NO: 47128), (ii) SSTAGGATNDNA (SEQ ID NO: 47131), (iii) SSTAGGSSNDNA (SEQ ID NO: 47134), or Atty Docket No.: 38053-57988/WO (112WO) (iv) SSTAGGSTNDNA (SEQ ID NO: 47137).
  • Embodiment 143 The modified AAV capsid protein of embodiment 142, wherein the peptide segment is SSTAGGASNDNA (SEQ ID NO: 47128).
  • Embodiment 144 is SSTAGGASNDNA (SEQ ID NO: 47128).
  • the modified AAV capsid protein of embodiment 142 wherein the peptide segment is SSTAGGATNDNA (SEQ ID NO: 47131).
  • Embodiment 145. The modified AAV capsid protein of any one of embodiments 1-113, wherein the peptide segment is NSTSGASTNDNA (SEQ ID NO: 48390).
  • Embodiment 146. The modified AAV capsid protein of any one of embodiments 1-113, wherein the peptide segment is selected from a peptide segment as shown in Tables 20 or 28.
  • Embodiment 147. The modified AAV capsid protein of any one of embodiments 1-146, wherein variable region I (VR I) corresponds to amino acid residues between about position 259 to about position 275 of the modified capsid protein.
  • VR I variable region I
  • Embodiment 148 The modified AAV capsid protein of any one of embodiments 1-146, wherein the peptide segment is at a position between S261 and Y274 of an AAV9 capsid protein (SEQ ID NO: 61).
  • Embodiment 149 The modified AAV capsid protein of any one of embodiments 1-146, wherein the peptide segment is at a position between S260 and Y273 of an Anc80 capsid protein (SEQ ID NO: 132).
  • Embodiment 150 The modified AAV capsid protein of any one of embodiments 1-146, wherein the peptide segment is at a position between S260 and Y273 of an Anc80L65 capsid protein (SEQ ID NO: 142).
  • Embodiment 151 The modified AAV capsid protein of any one of embodiments 1-146, wherein the peptide segment is at a position between S260 and Y273 of an AAV2 capsid protein (SEQ ID NO: 55).
  • Embodiment 152 The modified AAV capsid protein of any one of embodiments 1-146, wherein the peptide segment is at a position between S260 and Y273 of an AAV2 capsid protein (SEQ ID NO: 55).
  • the modified AAV capsid protein of any one of embodiments 1-151, wherein Atty Docket No.: 38053-57988/WO (112WO) (i) the targeting peptide has an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) the peptide segment has an amino acid sequence selected from SEQ ID NOs: 46026, 46029, 46031, 46035, 46073, 46076, 46079, 46082, 46505, 46508, 46511, 46514, 46554, 46560, 46609, 46600, 46603, 46606, 46650, 46653, 46656, 46659, 47128, 47131, 47134, 47137, and 48390.
  • Embodiment 153 The modified AAV capsid protein of any one of embodiments 1-152, wherein (i) the targeting peptide has an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) the peptide segment has the amino acid sequence SGTAGGASNDNT (SEQ ID NO: 46554).
  • Embodiment 154 Embodiment 154.
  • the targeting peptide has an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) the peptide segment has the amino acid sequence SGTSGGSTNDNA (SEQ ID NO: 46609).
  • Embodiment 155 Embodiment 155.
  • the targeting peptide has an amino acid sequence selected from SEQ ID NOs:44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391-157057; and (ii) the peptide segment has the amino acid sequence SGTTGGSTNDNT (SEQ ID NO: 46659).
  • Embodiment 156 Embodiment 156.
  • the modified AAV capsid protein of any one of embodiments 1-152, wherein Atty Docket No.: 38053-57988/WO (112WO) (i) the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391- 157057; and (ii) the peptide segment has the amino acid sequence SGTTGGSTNDNT (SEQ ID NO: 47128).
  • Embodiment 157 Embodiment 157.
  • the modified AAV capsid protein of any one of embodiments 1-152 wherein (i) the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391- 157057; and (ii) the peptide segment has the amino acid sequence NSTSGGSSNDNA (SEQ ID NO: 48388).
  • the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391- 157057; and (ii) the peptide segment has the amino acid sequence NSTSGGSSNDNA (SEQ ID NO: 48388).
  • Embodiment 158 Embodiment 158.
  • the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, 44918-44919, and 48391- 157057; and (ii) the peptide segment has the amino acid sequence NSTSGASTNDNA (SEQ ID NO: 48390).
  • Embodiment 160. The modified AAV capsid protein of any one of embodiments 1-152, wherein (i) the targeting peptide has the amino acid sequence ENRRGDFNNL (SEQ ID NOs: 44864); and Atty Docket No.: 38053-57988/WO (112WO) (ii) the peptide segment has the amino acid sequence SGTTGGSSNDNT (SEQ ID NO: 46656).
  • the modified AAV capsid protein of any one of embodiments 1-152 wherein (i) the targeting peptide has the amino acid sequence ENRRGDFNNL (SEQ ID NOs: 44864); and (ii) the peptide segment has the amino acid sequence SSTAGGASNDNA (SEQ ID NO: 47128).
  • Embodiment 162. The modified AAV capsid protein of any one of embodiments 1-152, wherein (i) the targeting peptide has the amino acid sequence ENRRGDFNNL (SEQ ID NOs: 44864); and (ii) the peptide segment has the amino acid sequence NSTSGASTNDNA (SEQ ID NO: 48390).
  • Embodiment 165 Embodiment 165.
  • Embodiment 167 The modified AAV capsid protein of any one of embodiments 1-152, wherein (i) the targeting peptide has the amino acid sequence ENRRGDFNNL (SEQ ID NOs: 44864); and (ii) the peptide segment has the amino acid sequence NSTSGASTNDNA (SEQ ID NO: 48390).
  • Embodiment 168 The modified AAV capsid protein of any one of embodiments 1-152, wherein (i) the targeting peptide has the amino acid sequence ENRRGDFQNT (SEQ ID NO: 44866); and (ii) the peptide segment has the amino acid sequence SSTAGGATNDNA (SEQ ID NO: 47131).
  • Embodiment 169 Embodiment 169.
  • Embodiment 172. The modified AAV capsid protein of any one of embodiments 1-152, wherein (i) the targeting peptide has the amino acid sequence SNRRGDFNNT (SEQ ID NO: 44883); and (ii) the peptide segment has the amino acid sequence SSTAGGASNDNA (SEQ ID NO: 47128).
  • a modified adeno-associated virus (AAV) capsid protein comprising: (i) a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919; and (ii) a peptide segment within VR I, wherein the peptide segment has a sequence selected from SEQ ID NOs: 46026, 46029, 46031, 46035, 46073, 46076, 46079, 46082, 46505, 46508, 46511, 46514, 46554, 46560, 46609, 46600, 46603, 46606, 46650, 46653, 46656, 46659, 47128, 47131, 47134, and 47137.
  • AAV adeno-associated virus
  • Embodiment 174 A polynucleotide encoding the modified AAV capsid protein of any one of embodiments 1-173.
  • Embodiment 175. A vector comprising the polynucleotide of embodiment 174. Atty Docket No.: 38053-57988/WO (112WO) Embodiment 176. The vector of embodiment 175, further comprising a promoter operably linked to the polynucleotide.
  • Embodiment 177 A host cell comprising the modified AAV capsid protein of any one of embodiments 1 to 172, the polynucleotide specified in embodiment 174, or the vector of embodiment 175 or 176.
  • a recombinant AAV virion comprising the modified AAV capsid protein of any one of embodiments 1-173.
  • the rAAV virion of embodiment 179, wherein the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA.
  • Embodiment 186 The rAAV virion of any one of embodiments 179-185, wherein the exogenous polynucleotide further comprises a regulatory sequence.
  • the rAAV virion of embodiment 187, wherein the EREs comprise a CAG promoter.
  • Embodiment 190 The rAAV virion of embodiment 187, wherein the EREs comprise a sequence having at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ IDs NO:171-173.
  • Embodiment 190. A pharmaceutical composition comprising the modified AAV capsid protein of any one of embodiments 1-173 or the AAV virion of any one of embodiments 178- 189.
  • Embodiment 191. A method for treating or ameliorating or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of the rAAV virion of any one of embodiments 178-189 or the pharmaceutical composition of embodiment 190.
  • said muscular disease is a muscular dystrophy, a cardiomyopathy, a myotonia, a muscular atrophy, a myoclonus dystonia, a mitochondrial myopathy, a rhabdomyolysis, a fibromyalgia, and/or a myofascial pain syndrome.
  • AAV modified adeno-associated virus
  • An AAV virion comprising the modified AAV capsid protein of any one of embodiments1-173 or the AAV virion of any one of embodiments 178-189 for use in treating and/or preventing a muscular disease and/or in muscle regeneration.
  • Embodiment 197 A pharmaceutical composition comprising the modified AAV capsid protein of any one of embodiments 1-159, and/or the AAV virion specified in any one of embodiments 178-189 for use in treating and/or preventing a muscular disease and/or in muscle regeneration.
  • a method of transferring an exogenous polynucleotide into a muscle cell comprising the step of administering the AAV virion specified in any one of embodiments 178-189 to a subject.
  • Embodiment 199. The method of embodiment 198, wherein the administration results in transfer of the exogenous polynucleotide in the muscle cell, at a muscle:liver infection ratio of greater than 1 when measured by genome copies of the AAV virion.
  • Embodiment 200 The method of embodiment 198, wherein the muscle:liver infection ratio ranges from 1 to 100.
  • Embodiment 201 wherein the muscle:liver infection ratio ranges from 2 to 8.
  • Embodiment 203 The method of any one of embodiments 198-202, wherein the administration results in expression of the exogenous polynucleotide in the muscle cell, at a muscle:liver expression ratio of greater than 10.
  • Embodiment 204 The method of embodiment 203, wherein the muscle:liver expression ratio ranges from 10 to 100.
  • the method of embodiment 204, wherein the muscle:liver expression ratio ranges from 20 to 80.
  • Embodiment 206 The method of any one of embodiments 198-205, wherein the muscle:liver expression ratio ranges from 50 to 80 when measured by mRNA transcript expression.
  • Embodiment 207 The method of any one of embodiments 198-205, wherein the muscle:liver expression ratio ranges from 50 to 80 when measured by mRNA transcript expression.
  • Embodiment 208 The method of any one of embodiments 198-206, wherein the muscle:liver expression ratio ranges from 10 to 50 when measured by protein expression. Atty Docket No.: 38053-57988/WO (112WO) Embodiment 208.
  • Embodiment 209 Use of the AAV capsid polypeptide of any one of embodiments 1-173, and/or the AAV virion specified in any one of embodiments 178-189 for transferring an exogenous polynucleotide into a muscle cell.
  • Embodiment 210 Use of the AAV capsid polypeptide of any one of embodiments 1-173, and/or the AAV virion specified in any one of embodiments 178-189 for transferring an exogenous polynucleotide into a muscle cell.
  • the experiment was designed to test the hypothesis that the liver toggle mutation provides a structure that can determine efficiency of liver gene delivery and that the peptide insertion into VR VIII can act independently and/or synergistically.
  • Two doses were also used, a low dose of 1x10 13 gc/kg and a high dose of 5x10 13 gc/kg, seeking to demonstrate possible equivalence of eGFP expression afforded by the modified AAV vector at a low dose with the eGFP expression observed with the unmodified vector at high dose.
  • Example 2 AAV variants
  • the polynucleotide encoding the wild-type AAV9 VP1 capsid protein (SEQ ID NO: 61) or AAV mut1 capsid protein (SEQ ID NO: 163) was modified by by inserting a Atty Docket No.: 38053-57988/WO (112WO) coding sequence for the 7-mer peptide RGDLLLS (SEQ ID NO: 1) at a position that effected insertion of the peptide between amino acid residues 588 and 589 of the encoded polypeptide.
  • modified polynucleotides encode modified VP1 proteins referred to as: AAV deco1 capsid protein (SEQ ID NO: 158) or AAV mut1_deco1 capsid protein (SEQ ID NO: 159).
  • AAV deco1 capsid protein SEQ ID NO: 158
  • AAV mut1_deco1 capsid protein SEQ ID NO: 159.
  • Corresponding AAV vectors were manufactured with the modified capsid proteins in the Affinia Therapeutics Vector Core facility via standard triple transfection into HEK293 cells.
  • the AAV vectors produced by the method include AAV9 CAG.GFP (CAG.GFP construct encapsulated in AAV9 capsid), AAV mut1 CAG.GFP (CAG.GFP construct encapsulated in a capsid comprising the AAV mut1 capsid protein), AAV9 deco1 CAG.GFP (CAG.GFP construct encapsulated in a capsid comprising the AAV deco1 capsid protein), and AAV mut1_deco1 CAG.GFP (CAG.GFP construct encapsulated in a capsid comprising the AAV mut1_deco1 capsid protein).
  • Successful gene transfer by these vectors was detected by GFP expression in target cells.
  • Example 3 Confirmed enhanced muscle tropism with limited liver tropism for AAV -mut1-deco1 in C57BL/6 mice at both low and high dosage regimes [00511] Gene transfer efficacy of each AAV vector prepared according to Example 1 was tested with three C57BL/6 mice, injected with one of the AAVs at one of the two different doses by intravenous tail vein injection. Total twenty-four mice were injected in total as summarized in the below table.
  • mice comprised the study.
  • Table 2 The low dose was 1x10 13 gc/kg (total 2x10 11 gc), and the high dose was 5 x 10 13 gc/kg (total 1x10 12 gc).
  • a control mouse was injected with vehicle (1X PBS, 35mM NaCl, 0.001% Pluronic) alone.
  • vehicle (1X PBS, 35mM NaCl, 0.001% Pluronic
  • AAV Vector (AAV) low dose (1x10 13 gc/kg) high dose (5 x 10 13 gc/kg)
  • the mice were sacrificed 28 days after the injection. Individual tissues, notably the liver, major skeletal muscles of the hind limb, heart, and diaphragm, were collected at the time of necropsy.
  • RNAlater Tissue were immediately placed into the preservative RNAlater, after which the RNAlater was removed and the tissue flash frozen. The same tissues were fixed and embedded for sectioning and anti-GFP staining by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • ddRT-PCR for the eGFP vector genome copies per DPG (DNA) and transcript (mRNA). The eGFR transcript level was compared against the transcript of a housekeeping standard RPP30. IHC was performed at Histoserv Inc. (Germantown, MD). ddRT-PCR was performed at Affinia Therapeutics (Waltham, MA).
  • FIGs.4A-4J Images of exemplary liver and skeletal muscle tissue cross-sections obtained from the anti-GFP IHC are provided in FIGs.4A-4J.
  • the tissue cross-sections were stained with an anti-GFP primary antibody followed by an HRP-linked secondary staining and substrate addition. Brown staining of cells above the counterstain for intact cells and nuclei indicates eGFP expression.
  • the vehicle control tissues from liver or skeletal muscle show the structure and organization expected from healthy tissues.
  • AAV9 at 5 x 10 13 gc/kg robustly transduces the liver and muscle cells (brown individual cells). GFP expression within the liver is reduced in mice injected with AAV-mut1-deco1, such that isolated individual cells are stained.
  • GFP expression within muscle tissue was significantly increased in the mice injected with AAV-mut1-deco1.
  • Transgene transfer and expression capabilities of administered AAVs were also evaluated with ddPCR, by measuring amounts of DNA and mRNA of the transgene (eGFP) in the various tissue samples 28 days after injection. DNA genome copies and mRNA transcript copies of the transgene (eGFP) were quantified in comparison to the amounts of DNA genome copies or mRNA transcript copies of a house keeping gene (RPP30), respectively. Specifically, DNA genome copies are reported as vector genomes copies per diploid genome (VGC/DG).
  • VGC/DG (eGFP cp/ ⁇ L ⁇ RPP30 cp/ ⁇ L) ⁇ 2.
  • % eGFP expression (eGFP cp/ ⁇ L ⁇ RPP30 cp/ ⁇ L) ⁇ 100.
  • Atty Docket No.: 38053-57988/WO (112WO) [00516] Tissues were homogenized in a Qiagen Tissuelyser II (20rps for 2 min) in lysis buffer from the Qiagen Dneasy Blood and Tissue Kit or the Qiagen RNeasy Lipid Tissue Mini Kit following the standard Qiagen protocol.
  • RNA samples were eluted in 50uL of buffer. Prior to analysis, DNA and RNA concentration and quality were determined using a NanoDrop One, using the nucleic acid (DNA or RNA) program. DNA samples were analyzed for biodistribution of vector genomes using a duplexed ddPCR method targeting the transgene (eGFP) and a reference gene (RPP30). RNA samples were analyzed for expression of the eGFP transgene using a duplexed, one-step RT-ddPCR method and a reference gene (RPP30). [00517] mRNA was extracted from 30 mg sections of liver, and quadriceps.
  • FIG.5A and FIG.5B show that AAV Mut1 reduces liver tropism but does not enhance muscle tropism, AAV Deco1 has high liver tropism and comparatively high muscle tropism, and that AAV Mut1_deco1 has decreased liver tropism and increased muscle tropism compared to AAV9 (WT).
  • eGFP mRNA expression in various tissues was measured by RT-ddPCR and presented as the ratio of eGFP transcripts over RPP30 transcripts, a rough indicator of eGFP mRNA copies per cell.
  • FIG.6A liver
  • FIG.6B heart
  • FIG.6C tricep surae
  • FIG.6D quadricep
  • FIG.6E diaphragm
  • results from three biological replicates are provided for each AAV variant at each dose (high or low dose).
  • FIG.6A provides the ratio of eGFP to RPP30 transcripts in the liver.
  • FIG.6B shows the ratio of eGFP to RPP30 transcripts in the heart. Both AAV deco1 and AAV mut1_deco1 had higher expression in the heart, although the difference and significance are reduced by a single outlier within the AAV9 at 5 x 10 13 gc/kg dose group, and possible signal saturation within the AAV deco and AAV mut1_deco1 high dose group.
  • FIG.6C shows the ratio of eGFP to RPP30 transcripts in the triceps surae. Within the 5 x 10 13 gc/kg groups, there was more than 1-log increase in the eGFP per RPP30 mRNA ratio in AAV9 deco1 and AAV mut1_deco1 compared to AAV9 in the calf muscle tissue of the study subjects.
  • FIG.6D shows the ratio of eGFP to RPP30 transcripts in the quadricep. Results were similar to the triceps surae, the other skeletal muscle tested in this study. Within the 5 x 10 13 gc/kg groups, there is more than 1 log increase in eGFP per RPP30 mRNA ratio in AAV deco1 and AAV mut1_deco1 compared to AAV9 in quadricep tissue of the study subjects.
  • FIG.6E shows the ratio of eGFP to RPP30 transcripts in the diaphragm. Increase of gene delivery efficacy in deco-containing vectors was also observed in the diaphragm, but in this study all but one comparison exceeded the threshold of significance: high dose AAV9 versus high dose AAV deco . 8.4.
  • Example 4 Enhanced muscle tropism with limited liver tropism for AAV- mut1-deco1 confirmed at the earlier d14 time point in C57BL/6 mice [00524] Gene transfer efficacy of AAV9 vector and AAV9 mut1_deco1 vector was tested with groups of three C57BL/6 mice, injected with one of the AAVs by intravenous tail vein injection. Total thirteen mice were injected in total as summarized in the below table. The dose was 1x10 13 gc/kg (total 2x10 11 gc). Additionally, a control mouse was injected with vehicle (1X PBS, 35mM NaCl, 0.001% pluronic) alone.
  • mice were sacrificed 14 or 28 days after the injection. Individual tissues, notably the liver and major skeletal muscles of the hind limb (quad), were collected at the time of necropsy. Tissues were immediately placed into the preservative RNAlater, after which the RNAlater was removed and the tissue flash frozen.
  • FIGs.7A-7B show eGFP vector genome (DNA) in liver and quad tissues of C57BL/6 mice 14 days (FIG.7A) or 28 days (FIG.7B) after treatment with vehicle, AAV Mut1 and AAV Mut1-deco1 AAV vectors.
  • FIGs.7C-7D show eGFP mRNA expression in liver and quad tissues of C57BL/6 mice 14 days (FIG.7C) or 28 days (FIG.7D) after treatment with vehicle, AAV Mut1 and AAV Mut1-deco1 AAV vectors.
  • AAV Mut1_Deco1 enhancement of muscle tropism is observable at d14; AAV Mut1 and AAV Mut1_Deco1 vector genome copies (VGs) are stable from d14 to d28; AAV Mut1_Deco1 enhancement leads to greater accumulation of eGFP signal; and liver tropism is consistently low through all samples.
  • Example 5 Confirmed enhanced muscle tropism with limited tropism to liver and other organs for AAV -mut1-deco1 in BALB/c mice
  • Gene transfer efficacy of AAV mut1 and AAV mut1_deco1 were tested with three or six BALB/c mice, injected with one of the vectors at 5 x 10 13 gc/kg (total 1x10 12 gc) by intravenous tail vein injection. Additionally, control mice were injected with vehicle (1X PBS, 35mM NaCl, 0.001% pluronic) alone. Total twelve mice were injected in total as summarized in the below table. Table 4.
  • AAV Vector
  • AAV Vector
  • AAV 5 x 10 13 gc/kg (total 1x10 12 gc)
  • Control vehicle
  • the mice were sacrificed 28 days after the injection. Individual tissues, notably the liver, major skeletal muscles of the hind limb, heart, diaphragm, brain, spinal cord, and spleen were collected at the time of necropsy.
  • DNA and RNA were extracted from 30 mg sections of liver and quadricep.
  • Example 6 Summary of Mouse data [00534] The below Table 5 exemplifies the Muscle:Liver infection ratios calculated for the DNA biodistribution data, the RNA expression data and the IHC expression data obtained for administration of AAV mut1_deco1 vector compared to AAV9 vector in Mice. Atty Docket No.: 38053-57988/WO (112WO) Table 5.
  • Example 7 Confirmed enhanced AAV -mut1-deco1 muscle tropism with limited liver tropism confirmed in NHP [00535]
  • the objective of this study is to confirm liver retargeting and muscle transduction superiority of AAV mut1_deco1 vector compared to AAV9 vector in non-human primates (NHP) as was observed in mice.
  • the results confirm enhanced muscle transduction superiority and liver de-targeting of AAV Mut1_Deco1 vector compared to AAV9.
  • AAV constructs were used in the experiment: (i) AAV -mut1.deco1 -CAG- GFP, and (ii) AAV9-CAG-GFP, each including an AAV genome construct containing a coding sequence of GFP. GFP was used to detect distribution of AAVs and expression of the transgene. Marmoset monkeys were used as the subject animals. [00537] Total of 7 animals were divided into 3 groups as summarized in the below TABLE #. Immunosuppression of the animals began 7 days prior to AAV administration. Group 1 is a control animal administered with vehicle.
  • IHC for GFP expression were scored (blinded) by a pathologist. A second pathologist peer reviewed the data.
  • Initial assessment for GFP expression by IHC was conducted on one section per tissue-referred to as Run 1 tissues and included liver, heart and skeletal muscle (right and left sides-tibialis, biceps, quadriceps, gastrocnemius. Two additional sections per muscle group were run to assess consistency of expression within each muscle group-referred to as Run 2.
  • FIGs.9A and 9B Analysis of Run 1 tissue samples is shown in FIGs.9A and 9B.
  • Exemplary IHC liver tissue is shown in FIG.9A, obtained from AAV9 treated animal on the left , and AAV mut1_deco1 treated animal illustrated on the right side of the chart.
  • Exemplary IHC quadriceps tissue is shown in FIG.9B, obtained from AAV9 treated animal on the left, and AAV mut1_deco1 treated animal on the right.
  • FIG.10 shows the % GFP positive cells in the liver tissue (right and left side of the organ) and quadriceps tissue (right and left leg) in slides obtained from Run 1 for each animal administered vehicle or AAV (AAV9 or AAV mut1deco1 ).
  • FIG.11 shows the % GFP positive cells in various skeletal muscle and liver tissue (average from Runs 1 and 2) for each animal administered vehicle and AAV (AAV9 or AAV mut1deco1 ).
  • FIG.12 shows the % GFP positive cells per animal in various skeletal muscle and liver tissue (average from Runs 1 and 2) for each animal administered vehicle and AAV (AAV9 or AAV mut1deco1 ).
  • FIG.13 shows the average combined quantification of % GFP positive cells per animal in various skeletal muscle and liver tissue (average from Runs 1 and 2) for each animal administered vehicle and vector(AAV9 or AAV mut1deco1 ).
  • FIG.14 shows the % GFP positive cells in various cardiac tissue (average from Runs 1 and 2) for each animal administered vehicle and AAV (AAV9 or AAV mut1deco1 ).
  • FIG.15 shows the % GFP positive cells per animal in various cardiac muscle (average from Runs 1 and 2) for each animal administered vehicle and AAV (AAV9 or AAV mut1deco1 ).
  • FIG. 16 shows the average % GFP positive cells per animal in various cardiac muscle (average from Runs 1 and 2) for each animal administered vehicle and AAV (AAV9 or AAV mut1deco1 ).
  • FIGs.17A-17C show the average % GFP positive cells per animal in various tissues (average from Runs 1 and 2) for vehicle, AAV9 and AAV mut1_deco1 .
  • FIG.17A shows average % GFP positive cells per animal in liver tissue.
  • FIG.17B shows average % GFP positive cells per animal in various skeletal muscle tissue.
  • FIG.17C shows average % GFP positive cells per animal in various cardiac tissue.
  • DNA samples were analyzed for biodistribution of vector genomes in the liver and quadriceps tissue using a duplexed ddPCR method targeting the transgene (eGFP) and a reference gene (RPP30).
  • eGFP transgene
  • RPP30 reference gene
  • FIG.18A liver
  • FIG.18B quaddriceps
  • FIG.18C biceps
  • FIG.18D heart
  • the x-axis represents AAV vectors (wild type AAV9 on the left and AAV mut1deco1 on the right) and whether the sample was taken from the left or right side of the organ/animal.
  • mRNA transcript amounts measured by eGFP copies of eGFP over RPP30 mRNA are shown in FIGs.19A (liver), FIG.19B (quadriceps), FIG.19C (biceps), and FIG. 19D (heart).
  • the x-axis represents AAV vectors (wild type AAV9 on the left and AAV mut1deco1 on the right) and whether the sample was taken from the left or right side of the organ/animal.
  • the DNA, RNA and IHC expression data obtained from NHP experiments are quantified and summarized in the below Table 7 where each IHC stain is a technical replicate, data from all tissues combined including left and right sides; averages of the data obtained for all three animals is shown.
  • heart data includes data from ventricles and atria but does not include septum.
  • Atty Docket No.: 38053-57988/WO (112WO) [00547]
  • the below Table 8 exemplifies the Muscle:Liver infection ratios calculated for the DNA biodistribution data, the RNA expression data and the IHC expression data obtained for administration of AAV mut1_deco1 compared to AAV9 in non-human primates (NHP) as shown in the table above.
  • Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei.
  • Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood.
  • the objective of the current study was to identify a promoter that provides a broad biodistribution of expression within skeletal muscle.
  • a nucleotide sequence was synthesized to include the untranslated first exon and a portion of the intron from the human Cytomegalovirus (hCMV) IE gene, a portion of the intron of the second intron of the human beta globin gene, a portion of the 3 rd exon of the human beta globin gene, a NotI restriction site, a predicted optimal Kozak sequence, a codon optimized human MTM1 CDS with a modified stop codon using a sequence provided by Genscript, a PacI restriction site which overlaps with the modified stop codon, the Rabbit beta-globin PolyA signal sequence, an AvrII site, and the first 10 bp of the AAV2 I
  • Portions of SA024 containing the first ITR and expression regulatory sequences 5’ to the gene of interest are provided as SEQ ID NO:204 and the portions of SA024 from the 3’ of the open reading frame of the gene of interest through the second ITR are provided as SEQ ID NO:205.
  • the 2443 bp long fragment containing MTM1 and the 6693 bp long fragment containing the plasmid elements were isolated by agarose gel electrophoresis and eluted from the agarose using NEB Monarch DNA Gel isolation kit. Ligations of the sticky end fragments were performed with T4 DNA ligase. Successful ligation products were isolated from E. coli transformants and confirmed by restriction digest and Sanger sequencing to confirm the insertion of the codon optimized Atty Docket No.: 38053-57988/WO (112WO) MTM1 sequence and the additional features. The portions of the vector within the ITRs (and including the ITRs) are provided as SEQ ID NO:181.
  • the CAG promoter was amplified from construct 7701591057 (the portion of which within (and including) the ITRs is provided as SEQ ID NO:201), which had been synthesized previously, using a 5’ primer with the sequence SEQ ID NO: 209 (ttttGGTACCgacattgattattgactagttatt) which contains a KpnI restriction site and a Poly T tag to aid in restriction digestion and a region matching the start of the CMV immediate early promoter in a linear amplification reaction.
  • the amplification product was isolated from the amplification mixture with NEB Monarch DNA Gel isolation kit.
  • a second amplification step was performed with a primer with the sequence SEQ ID NO: 210 (aaaaaaccatc cgcccgccgcgcgcgcgcgcgcgcgcgc) which contains a region matching the reverse complement of the Chicken beta actin promoter, an EcoRV restriction site, and a poly A sequence to aid in fragment digestion.
  • the 675 base pair fragment (SEQ ID NO:200) was isolated by agarose gel electrophoresis Atty Docket No.: 38053-57988/WO (112WO) and eluted from the agarose using NEB Monarch DNA Gel isolation kit.
  • the 675 bp fragment and SEQ ID NO:181 vector were digested with KpnI and EcoRV.
  • the 663 bp digested fragment of the amplification and the 8581 bp fragment of the SEQ ID NO:181 vector were isolated by agarose gel electrophoresis and eluted from the agarose using NEB Monarch DNA Gel isolation kit. Ligations of the sticky end fragments were performed with T4 DNA ligase. Successful ligation products were isolated from E. coli transformants and confirmed by restriction digest and Sanger sequencing to confirm the insertion of the CAG promoter sequence into the vector containing SEQ ID NO:181 resulting in a vector containing SEQ ID NO:186, which includes an MTM1 CDS with codon optimizations provided by Genscript.
  • the native MTM1 sequence was amplified from the vector containing SEQ ID NO:184 using a 5’ primer with the sequence TTTGAGCGGCCGCCA (SEQ ID NO: 215) which corresponds to the Kozak and start sequence of MTM1 and contains a NotI restriction site and a 3’ primer with the sequence GATCTTAATTAAAAGTGAGTTTGCACATGGG (SEQ ID NO: 216) which contains the reverse complement to the 3’ end of MTM1, an altered stop codon, and a PacI restriction site.
  • the 1837 base pair PCR product (SEQ ID NO:19) was isolated by agarose gel electrophoresis and eluted from the agarose using NEB Monarch DNA Gel isolation kit.
  • the purified amplicon and the vector containing SEQ ID NO:186 were digested with NotI and PacI.
  • the 1823 bp fragment containing the MTM1 CDS and the 7350 bp fragment containing the plasmid and ITR sequence and other SEQ ID NO:186 features were isolated by agarose gel electrophoresis and eluted from the agarose using NEB Atty Docket No.: 38053-57988/WO (112WO) Monarch DNA Gel isolation kit. Ligations of the sticky end fragments were performed with T4 DNA ligase.
  • a miniTK portion of the vector is provided as SEQ ID NO:190 and the portion of the vector containing the rabbit globin poly A and ITR is provided as SEQ ID NO:191.
  • This synthetic sequence was flanked by SalI site at the 5’ end and AscI at the 3’ end.
  • This fragment was introduced into the vector comprising SEQ ID NO:201 via restriction enzyme digestion, agarose gel fragment isolation, and T4 DNA ligase ligation.
  • Successful ligation products were isolated from E. coli transformants and confirmed by restriction digest and Sanger sequencing to confirm the insertion of self- complementary vector sequence into the vector comprising SEQ ID NO:201 resulting in a vector containing SEQ ID NOS:190 and 191.
  • the first ITR and miniTK portions of the vector are provided as SEQ ID NO:206 and the rabbit poly and second ITR portions of the vector are provided as SEQ ID NO:207.
  • the miniTK promoter was synthesized with KpnI restriction site at the 5’ end and a NotI site at the 3’ end. Additionally, bases were added to the synthesize product to enhance the efficiency of restriction digestion SEQ ID NO:192. The fragment containing SEQ ID NO:192 was digested with KpnI and NotI and inserted into a vector containing SEQ ID NO:184 via the same restriction sites following agarose gel electrophoresis, gel extraction, T4 ligation.
  • This vector (the portion of which within (and including) the ITRs is provided as SEQ ID NO:11B) after sequencing, was determined to have an undesired deletion in the 5’ ITR. However, the insert of SEQ ID NO:193 was identical to the desired sequence.
  • the miniTK-native MTM1 sequence was PCR amplified using the 5’ primer SEQ ID NO: 211 (tttttGtcGACTTCGCATATTAAGGTGACGCGT) Atty Docket No.: 38053-57988/WO (112WO) which contains a polyT sequence to aid in restriction digestion, the KpnI site, and the 5’ end of the miniTK promoter and the 3’ primer SEQ ID NO: 212 (ttttttt cctagg gagTGAGAGACACAAAAAATTCCAACACAC), which contains a polyT sequence to aid in restriction digestion, an AvrII site, and the reverse complement of the 3’ end of the Rabbit beta-globin PolyA signal creating SEQ ID NO:194.
  • SEQ ID NO:194 and the vector containing SEQ ID NOS:190 and 191 were digested with KpnI and SalI.
  • the 2024 bp fragment with SEQ ID NO:194 and the 6603 bp fragment comprising SEQ ID NO:190 and SEQ ID NO:191 were isolated by agarose gel electrophoresis and eluted from the agarose using NEB Monarch DNA Gel isolation kit. Ligations of the sticky end fragments were performed with T4 DNA ligase. Successful ligation products were isolated from E. coli transformants and confirmed by restriction digest and Sanger sequencing to confirm the insertion of the promoter and native MTM1 CDS into the vector with appropriate ITRs for creating a self-complementary AAV vector.
  • the vector created this way contains a full ITR, the miniTK promoter, the native MTM1 CDS, the Rabbit beta-globin Poly A signal and an ITR with an appropriate deletion to create a self-complementary AAV vector.
  • the portion of this vector within (and including) the ITRs is provided as SEQ ID NO:208.
  • the mini Desmin promoter was synthesized with KpnI site at the 5’ end and NotI site at the 3’ end (SEQ ID NO:195). Additional bases were added to the synthesized product to enhance the efficiency of restriction digestion (SEQ ID NO:196).
  • the fragment containing SEQ ID NO:196 was digested with KpnI and NotI and inserted into a vector containing SEQ ID NO:184 via the same restriction sites following agarose gel electrophoresis, gel extraction, T4 ligation.
  • This vector (the portion of which within (and including) the ITRs is provided as SEQ ID NO:197), after sequencing, was determined to have a undesired deletion in the 5’ ITR.
  • the insert of SEQ ID NO:197 was identical to the desired sequence.
  • the miniDes-native MTM1 sequence was PCR amplified using the 5’ primer SEQ ID NO: 213 (tttttGtcGACCCTCTATAAATACCCGCTCTGG) which contains a polyT sequence to aid in restriction digestion, the KpnI site, and the 5’ end of the miniDesmin promoter and the 3’ primer SEQ ID NO: 214 (tttttt cctagg gagTGAGAGACACAAAAAATTCCAACACAC) which contains a polyT sequence to aid in restriction digestion, an AvrII site, and the reverse complement of the 3’ end of the Rabbit beta-globin PolyA signal creating SEQ ID NO:198.
  • SEQ ID NO:198 and the vector comprising SEQ ID NO:190 and SEQ ID NO:191 were digested with KpnI and SalI.
  • the 2185 bp fragment containing SEQ ID NO:198 and the 6603 Atty Docket No.: 38053-57988/WO (112WO) bp fragment containing comprising SEQ ID NO:190 and SEQ ID NO:191 were isolated by agarose gel electrophoresis and eluted from the agarose using NEB Monarch DNA Gel isolation kit. Ligations of the sticky end fragments were performed with T4 DNA ligase. Successful ligation products were isolated from E.
  • the coli transformants and confirmed by restriction digest and Sanger sequencing to confirm the insertion of the promoter and native MTM1 CDS into the vector with appropriate ITRs for creating a self-complementary AAV vector.
  • the vector created this way contains a full ITR, the miniDesmin promoter, the native MTM1 CDS, the Rabbit beta-globin Poly A signal and an ITR with an appropriate deletion to create a self-complementary AAV vector.
  • the portion of this vector within (and including) the ITRs is provided as SEQ ID NO:199. 8.8.1.3 Expression studies by cell transfection [00558]
  • the RD cell line (ATCC CCL-136) was used for our in vitro expression studies.
  • RD cells are derived from patients with Rhabdomyosarcoma, a rare form of pediatric cancer that develops from skeletal muscles. RD cells were maintained in 10% FBS DMEM inside a humidified 37 degrees C incubator with 5% CO2 air with serial passage every three to four days following TrypLE non-enzymatic lifting and replating at 1/4th density. [00559] 24 h prior to transfection, RD cells were lifted with TrypLE, pelleted (7’, room temperature, 1400xg), and resuspended in media. Viability was determined by Trypan Blue exclusion using two chambers of a Countess automated cell counter (Thermo Fisher).
  • Average cell density was adjusted to 3.2E5 live cells per mL and 1.6E5 viable cells were plated in 500 uL media. In a 24 well plate. [00560] On the day of transfection, all reagents were warmed to room temperature before use. Plasmid DNA was diluted to 250 ng/uL in TE buffer. Enough reagent was used to transfect 4 wells per plasmid.100 uL OptiMEM (gibco) plus 6 uL Lipofectamine 3000 (Thermofisher, lot 2170726) was prepared per plasmid.
  • OptiMEM 100 uL of OptiMEM plus 6 ug of DNA (24 uL of 250 ng/uL diluted plasmid) plus 12 uL 3000 Reagent (Thermo Fisher) were combined.
  • the diluted DNA was then mixed with the diluted Lipofectamine 3000, spun down briefly, and incubated at room temperature for 16 minutes.57 uL of the mixture was added to each of 4 wells per plasmid. Some wells of cells were left untransfected to serve as a negative control.
  • Atty Docket No.: 38053-57988/WO (112WO) A 1 second exposure using the LED intensity setting of 10 was used. After imaging, the cells were washed with 500 uL DPBS. The DPBS was removed and 125 uL TrypLE was added and incubated for 5 minutes at 37 degrees C in a humidified incubator with 5% CO2.
  • Plasmids used In addition to the MTM1 expression constructs (Seq 1 to 9) certain reference plasmids were used.
  • Plasmid 7701591057 a fully-synthesized plasmid vector, which contains AAV2 ITRs and eGFP under the control of the CAG promoter and the Rabbit beta-globin PolyA signal was used as a transfection control for fluorescently visualizing eGFP and percent of cells successfully transfected as well as a negative control for antibody-mediated MTM1 detection.
  • pCDNA3.1+C/(K)DYK with human native MTM1 under the control of the CMV promoter, an in-frame DYK epitope tag, and a bovine Growth Hormone PolyA signal. This was obtained from Genscript.
  • Example 9 Anc80 variants
  • AAV capsids of the Anc80 library and 5 control capsids were systemically administered to four NHPs by i.v. injection at the dose of 1.6x10 12 gc/kg.
  • RNA RNA samples were immediately placed into the preservative RNAlater, after which the RNAlater was removed and the tissue flash frozen
  • tissues were homogenized in a Qiagen Tissuelyser II (20rps for 2 min) in lysis buffer from the Qiagen Dneasy Blood and Tissue Kit or the Qiagen Atty Docket No.: 38053-57988/WO (112WO) RNeasy Lipid Tissue Mini Kit following the standard Qiagen protocol. Samples were eluted in 50uL of buffer.
  • NGS was performed on RNA harvested from non-human primate tissue following IV infusion of the Anc80 library and reverse transcribed into cDNA for analysis.
  • tissue i.e., the liver and major skeletal muscles of the hind limb (quad)
  • Tissues were immediately placed into the preservative RNAlater, after which the RNAlater was removed and the tissue flash frozen.
  • the viral DNA and RNA of each of the Anc80 variants were measured and enrichment of Anc80 variants in the quadricep muscle and the liver was analyzed.
  • PCR amplified products corresponding to the bar code of the vector were analyzed via Illumina sequencers, either MiSeq or NovaSeq, to quantify the presence of each variant in tissue or test article. These counts were normalized to one million reads.
  • Tissue enrichment was calculated by dividing tissue counts per million by test article counts per million. The base two logarithm is often used to present this last calculation. Enrichment data of Anc80 variants in the quadricep muscle (y-axis) and the liver (x-axis) are presented as individual dots in the plot in FIG.22. [00570] In the previous study described in WO2019217911, which is incorporated by reference in its entirety herein, it was demonstrated that amino acid at the position “P3” corresponding to amino acid 266 of Anc80 capsid protein can affect liver tropism of the AAVs containing the capsid protein.
  • capsids having a glycine (G) at P3 tend to have “liver on” whereas capsids having alanine (A) at P3 tend to have “liver off” properties.
  • Anc80 capsid variants having G (“liver on”) or A (“liver off”) at P3 position respectively were separately analyzed and their log fold changes (log (FC)) in the quadricep muscle are plotted in FIG.23.
  • FIG.23 shows that most of the Anc80 variants, both liver on and liver off, have negative log fold changes in tissues compared to target input (the pool of AAV variants that were administered).
  • FIG.26 provides the data of two experimental animals on average, and FIG.24 (G62N) and FIG.25 (G66E) provide data from individual animals. Atty Docket No.: 38053-57988/WO (112WO) [00572]
  • the quadricep enrichment data for the two animals are also provided in the quadrant plots of FIG.27 (G62N) and FIG.28 (G66E).
  • the average data of the two animals are provided in FIG.30.
  • each dot represents the quadricep enrichment of an Anc80 variant with G (“liver on”) (y-axis) or A (“liver off”) (x-axis) at P3 position.
  • Dots on the upper left box (UL) represent Anc80 variants with higher quadricep enrichment with G (“liver on”)
  • dots in the upper right box (UR) represent Anc80 variants with high quadricep enrichment in both G (“liver on”) and A (“liver off”)
  • dots in the lower left box (LL) represent Anc80 variants with low quadricep enrichment in both G (“liver on”) and A (“liver off”)
  • dots in the lower right box (LR) represent Anc80 variants with lower quadricep enrichment with G (“liver on”).
  • FIG.33 shows exemplary amino acids at the P1 to P11 positions in three liver-toggle variant pairs. Two capsids in each pair differs only at one of the P1 to P11 positions, and the one amino acid difference makes one capsid liver-on and the other capsid liver-off.
  • 6 Anc80 variants (Anc80-55, Anc80-129, Anc80-156, Anc80-751, Anc80-1029, and Anc80-1712) were selected from the Anc80 library for the high muscle tropism in the quadricep.
  • Example 10 Deco1 targeting peptide enhances muscle delivery in Anc80 Variants [00575]
  • a targeting peptide having SEQ ID NO: 1 (“deco1”) was inserted into VR VIII of Anc80 variants (Anc80L65 (SEQ ID NO: 44895), Anc80L65 Mut1 (SEQ ID NO: 44896), Anc80-55 (SEQ ID NO: 44885), Anc80-129 (SEQ ID NO: 44887), Anc80-156 (SEQ ID NO: 44889), Anc80-751 (SEQ ID NO: 44916), Anc80-1029 (SEQ ID NO: 44917), and Anc80-1712 (SEQ ID NO: 44893).
  • Anc80 variants with the deco 1 insertion are referred to here as Anc80L65 deco1 (SEQ ID NO: 44897), Anc80L65 mut1deco1 (SEQ ID NO: 44898), Anc80-55 deco1 (SEQ ID NO: 44886), Anc80-129 deco1 (SEQ ID NO: 44888), Anc80-156 deco1 (SEQ ID NO: 44890), Anc80- 1712 deco1 (SEQ ID NO: 44894), and Anc80-751 deco1 (SEQ ID NO: 44891), Anc80-1029 deco1 (SEQ ID NO: 44892).
  • the Anc80 variants with or without deco1 peptide were then administered via IV injection to non-human primates at a concentration of 1x10 14 vg/ml.
  • the variants targeted to the muscles were quantified and the log2 fold changes of tissue enrichment are show in FIG.40.
  • the tissue enrichment is the ratio of the amount of each individual variant in the tissue divided by the amount of that same variant in the pool of AAV variants administered.
  • the results show that insertion of deco1 peptide increases muscle tropism of Anc variants. Six (6) out of 7 Anc80 variants with a deco1 insertion at VR VIII enhanced muscle delivery of Anc80 variants (FIG.40).
  • Tissue enrichment is the ratio of the amount of each individual variant in the tissue divided by the amount of that same variant in the pool of AAV variants administered.
  • Example 11 Muscle targeting of AAV capsids including RGD peptides (“VRVIII library”)
  • a library of AAVs (VRVIII library) containing capsids modified to comprise a unique targeting peptide inserted into the VR VIII region of the capsid protein was generated.
  • the modified capsid proteins include one of 44,617 unique 7-mer peptides between Q588 and A589 of AAV9 mut1 (SEQ ID NO: 163) (“VRVIII library”).
  • the sequences of the 7-mer peptides are provided as SEQ ID NOs: 238-44,853 (the “RGD peptide library” of as described in Table 27). Atty Docket No.: 38053-57988/WO (112WO) [00579]
  • the seven amino acid positions of the targeting peptides inserted between Q588 and A589 of AAV9 mut1 VP1 capsid sequence are identified as “Y1, Y2, Y3, Y4, Y5, Y6, and Y7” in FIG.34.
  • the 7 amino acid positions (P1, P2, P3, Y4, P5, P6, P7) of FIG.34 correspond to RGDX7X8X9X10 within X1X2X3RGDX7X8X9X10 described as a targeting peptide in the present disclosure, where “Y1” is “R”, “Y2” is “G”, “Y3” is “D”, “Y4” is “X 7 ”, “Y5” is “X 8 ”, “Y6” is “X 9 ”, and “Y7” is “X 10 ” of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 (SEQ ID NO: 44915).
  • capsid proteins in the VRVIII library include a targeting peptide having a sequence of “SAQRGD X7X8X9X10” at VRVIII.
  • the targeting peptides in the library include one of 15 amino acids (A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V) at Y4, Y5, Y6, and Y7 positions as illustrated in FIG.34. Random combinations of the 15 amino acids at Y4, Y5, Y6, and Y7 positions allow creation of 50,625 (15 ⁇ 4) unique targeting peptide sequences. The 50,625 unique targeting peptide sequences were then quality filtered to remove variation, resulting in 40,617 unique targeting peptide sequences.
  • AAVs containing the modified AAV capsid proteins were produced using HEK293 cells containing an adenoviral helper gene plasmid construct and a plasmid containing the AAV rep expression cassette and a capsid coding sequence.
  • the modified capsid coding sequence was flanked by the AAV ITR sequences to generate AAVs containing the modified capsid coding sequence encapsidated in the modified capsid proteins.
  • the AAV samples were analyzed by next-generation sequencing using Illumina reagents and hardware. Viral genomic DNA containing the capsid coding sequence was isolated from viral supernatant and sequenced. The analysis confirmed the presence of 44,617 capsids.
  • AAVs containing the modified AAV capsids were administered to cynomolgus monkeys by IV administration.
  • AAV containing the wild-type AAV9 capsids was included in the mixture for comparison.
  • a control animal treated with a buffer was also included.
  • the AAV variants including each targeting peptide were ranked based on a mean log fold-change tissue score (Column C of Table 27).
  • Table 27 also includes tissue enrichment scores for each individual tissue analyzed.
  • FASTQ sequence read data
  • AAV variants containing a unique targeting peptide were counted and normalized, followed by performing a tissue enrichment analysis for each tissue.
  • Tissue enrichment analysis included Sequence Activity Relationship Atty Docket No.: 38053-57988/WO (112WO) (SAR) analysis, Network analysis, and structural modeling. SAR analysis identified particular amino acid at a specific capsid position or sequence motifs (combination of amino acids at several positions) that significantly affects tissue tropism.
  • a mean log fold-change tissue enrichment score is based on the combination of amino acid residues present in the modified sequence within VR VIII.
  • the density plot shows highest tissue enrichment when Y4 is “R” as compared to other amino acid residues F, K, D, E, S, G, I, or V. Additionally, after fixing R at position 4, amino acid residues S or G at position 5 (or “X8” in X1X2X3RGDX7X8X9X10) (P5) and I or V at position 7 (or “X10” in X1X2X3RGDX7X8X9X10) were found to lead to higher muscle enrichment scores. [00591] Based on the analysis the muscle-tropic sequence motif was identified as provided in FIG.36A.
  • FIG.36B is a scatter plot showing muscle enrichment scores of variants (an inverse coefficient of variation, which is the mean divided by the standard Atty Docket No.: 38053-57988/WO (112WO) deviation). Each dot represents a single variant with a unique targeting peptide, and dots corresponding to variants with the muscle-tropic motif of FIG.36A are colored in red. The muscle enrichment scores of the variants with and without the muscle-tropic motif were further analyzed as provided in FIG.36C, which shows that there was significant separation between targeting peptide with the muscle-tropic motif and targeting peptide without the motif. [00592]
  • FIG.37A shows amino acid probability distribution of the targeting peptides that provided top 10 muscle enrichment (see Table 27).
  • top 10 peptides included amino acid residue “R” at position 4, amino acid residues S or G at position 5 and I or V at position 7 showing significant sequence similarity to the muscle-tropic motif in FIG.36A.
  • the top targeting peptides (FIG.37B) belong to a muscle-tropic sequence motif by having R, G, Q, and I, at Y4, Y5, Y6, and Y7 position, respectively.
  • the top 10 peptides had highly similar sequences to each other.8 out of 10 top variants were connected in a sequence similarity network.
  • the network plot (FIG.37C) shows a capsid represented by a “dot”, where the lines connecting various dots represent sequence connectivity between the capsids.
  • the sequence similarity network shows that SEQ ID NO: 238 (“ATLVT013XX38181”) was the top targeting peptide for muscle tropism and has a sequence similar to other targeting peptides providing high muscle enrichment.
  • each dot/line is a AAV variant.
  • the radial axis represents the tissue enrichment score for a particular tissue.
  • the most inner circle represents a “low tissue enrichment”, while an outer circle represents a “high tissue enrichment”.
  • Network plots are useful for comparing the performance of a small number of variants in multiple tissues.
  • FIG.38A shows enrichment of four AAV variants (AAV9 mut1 containing SEQ ID NO: 238 (“ATLVT013XX38181”); wild-type AAV9, AAV9 deco1 (AAV9 capsid with the deco1 peptide), and AAV9 with SEQ ID NO: 44880).
  • FIG.38B is a scatter plot showing tissue enrichment scores for each tissue region assessed.
  • AAV9 mut1 containing SEQ ID NO: 238 (“ATLVT013XX38181”) outperformed wild-type AAV9, AAV9 deco1 , and AAV9 with SEQ ID NO: 44880 for muscle tropism. Further, AAV9 mut1 containing SEQ ID NO: 238 maintained the liver-detargeting phenotype AAV9 mut1 as compared to wild-type AAV9, showing lower liver enrichment.
  • the Atty Docket No.: 38053-57988/WO (112WO) targeting peptide SEQ ID NO: 238 (“ATLVT013XX38181”) consistently ranked at the top for target enrichment in all muscles except for the triceps.
  • FIG.38C shows a plot of tissue enrichment scores for liver and the indicated muscle tissues for AAV9 and AAV9 comprising an M1 (RGDRGQI (SEQ IDNO: 238) targeting peptide located in VR VIII between amino acids at positions 588 and 589 or an M2 (RGDRSVV (SEQ ID NO: 239)) targeting peptide located in VR VIII between amino acids at positions 588 and 589.
  • M1 RDRGQI
  • M2 RGDRSVV
  • capsids comprising either an M3 targeting peptide or an M2 targeting peptide also showed increased tissue enrichment (i.e., increased tropism) in each of the muscle tissues analyzed as compared to AAV9 (see FIG.38C).
  • the increased muscle tropism in capsids comprising either an M1 targeting peptide or an M2 targeting peptide ranged from 10-100 fold increased expression compared to AAV9.
  • FIG.39 shows amino acid probability distribution of the targeting peptides that provided top 100 muscle enrichment. Similar to the earlier data with top 10 variants, amino acid “R” is enriched at position 4 (Y4), and amino acids “V, I, and L” are enriched at position 7 (Y7).
  • Example 12 Muscle targeting of AAV capsids including RGD peptides (VRVIII mini-library) [00597] A library of AAVs (VRVIII mini-library) containing capsids modified to comprise a different set of targeting peptides inserted into the VR VIII region of the capsid protein was generated.
  • the modified capsid proteins include one of 22 unique targeting peptides (SEQ ID NO: 44859-44878, 44879, and 44883) between Q585 and A589 of AAV9 (SEQ ID NO: 61).
  • the modified capsid proteins include the targeting peptide having the sequence of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 in place of “SAQ”, the original amino acids at positions 586, 587 and 588 in AAV9 VP1 capsid protein.
  • X1X2X3 has the original sequence, “SAQ” of the AAV9 capsid protein (SEQ ID NOs.: 44881-44882, 44911, Atty Docket No.: 38053-57988/WO (112WO) 44912, 44913, 44918-44919), whereas in other modified capsid proteins, X1X2X3 has a sequence different from the original “SAQ” (SEQ ID NOs: 44859-44878, 44879, 44880, 44883, and 44910). [00599] AAVs containing the modified capsid proteins of the VRVIII mini-library were generated and tested as described in Example 11.
  • FIG.41 shows a list of 20 targeting peptides (myoDV1-myoDV10 and myoCD5-myoCD14) in the VRVIII mini-library.
  • FIG.41 shows tissue enrichment scores for each of the 20 targeting peptides in muscle tissue (diaphragm, flexor digitorum profundus, heart left ventricle wall, tibialis anterior, and triceps brachii). Higher tissue enrichment score values represent greater muscle tropism, with MYODV6 and MYODV8 having the highest averages tissue enrichment score for all muscle tissue regions.
  • FIG.42 shows a network plot showing log2 fold change of tissue enrichment for each of the muscle tissues analyzed for the targeting peptide having an amino acid sequence of SEQ ID NO: 44880, MYODV6, MYODV8, MYOCD10, and MYOCD12 targeting peptides.
  • MYODV6 and MYODV8 each have the highest overall log2 fold change of tissue enrichment in each muscle tissue compared to MYOCD10 and MYCD12 (which contain amino acid residues “SNR” (triplet) preceding “RGD” within the targeting peptide.
  • MYODV6 and MYODV8 each have a higher overall log2 fold change of tissue enrichment in each muscle tissue compared to a targeting peptide having an amino acid sequence of SEQ ID NO: 44880. 8.13.
  • Example 13 SAR analysis of modified AAV capsid proteins identifies cardiac-tropic capsids [00602] Using the data from Example 11, SAR analysis was performed with the aim of identifying modified AAV capsid proteins having different tropism for different types of muscle tissue. For example, SAR was used to identify modified AAV capsid proteins that had increased enrichment in cardiac muscle tissue as compared to skeletal muscle tissues.
  • SAR analysis identified at least two modified AAV capsid proteins (i.e., modified AAV capsid proteins comprising either targeting peptide H1 Atty Docket No.: 38053-57988/WO (112WO) (RGDLIGR (SEQ ID NO: 1422)) or targeting peptide H2 (RGDQSTL (SEQ ID NO: 3052)) inserted into VR VIII of AAV9 between amino acids at positions 588 and 589)) that increased tissue enrichment in cardiac muscle tissue as compared to skeletal muscle tissue.
  • modified AAV capsid proteins comprising either targeting peptide H1 Atty Docket No.: 38053-57988/WO (112WO) (RGDLIGR (SEQ ID NO: 1422)) or targeting peptide H2 (RGDQSTL (SEQ ID NO: 3052)
  • SAR analysis also identified at least two modified AAV capsid proteins (i.e., modified AAV capsid proteins comprising either targeting peptide S1 (RGDISRT (SEQ ID NO: 263)) or targeting peptide S2 (RGDRSQT (SEQ ID NO: 251)) inserted into VR VIII of AAV9 between amino acids at positions 588 and 589) that increased tissue enrichment in skeletal tissue as compared to cardiac muscle tissue. 8.14.
  • modified AAV capsid proteins comprising either targeting peptide S1 (RGDISRT (SEQ ID NO: 263)
  • targeting peptide S2 RGDRSQT (SEQ ID NO: 251)
  • Example 14 Clonal analysis of a subset of modified AAV capsid proteins in non-human primates [00605] The objective of this study was a clonal assessment of selected modified AAV capsid proteins comprising different targeting peptides in VR VIII in non-human primates.
  • rAAVs comprising modified capsid proteins having a targeting peptide having a sequence of SEQ ID NO: 44864 (ENRRGDFNNL) (M3 or MYODV6) or a targeting peptide having a sequence of SEQ ID NO: 44911 (SAQRGDRGQI)) in VR VIII of AAV9 were constructed as described herein (i.e., the targeting peptide was inserted between 585 and 589 such that amino acids at positions 586, 587 and 588 were replaced with three amino acids from the targeting peptide in AAV9 VP1 capsid protein).
  • rAAVs comprising an M1 targeting peptide in VR VIII of AAV9 were constructed as described herein (i.e., the targeting peptide was inserted between 585 and 589 such that amino acids at positions 586, 587 and 588 were replaced with three amino acids from the targeting peptide in AAV9 VP1 capsid protein) and a mut1 VR I were also tested.
  • rAAV comprising capsid proteins having a targeting peptide with the amino acid sequence of SEQ ID NO: 44880 (ENRRGDFNNT) was used as a control.
  • AAV9 with no targeting peptide in VR VIII was also used as a control.
  • control rAAV and rAAV comprising the modified capsid proteins were administered to the NHPs according to the experimental design in the table 13 below. Atty Docket No.: 38053-57988/WO (112WO) Table 13. Experimental design for Example 14.
  • RNA RNA-derived tissue
  • DNA DNA
  • tissues were homogenized in a Qiagen Tissuelyser II (20rps for 2 min) in lysis buffer from the Qiagen Dneasy Blood and Tissue Kit or the Qiagen RNeasy Lipid Tissue Mini Kit following the standard Qiagen protocol.
  • the same tissues were fixed and embedded for sectioning and anti-GFP staining by immunohistochemistry (IHC) as described herein.
  • FIG.45 shows the median percent GFP + cells for liver and the indicated muscle tissues for AAV9 and AAV9 comprising an M3 (“DV6” ENRRGDFNNL (SEQ ID NO: 44864)) targeting peptide located in VR VIII (M3 targeting peptide was inserted between 585 and 589 such that amino acids at positions 586, 587 and 588 were replaced with three amino acids from the targeting peptide in AAV9 VP1 capsid protein); an M1 (“38181” SAQRGDRGQI (SEQ ID NO: 44911)) targeting peptide located in VR VIII (M1 targeting peptide was inserted between 585 and 589 such that amino acids at positions 586, 587 and 588 were replaced with three amino acids from the targeting peptide in AAV9 VP1 capsid protein) and a mut1 VR I substitution, or an M1 (“38181” SAQRGDRGQI (SEQ ID NO: 44911)) targeting peptide located in VR VIII (M1 targeting peptide
  • Capsids comprising either the M1 targeting peptide or the M3 targeting peptide also showed increased tissue enrichment (i.e., increased tropism) in each of the muscle tissues analyzed as compared to AAV9 (see FIG.45).
  • FIG.46 shows representative images of IHC quantified in FIG.45.
  • Results for ddRT-PCR shown eGFP RNA copies per copies of NHP RPP30 RNA is shown in the table 14 below) and FIG.47. ddRT-PCR was performed at Affinia Therapeutics (Waltham, MA). Table 14. ddRT-PCR data.
  • FIG.89 shows the percent GFP+ cells in cervical DRG, thoracic DRG, and lumbar DRG for wild type AAV9 and AAV9 comprising an M3 targeting peptide, an M1 targeting peptide with wild type VRI, or an M1 targeting peptide with mut1 VRI substitution.
  • AAV9 comprising a targeting peptide with the amino acid sequence of SEQ ID NO: 44880 was used as a control. Both M3 and M1 targeting peptide resulted in DRG detargeting in in non-human primates compared to wild type AAV9.
  • FIGs.90A and 90B show the levels of liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST) for wild type AAV9 and AAV9 comprising an M3 targeting peptide, an M1 targeting peptide with wild type VRI, or an M1 targeting peptide with mut1 VRI substitution.
  • the ALT and AST levels were measured at Day 8, peak of liver inflammation.
  • the data show that M1 (AAV9-38181-mut1) did not cause liver enzyme elevation.
  • Example 15 Analysis of AAV VRI (AAV- Capsid Library [00614] Experiment AFT-MR0026 was designed to identify one or more amino acid modifications in variable region I (VR I) of an AAV capsid protein that decreases liver tropism and increases muscle tropism (or do not effect reduced muscle tropism as compared to a control capsid, such as the reference capsid).
  • VR I variable region I
  • Example 16 Wild type AAV9 and AAV mut1 tropism in liver and muscle in C57BL/6 mice [00615] As a preliminary assessment for determining whether mice were a suitable system for an initial screen of the AAV- Lib1 capsid proteins, an analysis of AAV vector tissue distribution was performed in mice following IV injection of AAV mut1 and wild type AAV9.
  • a low dose of 1x10 13 gc/kg and a high dose of 5x10 13 gc/kg were IV administered for AAV capsid to 10 week old C57BL/6 mice (see Table below). Additionally, a control mouse was injected with vehicle (1X PBS, 35mM NaCl, 0.001% pluronic) alone. Thus, a total 14 mice comprised the study. Table 15.
  • the low dose was 1x10 13 gc/kg (total 2x10 11 gc), and the high dose was 5 x 10 13 gc/kg (total 1x10 12 gc).
  • GFP expression was assessed by ddRT-PCR for the eGFP vector genome copies per DPG (DNA) and transcript (mRNA). The eGFP transcript level was compared against the transcript of a housekeeping standard RPP30. IHC was performed at Histoserv Inc. (Germantown, MD). ddRT-PCR was performed at Affinia Therapeutics (Waltham, MA).
  • % eGFP expression (eGFP cp/ ⁇ L ⁇ RPP30 cp/ ⁇ L) ⁇ 100.
  • Tissues were homogenized in a Qiagen Tissuelyser II (20rps for 2 min) in lysis buffer from the Qiagen Dneasy Blood and Tissue Kit or the Qiagen RNeasy Lipid Tissue Mini Kit following the standard Qiagen protocol. Samples were eluted in 50uL of buffer. Prior to analysis, DNA and RNA concentration and quality were determined using a NanoDrop One, using the nucleic acid (DNA or RNA) program.
  • RNA samples were analyzed for biodistribution of vector genomes using a duplexed ddPCR method targeting the transgene (eGFP) and a reference gene (RPP30).
  • RNA samples were analyzed for expression of the eGFP transgene using a duplexed, one-step RT-ddPCR method and a reference gene (RPP30).
  • mRNA was extracted from 30 mg sections of liver, and quadriceps.
  • FIG.48 The results of the ddPCR assays are shown in FIG.48 which show that AAV Mut1 has low liver tropism but does not have high muscle tropism, AAV Deco1 has high liver tropism and comparatively high muscle tropism, and that AAV Mut1_deco1 has decreased liver tropism and increased muscle tropism compared to AAV9 (WT).
  • eGFP mRNA expression in various tissues was measured by RT-ddPCR and presented as the ratio of eGFP transcripts over RPP30 transcripts, a rough indicator of eGFP mRNA copies per cell.
  • FIG.48 provides the eGFP mRNA per murineRPP30 transcript in the liver, quadriceps, triceps surae, heart, and diaphragm. For each dose group, mice treated with AAV mut1 had lower eGFP expression compared to mice treated with AAV9 for each of the five tissues measured (see FIG.48).
  • FIG.48 also provides the vector genomes per DPG in the liver, quadriceps, triceps surae, heart, and diaphragm.
  • mice treated with AAV mut1 had fewer vector genomes per DPG compared to Atty Docket No.: 38053-57988/WO (112WO) mice treated with AAV9 for ease dose group (see FIG.48).
  • AAV mut1 and AAV9 showed similar vector genomes per DPG for each dose group (FIG.48). 8.17.
  • Example 17 AAV VRI (AAV -Lib1 ) Capsid Libraries
  • SEQ ID NO: 61 The polynucleotide encoding the wild-type AAV9 VP1 capsid protein (SEQ ID NO: 61) was modified by insertion, deletion or substitution of one or more amino acid residues between S261 and Y274 in the VR I region of AAV9.
  • the amino acid positions between S261 and Y274 of AAV9 capsid are represented as “P 1 , P 2 , P 3 , P 4 , P 5 , P 6 , P 7 , P 8 , P 9 , P10, P11, and P12” as illustrated in FIG.49A.
  • AAV -Lib1 library a library containing 3,456 unique sequences of modified AAV9 VP1 capsid proteins was produced, which is referred to herein as the AAV -Lib1 library or AAV -Lib1 capsid library.
  • the amino acid sequences (a peptide segment in the VR I region) corresponding to the 12 amino acids between S261 and Y274 for each of the 3,456 sequences are as described in SEQ ID NOs: 251-3703.
  • the amino acid modifications (insertion, deletion, substitution) in the VR I region of AAV9 include those described herein as shown in FIG.49A.
  • FIG.49B a sequence alignment of variable region I for certain AAV variants is shown in FIG.49B.
  • the construct used for generating each of the AAVs for the AAV library included a polynucleotide sequence located between the ITRs that encoded a sequence that was reflective of the genome of the virus that transduced the cell.
  • the AAV9 control construct included a polynucleotide sequence encoding the wild type AAV9 capsid protein (e.g., SEQ ID NO: 61). This polynucleotide was then encapsulated in an AAV virion that included an AAV9 capsid protein.
  • the construct included a polynucleotide sequence encoding the AAV mut1 capsid protein (e.g., SEQ ID NO: 163). This polynucleotide was then encapsulated in an AAV virion that included an AAV mut1 capsid protein.
  • each construct included polynucleotide sequence encoding a modified AAV capsid protein. This polynucleotide was then encapsulated in an AAV virion that included the same modified AAV capsid protein.
  • An AAV library was generated by transfecting the pooled plasmid library into HEK293 cells together with an adenoviral helper gene plasmid construct and a plasmid containing the AAV rep expression cassette. After AAV production, the AAV library was assessed for its diversity using sequencing (i.e., next-generation sequencing using Illumina reagents and hardware). DNA was isolated from viral supernatant and assessed for the Atty Docket No.: 38053-57988/WO (112WO) presence of each of the 3,456 modified capsids based on the presence of the polynucleotide sequence encoding the same modified capsid variant present in the AAV virion.
  • sequencing i.e., next-generation sequencing using Illumina reagents and hardware.
  • mRNA based-detection of a modified capsid protein using NGS was more specific and effective to determine a virus particle capable of functionally transducing a cell because it was based on the functional product produced from the AAV genome. Viral DNA using NGS was also used to detect the presence of a virus particle in the cell.
  • Experimental design [00627] Experimental designed for testing the AAV -Lib1 library is as described in the Table 16 below. Group 1 included a control mouse injected with vehicle (1X PBS, 35mM NaCl, 0.001% pluronic) alone. For Group 2, 5x10 13 gc/kg of AAV -Lib1 Library was administered intravenously to each 10 week old C57BL/6 mice.
  • RNA For DNA, tissues were homogenized in a Atty Docket No.: 38053-57988/WO (112WO) Qiagen Tissuelyser II (20rps for 2 min) in lysis buffer from the Qiagen Dneasy Blood and Tissue Kit or the Qiagen RNeasy Lipid Tissue Mini Kit following the standard Qiagen protocol. Samples were eluted in 50uL of buffer. For RNA, tissues were immediately placed into the preservative RNAlater, after which the RNAlater was removed and the tissue flash frozen. Variations to these general protocols are as described below.
  • NGS Next generation sequencing
  • AAV vector genomes i.e., DNA
  • AAV components i.e., viral RNA
  • SeqCount QC i.e., quality control of sequencing reads
  • Variant Counts represent the total Variant Count (i.e., sequencing reads associated with a particular capsid variant) in a sample or input test article. Prior to determining tissue enrichment, Variant Count data was normalized.
  • Step 1 included sequencing depth normalization: ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ [00631]
  • tissue enrichment measurements were used for functional interpretation of the data as well as for variant ranking and selecting capsids from the AAV -Lib1 library.
  • tissue enrichment analysis identified numerous peptides segments having the desired tropism (e.g., decreased liver tropism and increased muscle tropism (or do not effect Atty Docket No.: 38053-57988/WO (112WO) reduced muscle tropism as compared to a control capsid, such as the reference capsid (e.g., AAV9)).
  • the tissue enrichment scores were also used for sequence activity relationship (SAR) analysis, network analysis, and structural modeling.
  • SAR sequence activity relationship
  • Sequence activity relationship (SAR) analysis of the AAV -Lib1 data was used to identify particular amino acid residues at specific amino acid positions and/or peptide segments (i.e., combinations of specific amino acid residues at specific amino acid positions) within VRI of the AAV capsid protein that significantly impact tissue tropism. Tissue enrichment scores from Table 28 were used for SAR analysis.
  • Example 13 provides an example SAR analysis performed using the AAV -Lib1 data that identified particular amino acid residues at specific amino acid position and/or peptide segments within VRI of the AAV capsid proteins that significantly impacted tissue tropism (see, e.g., peptide segments 1-5 corresponding to SEQ ID NO: 44921-44926).
  • AAV -Lib1 data was used to identify AAV capsid variants (from the 3,456 capsids of AAV -Lib1 ) that shared amino acid residues at specific amino acid positions and/or peptide segments within VRI based. Tissue enrichment scores from Table 28 were used for the network analysis. For example, network analysis was used to identify the peptide segments (i.e., amino acid sequences) within VRI for the top performing capsids from the AAV -Lib1 library data (i.e., the AAV -Lib1 tissue enrichment data provided in Table 28).
  • tissue enrichment scores were used along with SAR analysis, network analysis, and structural modeling to interpret the data generated by the AAV -Lib1 library (e.g., data provided in Table 28).
  • the Examples below show how analysis of tissue enrichment Atty Docket No.: 38053-57988/WO (112WO) scores, SAR analysis, network analysis, and structural modeling were used to identify particular amino acid residues at particular amino acid positions that impact tropism. 8.18.
  • Example 18 Analysis of AAV -Lib1 data showed capsid variants with enhanced muscle tropism with limited liver tropism in C57BL/6 mice
  • NGS was performed on DNA and/or RNA harvested from mouse tissues 28 days after IV injection of the AAV -Lib1 library according to the methods described herein.
  • gene transfer efficacy of each AAV within the AAV -Lib1 library was analyzed in liver and muscle (quadriceps) harvested 28 days after administration.
  • DNA and RNA were isolated as described above and NGS was performed to determine levels of expression (RNA) and vector genomes (DNA) present in liver and muscle.
  • FIG.50 shows DNA levels of each capsid variant from the AAV -Lib1 library (variant ID on x-axis) in the liver represented as Log2 Fold Change over test article (see AAV- Lib1 Library Screen Data Informatics Analysis in Example 11 above forLog2Fold Change calculation).
  • the AAV -Lib1 library included AAV mut1 and wild type AAV9 as controls.
  • AAV mut1 had lower liver tropism than wild type AAV9 (as described in PCT Application No.
  • FIG.50 shows the AAV -Lib1 library includes modified capsid proteins having a range of liver tropic capsid variants.
  • the AAV -Lib1 library includes modified capsids having one or more amino acid modifications in VR I that decrease liver tropism.
  • Applicant hypothesized that one or more of these modified capsid proteins would not only reduce liver tropism but also potentially increase muscle tropism (or do not effect reduced muscle tropism as compared to a control capsid, such as the reference capsid).
  • the AAV -Lib1 library includes capsid having an alanine (A) or a glycine (G) at amino acid position P6 (FIG.51A). This includes the alanine (A) found in the control AAV mut1 capsid (e.g., G266A) and the guanine (G) found in AAV9 (e.g., G266). Twenty-eight days after injection, DNA was isolated from mouse liver according to the methods described herein and sequenced to determine the amount of DNA from each capsid variant from AAV -Lib1 present in the liver.
  • an alanine (A) at P 6 resulted in less AAV DNA in the liver than compared to when a glycine (G) was present at P6 (amino acid position 266).
  • SAR could be used to identify amino acid residues in VRI that reduce liver tropism—an integral step in optimizing capsid amino acid sequences for suitability as a gene therapy vector.
  • modification to an alanine (A) or a glutamate (E) at P 3 reduce liver tropism as compared to capsids having a glutamine (Q) or a threonine (T) at P3.
  • Amino acid modifications that were tested at P 5 included an alanine (A) and a glycine (G).
  • modification to an alanine (A) at P 5 reduced liver tropism compared to capsids having a glycine (G) at P5.
  • Amino acid modifications that were tested at P6 included alanine (A) and glycine (G).
  • FIG.53A confirms previous SAR analysis showing that an analysis of all capsids in AAV -Lib1 having an alanine (A) at P 6 decreased liver tropism Atty Docket No.: 38053-57988/WO (112WO) (compared to capsids having a glycine (G) at P6).
  • FIG.53B illustrates how different combinations of amino acid substitutions at P5 and P6 impact liver tropism.
  • An analysis of all capsids in the AAV -Lib1 library having a P 5 P 6 combination of AA, AG, and GA showed that these combinations reduced liver tropism compared to a P5P6 combination of GG.
  • FIG.53C provides an analysis of all capsids in the AAV -Lib1 library having a P 3 , P 5 , and P 6 combination of: AAA, AAG, AGA, AGG, EAA, EAG, EGA, EGG, QAA, QAG, QGA, QGG, TAA, TAG, TGA, and TGG (appearing from left to right on the x-axis in FIG. 53C).
  • this Example showed that sequence activity relationship (SAR) analysis of the AAV -Lib1 library data could be used to determine which amino acid modifications at which residues or which peptide segments in VRI impact liver tropism.
  • SAR sequence activity relationship
  • Example 20 Network analysis and structural modeling of AAV -Lib1 library data identified positions P 3 , P 5 , and P 6 as important for liver tropism
  • Network analysis of AAV -Lib1 library data identified P 3 , P 5 , and P 6 as important for liver tropism.
  • network analysis revealed that modified capsid proteins having a VRI region having a threonine (T) at P3, a glycine (G) at P5, and a glycine (G) at P 6 were enriched in capsid variants from the AAV -Lib1 library that exhibited greater liver tropism than AAV9.
  • FIG.54 where each circle indicates a capsid variant and a line connecting the capsids indicates sequence similarity, network analysis revealed, for example, that NSTSGGSTNDNT (SEQ ID NO: 1351) had greater liver tropism than AAV9.
  • the network analysis supported the structural modeling data in FIG.55.
  • P3, P5, P6, and P8 correspond to amino acid residues TGGT, respectively, where modelling predicted that P 3 T is involved in internal stabilization, P 5 G and P 6 G maintain loop flexibility, and P 7 and P 8 form direct Hydrogen bonds with AAV receptor (AAVR), where a P8T resulted in 2 more Hydrogen bonds than other amino acids at P8.
  • AAVR AAV receptor
  • network analysis and structural modeling of the AAV -Lib1 library data provided an independent means by which to identify amino acid modifications within VRI that impact liver tropism.
  • the overall aim of the AAV -Lib1 library study was to identify AAV capsid proteins that decrease liver tropism and increase muscle tropism (or do not effect reduced muscle tropism as compared to a control capsid, such as the reference capsid (e.g., AAV9))). Therefore, additional analysis (see Examples 21 and 22) was performed to determine which peptide segments harbored the desired tropism. 8.21.
  • Example 21 Analysis of RNA levels from AAV -Lib1 showed enrichment variation in liver and heart tropism based on amino acid modificationP 3 , P 5 , and P 6 [00650] With one of the aims of the AAV -Lib1 library study to identify AAV capsid proteins that decrease liver tropism and increase muscle tropism (or at least have comparable muscle tropism to a control (e.g., AAV9)), expression data from mouse heart and mouse liver were analyzed to identify peptide segments that harbored the desired tropism. [00651] For expression analysis, NGS was performed on RNA harvested from mouse tissue following IV infusion of the AAV -Lib1 library.
  • RNA isolated from liver and heart revealed that particular AAV capsids from the AAV -Lib1 library exhibited residue-specific variation in tropism based on the particular amino acid modifications at P3, P5, and/or P6. In some instances, the amino acid residues that showed increased muscle tropism also exhibited liver tropism.
  • Example 22 Sequence Activity Relationship (SAR) analysis comparing heart and liver data for amino acid modifications at P 3 , P 5 , and P 6 showed specific amino acid combinations enhanced muscle tropism but reduced liver tropism
  • SAR Sequence Activity Relationship
  • FIG.57A shows enrichment in liver DNA (x-axis) plotted against enrichment in liver RNA (y-axis) for capsids comprising various combination of amino acid modifications at P3, P5, and P6.
  • FIG.57A shows a range of liver detargeting (i.e., capsids having a TGG at positions P 3 , P 5 , and P 6 , respectively) whereas most other sequence combinations detarget the liver.
  • FIG.57B shows enrichment in liver DNA (x-axis) plotted against enrichment in heart RNA (y-axis) for capsids comprising various combination of amino acid modifications at P 3 , P 5 , and P 6 .
  • some sequence combinations, such as those including QGG and TGG were enriched in both liver and muscle.
  • FIG.58A shows enrichment in liver DNA (x-axis) plotted against enrichment in liver RNA (y-axis) for AAV capsids having a TGG combination of amino acid modifications at P 3 , P 5 , and P 6 , respectively.
  • Overlaying the data for AAV mut1 and AAV9 onto the plot in FIG.58A shows AAV mut1 liver tropism compared to AAV9 liver tropism and the liver tropism for a subset capsid variants in the AAV -Lib1 library.
  • FIG.58B shows enrichment in liver DNA (x-axis) plotted against enrichment in heart RNA (y-axis) for AAV capsids having a threonine (T), a glycine (G), and a glycine (G) at P3, P5, and P6, respectively.
  • T threonine
  • G glycine
  • G glycine
  • modified capsid proteins having a threonine (T) at P1 exhibited heart enrichment levels below that of the control AAV mut1 (FIG.58C, yellow dots).
  • modified capsid proteins having an asparagine (N) or a serine (S) at P 1 exhibited increased heart and increased liver enrichment compared to AAV mut1 (FIG.58C, blue dots).
  • FIG.58D shows enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid residues at P 3 , P 5 , and P 6 , respectively.
  • T threonine
  • G glycine
  • G glycine
  • Capsids comprising a peptide segment having a TGG at P3, P5, and P6 and SGAT at positions P 1 , P 2 , P 4 , and P 12 were identified as capsids of interest based on mid to low levels of liver tropism (compared to AAV9) and increased muscle tropism (compared to AAV9). Positions of AAV mut1 and AAV9 controls are indicated by arrows and corresponding text.
  • FIG.58E shows enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for capsids having a threonine (Q), a glycine (G), and a glycine (G) combination of amino acid residues at P3, P5, and P6, respectively.
  • Particular subpopulations having SGTH at positions P 1 , P 2 , P 4 , and P 12 are identified.
  • Capsids comprising a peptide segment having a TGG at P 3 , P 5 , and P 6 and SGTH at positions P 1 , P 2 , P 4 , and P 12 were identified as capsids of interest based on mid to low levels of liver tropism (compared to AAV9) and increased muscle tropism (compared to AAV9). Positions of AAV mut1 and AAV9 controls are indicated by arrows and corresponding text.
  • FIG.58F shows average enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for all AAV-Lib1 capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid residues at P3, P5, and P6, respectively. Positions of AAVmut1 and AAV9 controls are indicated by corresponding text.
  • FIG.58G shows enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for 24 selected capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid residues at P 3 , P 5 , and P 6 , respectively.
  • T threonine
  • G glycine
  • G glycine
  • the 24 selected capsids correspond to groups: 1 NSTSGGP7P8NDNH (e.g., SEQ ID NO: 44927); group 2: NSTTGGP 7 P 8 NDNH (SEQ ID NO: 44928); group 4: SGTAGGP 7 P 8 NDNT (SEQ ID Atty Docket No.: 38053-57988/WO (112WO) NO: 44930); group 5: SGTSGGP7P8NDNA (SEQ ID NO: 44931); group 6: SGTTGGP7P8NDNT (SEQ ID NO: 44932); and group 7: SSTAGGP7P8NDNA (SEQ ID NO: 44933).
  • NSTSGGP7P8NDNH e.g., SEQ ID NO: 44927
  • group 2 NSTTGGP 7 P 8 NDNH (SEQ ID NO: 44928);
  • group 4 SGTAGGP 7 P 8 NDNT (SEQ ID Atty Docket No.: 38053-57988/WO (112
  • FIG.58G shows that capsids comprising peptides segments sequences in groups 1 to 7 (i.e., SEQ ID NOs: 44927-44933) have detarget the liver while not effecting significant reduction muscle tropism.
  • 3X reduction in muscle (e.g., heart) tropism corresponds to 100X detargeting in the liver.
  • FIG.58H and FIG.58I shows enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for 6 groups where each group has a threonine (T), a glycine (G), and a glycine (G) combination of amino acid residues at P 3 , P 5 , and P 6 , respectively.
  • T threonine
  • G glycine
  • G glycine
  • AAV capsids comprising peptide segments having sequences in groups 1 to 7 (i.e., SEQ ID NOs: 44927-44933) include capsids having the desired tropism. 8.23.
  • Example 23 Summary of Mouse data [00662] Overall, the AAV -Lib1 library data showed that AAV capsids comprising particular peptide segments in variable region I (VRI) had decreased liver tropism and increased muscle tropism (or at least had comparable muscle tropism to the AAV9 control). For example, Table 17 lists data for 28 AAV capsids selected from the AAV -Lib1 library data (see Table 28) with each AAV capsid having a different peptide segment within VRI.
  • VRI variable region I
  • AAV capsids met the criteria of having reduced liver tropism and had increased muscle tropism (or do not effect reduced muscle tropism as compared to a control capsid, such as the reference capsid (e.g., AAV9)).
  • the Liver DNA, Liver RNA, Heart DNA, and Heart RNA values in Table 10 and Table 28 are tissue enrichment scores shown as Log2Fold Change (FC) to test article.
  • FC Log2Fold Change
  • Tissue enrichment data for AAV capsids from AAV -Lib1 Library comprising the indicated peptide segment in VRI SEQ ID Sequence (peptide Liver Liver Heart Heart variant_id NO: segment) DNA RNA DNA RNA ATLVT012XX1092 46026 NSTSGGASNDNH 7.98 9.47 1.07 3.41 ATLVT012XX1095 46029 NSTSGGATNDNH 8.8 10.51 1.1 4.12 ATLVT012XX1097 46031 NSTSGGSSNDNH 8.46 9.88 1.13 3.37 ATLVT012XX1100 46034 NSTSGGSTNDNH 9.42 10.77 0.79 3.51 ATLVT012XX1139 46073 NSTTGGASNDNH 5.29 6.39 0.68 3.29 ATLVT012XX1142 46076 NSTTGGATNDNH 7.26 8.16 0.93 4.25 ATLVT012XX1145 46079 NSTTGGSSNDNH 7.06 7.38 0.79 3.51 ATLVT012X
  • Tissue enrichment data for AAV capsids from AAV -Lib1 Library comprising the indicated peptide segment in VRI SEQ ID Sequence (peptide Liver Liver Heart Heart variant_id NO: segment) DNA RNA DNA RNA ATLVT012XX1571 46505 SGQTGGASNDNH 6.54 8.15 1.31 3.5 ATLVT012XX1574 46508 SGQTGGATNDNH 9.6 12.38 0.01 4.01 ATLVT012XX1577 46511 SGQTGGSSNDNH 3.37 4.17 1.49 2.66 ATLVT012XX1580 46514 SGQTGGSTNDNH 8 8.23 0.82 4.32 ATLVT012XX1620 46554 SGTAGGASNDNT 6.3 9.6 0.57 4.51 ATLVT012XX1626 46560 SGTAGGSSNDNT 5.86 5.69 2.35 4.55 ATLVT012XX1666 46600 SGTSGGASNDNA 4.83 5.82 2.01 3.56 ATLVT012
  • Example 24 Analysis of AAV- Lib1 in non-human primate to identify VRI variants with enhanced muscle tropism with limited liver tropism
  • the objective of this study was to assess the 3,456 modified capsid proteins from the AAV -Lib1 in non-human primates (NHP) to identify capsids having VRI variants that enhance muscle tropism and limit liver tropism.
  • NHPs were administered the same AAV -Lib1 library as described in Example 15. Briefly, a library containing 3,456 unique sequences of modified AAV9 VP1 capsid proteins was produced, which is referred to herein as the AAV -Lib1 library or AAV -Lib1 capsid library.
  • the peptide segment of 12 amino acids positioned between S261 and Y274 for each of the 3,456 modified AAV9 VP1 capsid sequence are as described in SEQ ID NOs: 251- 3703.
  • the amino acid modifications (insertions, deletions, substitutions) in the VRI region of AAV9 included those described herein as shown in FIG.49A.
  • wild-type Atty Docket No.: 38053-57988/WO (112WO) AAV9 and AAV mut1 capsid proteins are used as controls. Wild-type AAV9 is known to target the liver whereas AAV mut1 has a liver detargeting phenotype.
  • Experimental design [00665] A total of 6 animals were divided into 2 groups as summarized in the below Table 18.
  • ATV-0071-001 and ATV-0071-002 represent different lots of the AAV -Lib1 library. Immunosuppression of the animals began 7 days prior to vector administration. Group 1 and 2 were administered with the indicated amounts or concentrations of AAV- Lib1 (comprising the 3,456 modified capsid proteins, an AAV9 capsid protein, AAV mut1 capsid protein) by ICM or IV. Animals were sacrificed on day 32 after AAV vector administration and their organ samples are collected for analysis. Table 18. Experimental design for non-human primate study. Dose Study end point Group Dose Dose (vg / Dose N o.
  • tissues were homogenized in a Qiagen Tissuelyser II (20rps for 2 min) in lysis buffer from the Qiagen Dneasy Blood and Tissue Kit or the Qiagen RNeasy Lipid Tissue Mini Kit following the standard Qiagen protocol. Samples were eluted in 50uL of buffer.
  • RNA tissues were immediately placed into the preservative RNAlater, after which the RNAlater was removed and the tissue flash frozen. Variations to these general protocols are as described below.
  • NGS Next generation sequencing
  • AAV vector genomes i.e., DNA
  • AAV components i.e., viral RNA
  • SeqCount QC i.e., quality control of sequencing reads
  • Table 30 reports tissue enrichment scores for Liver DNA, Heart DNA, and Diaphragm DNA as Log2 Fold Change (FC) to test article.
  • the tissue enrichment measurements were used for functional interpretation of the data as well as for variant ranking and selecting capsids from the AAV- Lib1 library.
  • DNA samples were analyzed for biodistribution of vector genomes in the liver, diaphragm, and hear using NGS.
  • Data i.e., AAV -Lib1 library data
  • AAV mut1 and AAV9 is found in Table 30, which is herein incorporated by reference in its entirety.
  • Table 30 shows tissue enrichment scores Log2 Fold Change (Fc) compared to test article.
  • top 200 modified AAV capsids from Table 30 are shown in Table 20. Atty Docket No.: 38053-57988/WO (112WO) Table 19. Top 200 Modified AAV Capsids from Appendix E. (AAV-Lib1 in NHP) SEQ Diaphrag Rank variant_id ID NO: Sequence m Heart Liver 1 ATLVT012XXAAV9 48388 NSTSGGSSNDNA 11.26 21.08 19.61 2 ATLVT012XX1099 46033 NSTSGGSTNDNA 8.69 11.7 18.88 3 ATLVT012XX1094 46028 NSTSGGATNDNA 9.77 11.36 16.69 4 ATLVT012XX1100 46034 NSTSGGSTNDNH 8.96 12.75 16.17 5 ATLVT012XX1097 46031 NSTSGGSSNDNH 8.93 11.1 17.04 6 ATLVT012XX1098 46032 NSTSGGSSNDNT 7.1 12.65 17.96 7 ATLVT012X1054 4
  • AAV-Lib1 in NHP SEQ Diaphrag Rank variant_id ID NO: Sequence m Heart Liver 4 2 ATLVT012XX1529 46463 SGQSGGSSNDNH 10.13 6.32 9.6 43 ATLVT012X1666 46600 SGTSGGASNDNA 8.04 8.48 10.31 44 ATLVT012XX1143 46077 NSTTGGATNDNT 6.1 7.87 14.37 45 ATLVT012XX1580 46514 SGQTGGSTNDNH 7.4 9.22 10.25 46 ATLVT012XX1574 46508 SGQTGGATNDNH 6.97 7.32 13.42 47 ATLVT012XX2054 46988 SSQAGGATNDNH 5.35 9.86 12.93 48 ATLVT012XX2251 47185 SSTSGGSTNDNA 5.82 10.22 11.43 49 ATLVT012XX2248 47182 SSTSGGSSNDNA 7.39 9.43 9.71
  • AAV-Lib1 in NHP SEQ Diaphrag Rank variant_id ID NO: Sequence m Heart Liver 8 3 ATLVT012XX1528 46462 SGQSGGSSNDNA 7.93 6.53 8.46 84 ATLVT012XX1382 46316 SGESGGATNDNH 8.11 6.14 8.62 85 ATLVT012XX3395 48329 TSTSGGASNDNH 4.27 10.33 9.68 86 ATLVT012XX1523 46457 SGQSGGASNDNH 5.7 7.33 10.93 87 ATLVT012XX960 45894 NSQSGGSTNDNT 8.43 5.47 8.52 88 ATLVT012XX1238 46172 SGASGGATNDNH 5.37 7.89 10.65 89 ATLVT012XX2300 47234 SSTTGGSTNDNH 7.18 8.2 7.07 90 ATLVT012XX1714 46648 SGTTGGASNDNA 6.11
  • AAV-Lib1 in NHP SEQ Diaphrag Rank variant_id ID NO: Sequence m Heart Liver 1 24 ATLVT012XX573 45507 NGTTGGSSNDNT 4.16 7.38 10.63 125 ATLVT012XX1430 46364 SGETGGATNDNH 4.68 7.69 9.3 126 ATLVT012XX2247 47181 SSTSGGATNDNT 3.72 6.99 11.63 127 ATLVT012XX1627 46561 SGTAGGSTNDNA 4.84 5.43 11.7 128 ATLVT012XX1725 46659 SGTTGGSTNDNT 3.93 7.56 10.49 129 ATLVT012XX2293 47227 SSTTGGATNDNA 4.5 9.42 6.94 130 ATLVT012XX3302 48236 TSQTGGATNDNH 3.59 6.48 12.1 131 ATLVT012XX1721 46655 SGTTGGSSNDNH
  • AAV-Lib1 in NHP SEQ Diaphrag Rank variant_id ID NO: Sequence m Heart Liver 1 65 ATLVT012XX1196 46130 SGAAGGSTNDNH 2.79 7.46 10.12 166 ATLVT012XX1573 46507 SGQTGGATNDNA 7.22 3.74 7.2 167 ATLVT012XX2296 47230 SSTTGGSSNDNA 6.39 5.03 6.97 168 ATLVT012XX569 45503 NGTTGGATNDNH 5.06 5.79 8.16 169 ATLVT012XX1235 46169 SGASGGASNDNH 6.83 6.1 4.67 170 ATLVT012XX958 45892 NSQSGGSTNDNA 7.97 5.14 3.95 171 ATLVT012XX2109 47043 SSQSGGSTNDNT 6.08 6.47 5.47 172 ATLVT012X567 45501 NGTTGGASNDNT 3.
  • the data in Table 30 was used to identify interesting peptide segments, for example, peptide segments with liver enrichment between AAV9 and AAV9-mut 1 (see FIG. 59).
  • enrichment scores for each tissue were calculated for each AAV variant containing a unique targeting peptide.
  • the AAV variants including each targeting peptide were ranked based on a mean log fold-change tissue score (see, e.g., Table 30).
  • FASTQ sequence read data
  • Tissue enrichment analysis included Sequence Activity Relationship (SAR) analysis, Network analysis, and structural modeling.
  • SAR analysis identified particular amino acid at a specific capsid position or sequence motifs (combination of amino acids at several positions) that significantly affects tissue tropism.
  • Network analysis identified modules of variants that shared amino acid or peptide sequences of top performing variants.
  • Structural modeling provided understanding of the structural effect of significant amino acids at specific capsid positions for mechanistic hypothesis formulation.
  • SAR analysis was performed on the peptide segments having liver enrichment between AAV9 and AAV9-mut1. As shown in FIG.60, this identified sequence motifs at four different positions. SAR analysis was reproducible (i.e., showed good correlation) between NHP and mice (see FIG.61).
  • Example 25 Assessment of AAVs having capsids a targeting peptide in VR VIII and a peptide segment within VR I in mice and non-human primates
  • SAR sequence activity relationship
  • the objective of this study was to assess AAVs comprising a capsid protein having a targeting peptide in VR VIII and a tropism-altering peptide segment within VR I in non-human primates (NHP) to identify heart neutral, liver detargeted, AAVs (e.g., moderate to low liver expression compared to a control (e.g., AAV9)) with potential for muscle targeting.
  • SAR sequence activity relationship
  • the modified AAV capsid protein included peptide segments (e.g., the peptide segments described in Table 20) positioned between S261 and Y274 in VR I of AAV9.
  • 28 peptide segments were selected based at least in part, on the data in Table 30, which is herein incorporated by reference in its entirety, including amino acid sequences selected from SEQ ID NO: 46026, 46029, 46031, 46035, 46073, 46076, 46079, 46082, 46505, 46508, 46511, 46514, 46554, 46560, 46609, 46600, 46603, 46606, 46650, 46653, 46656, 46659, 47128, 47131, 47134, and 47137.
  • the 28 peptide segments can be divided into seven groups that are represented by SEQ ID NOs: 44927-44933, where each SEQ ID NO represents one of the seven groups.
  • Six of the seven group include a threonine (T), a glycine (G), and a glycine (G) at positions P 3 , P 5 , and P 6 , respectively, within the peptide segment.
  • One group includes a glutamine (Q), a glycine (G), and a glycine (G) at positions P3, P5, and P6, respectively, within the peptide segment.
  • amino acid position P 7 is varied between an alanine (A) and a serine (S) and amino acid position P8 is varied between a serine (S) and a threonine (T).
  • SEQ ID NOs: 44927-44933 Four of the seven groups (i.e., SEQ ID NOs: 44927-44933) were selected for testing in a modified AAV capsid protein that also includes a targeting peptide.
  • the eight targeting peptides selected for testing in this example included targeting peptides having an amino acid sequence selected from SEQ ID NO: 44864-44867, 44879-44883, 44911, 44912, 44913, 44918, 44919.
  • SEQ ID NO: 44864-44867, 44879-44883 44911, 44912, 44913, 44918, 44919.
  • AAV9 capsids were modified to include (i) the targeting peptide at a site within VR VIII of the reference AAV capsid protein and (ii) one or more modifications to comprise a peptide segment within variable region I (VR I) of the reference AAV capsid protein as described in Table 20.
  • Table 20 Combinations of Targeting Peptides and Peptide Segments Table 20.
  • AAV -mini library This library is referred to herein as the AAV -mini library or AAV -mini capsid library. Additionally, wild-type AAV9 and AAV mut1 capsid proteins were used as controls. Wild-type AAV9 is known to target the liver whereas AAV mut1 has a liver detargeting phenotype.
  • the AAV -mini library was assessed in mice and non-human primates (NHPs). 8.25.1.
  • NHPs were administered with the indicated amounts or concentrations of AAV -mini library (comprising the 116 modified capsid proteins of Table 20) by IV.
  • Table 21 Experimental design for non-human primate study. Table 21. Dose Study end point Group Dose Dose (vg / N o. Treatment (necrops Route Vol y day 28) animal or kg) (ml) 1 Mini-Library IV 10.0 5E13vg/kg 3 [00680] Animals were sacrificed on day 28 after AAV vector administration and heart, liver, diaphragm, and quadricep samples were collected for analysis. Tissues were immediately placed into the preservative RNAlater, after which the RNAlater was removed and the tissue flash frozen.
  • RNA was isolated from the tissue samples and sequenced to identify the AAVs (AAVs from AAV -mini library, each AAV containing a unique targeting peptide in VR VIII and a unique peptide segment in VR I) localized in each tissue. Using the sequence data, enrichment scores for each tissue were calculated for each AAV variant (calculated as described in Example 11). After determining a sequence count from sequence read data (FASTQ) following sequencing, AAV variants were counted and normalized. The AAV variants were assigned tissue enrichment scores for each individual tissue analyzed (i.e., heart, liver, diaphragm, and quadriceps, biceps, gastrocnemius, and Tibialis). Tissue enrichment data for the AAV -mini library in NHP is shown in Table 23 with tissue enrichment scores in columns F-L for the respective tissues.
  • AAV capsids with “variant-ids” ATLVT019XX10 to ATLVT019XX20 exhibited the greatest reductions in liver tropism and the greatest increases in muscle tropism (or at least not effecting significant reduction in muscle tropism).
  • DNA samples are analyzed for biodistribution of vector genomes in the liver and quadriceps tissue using NGS. All analytical work is conducted, using an analytical method developed and qualified by that laboratory. Samples were collected at 4 weeks pre-dose and on day 28 following administration. 8.25.2.
  • AAV -mini library in mice Experimental design [00683] A total of 5 C57BL/6 mice were used for this study according to the experimental design in Table 23.
  • mice were administered with the indicated amounts or concentrations of AAV -mini library (comprising the 116 modified capsid proteins of Table 20) by intravenous tail vein injection.
  • Table 23 Experimental design for mouse study. Study end point Group Dose Dose D N o. Treatment Route Vol ose (vg / (necropsy day 28) (ml) animal or kg) 1 Mini-Library IV 10.0 5E13vg/kg 5 [00684] The mice were sacrificed 28 days after the injection. Individual tissues, notably the liver, heart, and diaphragm, were collected at the time of necropsy. Tissue were immediately placed into the preservative RNAlater, after which the RNAlater was removed and the tissue flash frozen.
  • VR VIII VR I SEQ SEQ ID variant_id VR1_ mutation ID NO: VR8_ insertion NO: Diaphragm Heart Liver ATLVT019XX67 NSTSGGSSNDNA 48388 ENRRGDFNNL 44864 0.21 0.50 1.49 ATLVT019XX68 NSTSGGSSNDNA 48388 SAQRGDRGVV 44918 0.18 -0.11 1.54 ATLVT019XX69 NSTSGASTNDNA 48390 ENRRGDFNNL 44864 -0.39 0.01 -0.38 ATLVT019XX7 SGTTGGSSNDNT 46656 ENRRGDFNNT 44880 0.99 0.96 0.34 ATLVT019XX70 NSTSGASTNDNA 48390 SAQRGDRGVV 44918 1.62 1.57 1.98 ATLVT019XX71 NSTSGGSSNDNA 48388 SAQRGDHVNL 44919 1.33 0.65 2.20 ATLVT019XX72 NSTSGASTNDNA 48390 SAQRGDHVNL 4
  • modified AAV capsids with “variant-ids” ATLVT019XX10 to ATLVT019XX20 exhibited the greatest reductions in liver tropism and the greatest increases in muscle tropism (or at least not effecting significant reduction in muscle tropism).
  • DNA samples are analyzed for biodistribution of vector genomes in the liver and quadriceps tissue using NGS. All analytical work is conducted using an analytical method developed and qualified by that laboratory. Samples were collected at 4 weeks pre-dose and on day 28 following administration. 8.26.
  • Example 26 Analysis of AAV capsids including X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 targeting peptides
  • the objective of this study was to assess the 50,500 modified AAV capsid proteins from AAV -Lib2 library in non-human primates (NHP) to identify capsids having targeting peptides that enhance muscle tropism.
  • NEP non-human primates
  • a library containing the 50,500 modified AAV capsid proteins was produced, which was referred to herein as the AAV -Lib2 library.
  • the library was constructed as described elsewhere herein.
  • the targeting peptides were positioned in VR VIII of AAV9 between amino acid residues 585 and 589, replacing amino acids 586, 587, and 588.
  • Targeting Atty Docket No.: 38053-57988/WO (112WO) peptides included the sequences as described in SEQ ID NOs:48391-98943 (see Table 29), which is hereby incorporated by reference in its entirety).
  • Controls include AAV9. See also FIGs.62A-62C.
  • Three animals were treated as summarized in the table below. The animals were administered with the indicated amounts or concentrations of AAV -Lib2 (comprising the about 50,500 modified capsid proteins and controls) (see Table 26 below) by IV. Table 26.
  • DNA samples will be analyzed for biodistribution of vector genomes in biceps femorris, quadriceps, diaphragm, heart aria, heart ventricle wall, and liver.
  • RNA samples were analyzed for vector expression in the each of the six tissue types collected at day 28 (biceps femorris, quadriceps, diaphragm, heart aria, heart ventricle wall, and liver).
  • AAV -Lib2 library data was analyzed as described elsewhere herein (see, e.g., Example 17 “AAV VRI (AAV -Lib1 ) Capsid Libraries”).
  • FIG.63A The AAV -Lib2 library data (see FIG.63A) was analyzed to determine how targeting peptides having a particular quad sequence performed.
  • FIG.63B shows performance of capsids having a targeting peptide where the targeting peptide had a quad selected from FNNL, FNNT, and RGQI.
  • AAV -Lib2 included targeting peptides having one of 20 different quads.
  • FIG. 64 shows the amount of RNA in muscle tissue for the sum of all the capsids in the AAV -Lib2 library having the indicated quad sequences.
  • Targeting peptides having these quad sequences corresponded to X 1 X 2 X 3 RGDYTSV (SEQ ID NO: 98941), X1X2X3RGDYTSM (SEQ ID NO: 98942), X1X2X3RGDRGVV (SEQ ID NO: 98928), X1X2X3RGDRSVV (SEQ ID NO: 98931), X1X2X3RGDYSSV (SEQ ID NO: 98937), and X1X2X3RGDHGVL (SEQ ID NO: 98938), respectively.
  • FIG.65 is a heatmap showing enrichment scores averaged across all muscle tissues for each of the capsids in the AAV -Lib2 library. Hierarchical clustering of the data revealed at least three groups of capsids performed similarly: Group 1, Group 2, and Group 3 (see FIG.65). Data associated with FIG.65 can be found in Table 29.
  • Targeting peptides with quads in group 2 have enhanced muscle tropism when combined with certain X1X2X3, e.g., APW, TEL, TDA, QPY, SPN, EHY, DWK, DLK, DFK, DVK, NSI, DIR, SPF, SEL, DRT, DRF, ADL, TDL, SDL, DNY, DKI, NDV, DKM, DNH, DNF, DSS, EST, EWT, DKN, DKS, SEH, ESQ, ESL, QND, EAH, AIF, AVF, QVF, TMY, ALY, NNG, NIF, NTF, NFF, AWF, NPY, SWF, AII, AYF, AQW, NFY, AGP, QQF, TKE, TNG, NSF, NAW, QAG, ERG, NKD, QSG, QNG, EAK, QWF, SWY, TFF, TYF
  • Targeting peptides with quads in group 3 have enhanced muscle tropism when combined with certain X 1 X 2 X 3 e.g., DMK, ATD, EEK, QMD, EFS, ERD, DDR, TDM, SAE, EHS, ENH, SWE, SNE, NNG, QAG, ERG, QSG, QNG, ASG, QFG, AMF, ELR, NFM, NNS, NNI, SDR, EQR, EHR, EWR, EQK, ESR, EKQ, EYR, ENR, EMK, EYK, EHK, EWK, QNI, TNI, TYI, SNY, DRQ, AWI, QWI, DFR, EKG, QYG, QWQ, EKN, EKF, EKT, AFH, ENK, NYS, DKH, AAG, QMF, QFH, QKD, ARE, AHQ, ADK, ADR, AHN, QNS
  • FIGs.66A-66C Analysis of capsids in Group 1 is shown in FIGs.66A-66C.
  • Group 1 included capsids comprising targeting peptides having a quad selected from: RGQI, RSVV, and RGVV.
  • FIG.66A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV -Lib2 library having a quad selected from: RGQI, RSVV, and RGVV.
  • the top 100 Atty Docket No.: 38053-57988/WO (112WO) capsids are identified by the larger of the two dots on the plot.
  • a motif analysis was performed to determine if there was a pattern of motifs enriched in the top 100 capsids in FIG.66A.
  • FIG.66B shows the motif plot that identifies the enriched motifs in the top 100. Additional analysis of the triplets for the top 100 capsids was provided in FIG.66C. For example, FIG. 66C shows the unique variant count of the triplet sequences present in the top 100 capsids in FIG.66A. Interestingly, this data shows that for Group 1 the X1 position favors amino acid residues D or E and the X 3 position favors amino acid residues Y, V, or F. [00697] Analysis of capsids in Group 2 is shown in FIGs.67A-67C. Group 2 included capsids comprising targeting peptides having a quad selected from: HGVL, YSSV, YSTM, and YTSV.
  • FIG.67A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV -Lib2 library having a quad selected from: HGVL, YSSV, YSTM, and YTSV.
  • the top 100 capsids are identified by the larger of the two dots on the plot.
  • a motif analysis was performed to determine if there was a pattern of motifs enriched in the top 100 capsids in FIG. 67A.
  • FIG. 67B shows the motif plot that identifies the enriched motifs in the top 100. . Additional analysis of the triplets for the top 100 capsids was provided in FIG. 67C.
  • FIG.67C shows the unique variant count of the triplet sequences present in the top 100 capsids in FIG.67A.
  • this data shows that for Group 2 the X 1 position favors amino acid residues S, A, or T and the X 3 position favors amino acid residues N, A, or Y.
  • FIGs.68A-68C Analysis of capsids in Group 3 is shown in FIGs.68A-68C.
  • Group 3 included capsids comprising targeting peptides having a quad selected from: HGVL, YSSV, YSTM, and YTSV.
  • FIG.68A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV -Lib2 library having a quad selected from: FNNT, FNNL, FQNT, and YNSL.
  • the top 100 capsids are identified by the larger of the two dots on the plot.
  • a motif analysis was performed to determine if there was a pattern of motifs enriched in the top 100 capsids in FIG. 68A.
  • FIG. 68B shows the motif plot that identifies the enriched motifs in the top 100. .
  • Additional analysis of the triplets for the top 100 capsids was provided in FIG. 68C.
  • FIG.68C shows the unique variant count of the triplet sequences present in the top 100 capsids in FIG.68A.
  • FIG.69 shows the amount of vector RNA in liver tissue for the sum of all the capsids in the AAV -Lib2 library having the indicated quad sequences. This analysis revealed that targeting peptides having RGVV, RSVV, RGQI, RSQT, and RQGI were the most liver tropic quads.
  • FIGs. 70A-70B Additional analysis of capsids with highest liver enrichment is shown in FIGs. 70A-70B.
  • enrichment scores averaged across all liver tissue for each of the capsids in the AAV -Lib2 library enabled identification of the top 100 liver tropic capsids.
  • the top 100 capsids are identified by the larger of the two dots on the plot (See FIG.70A).
  • a motif analysis was performed on the top 100 liver tropic capsids to determine if there was a pattern of motifs enriched in the top 100 capsids.
  • FIG.70B shows the enriched motifs in the top 100 capsids. Interestingly, this data shows that the triplets enriched in the muscle are different from the triplets enriched in the liver. 8.26.1.
  • FIG.71A shows enrichment score plots (inverse CV versus AverageMN_FC) for biceps femoris, quadriceps, diaphragm, heart – atria, heart – ventricle, and liver for each of the capsids in the AAV -Lib2 library.
  • the top 10 performing capsids are identified by the darker shaded circles. Tissue enrichment scores for each capsid in the tissues from FIG.71A are shown in FIG.
  • FIG.72A-72B looked at the how the triplets from the top 10 targeting peptides performed when combined with various quads.
  • FIG. 72A shows the enrichment scores for capsids having quads selected from RGQI, RGVV, and RSVV and targeting peptides having the triplets identified in the top 10 targeting peptides.
  • FIG.72B provides the amino acid residues for the relevant targeting peptides in FIG. 72A.
  • FIG.73A-73B looked at the how the triplets from the top 10 targeting peptides performed when combined with other quads.
  • FIG.73A shows the enrichment scores for capsids having quads selected from HGVL, YTSM, YSSV, and YTSV and targeting peptides having the triplets identified in the top 10 targeting peptides.
  • FIG.73B provides the amino acid residues for the relevant targeting peptides in FIG.73A.
  • FIG.74A-74B looked at how the top 10 targeting peptides performed compared to capsids : targeting peptide combinations identified in other studies (see, Example 25). As shown in FIG.74A, the top 10 targeting peptides performed similarly to previously identified targeting peptides when comparing enrichment in skeletal muscle versus heart. FIG.74B shows how the top 10 targeting peptides performed when comparing enrichment in muscle versus liver. Overall, this data showed that the current study could successfully identify targeting peptides with muscle tropism and that these new targeting peptides performed as well or better than targeting peptides identified elsewhere herein. 8.26.2.
  • the AAV -Lib2 library data was also analyzed to assess whether certain capsids had increased tropism for a particular subset of muscle tissue, for example, increased tropism in the heart over skeletal muscle or increased tropism in skeletal muscle over the heart.
  • the present study included capsids having a variety of tissue enrichment profiles as some capsids favored skeletal muscle over heart muscle and vice versa.
  • targeting peptides having quads selected from: QSTL (36653), LIGR (28422), and RGVV were the most “heart-specific” quads.
  • FIG.75B shows enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV -Lib2 library having the indicated quad (“LIGR”).
  • FIG.75C shows enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV -Lib2 library having the indicated quad (“QSTL”).
  • FIG.75D shows enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV -Lib2 library having the indicated quad (“RGVV”).
  • FIG.75E shows enrichment scores for heart over skeletal muscle for each of the capsids in the AAV -Lib2 library having the indicated quads. 8.26.3.
  • Conclusion [00712] Overall, this data shows that a subset of the modified AAV capsid comprising various targeting peptides tested in NHP have increased muscle tropism. Further analysis revealed that a subset of the targeting peptides were enriched in skeletal muscle over heart muscle and a second subset of the targeting peptides were enriched in heart muscle over skeletal muscle. 8.27.
  • Example 27 Assessment of AAVs having capsids a targeting peptide in VR VIII and a peptide segment within VR I in mice and non-human primates
  • the objective of this study was to assess AAVs comprising a capsid protein having a targeting peptide in VR VIII and a tropism-altering peptide segment within VR I in non-human primates (NHP) to identify modified AAV capsid proteins with potential for muscle targeting (e.g., cardiac tissue, skeletal muscle, or cardiac tissue and skeletal muscle).
  • muscle targeting e.g., cardiac tissue, skeletal muscle, or cardiac tissue and skeletal muscle.
  • FIGs.76A-76C The combination of targeting peptides and peptide segments tested in this example are as shown in FIGs.76A-76C.
  • the targeting peptides included a variable triplet, an RGD, and a variable quad (see FIG. 76-76C).
  • AAV-Lib3 library a library containing the 58,129 modified AAV capsid proteins was produced, which was referred to herein as the AAV-Lib3 library (“AAV -Lib3 library”).
  • the targeting peptides were positioned in VR VIII of AAV9 between amino acid residues 585 and 589, replacing amino acids 586, 587, and 588.
  • the peptide segments were positioned in VR I.
  • the amino acid sequences for the peptide segments in VR I corresponded to the 12 amino acids between S261 and Y274.
  • Controls included AAV9.
  • Three animals were treated as summarized in Table 25 below. Immunosuppression of the animals began 7 days prior to vector administration. The animals were administered with the indicated amounts or concentrations of AAV -Lib3 (comprising the about 58,129 modified AAV capsid proteins and controls) by IV. Atty Docket No.: 38053-57988/WO (112WO) Table 25. Experimental design for non-human primate study with AAV -Lib3 Library Study end point Group Dose Dose Vol Dose (vg / anim N o.
  • FIG. 77 shows the performance of capsids having a targeting peptide where the targeting peptide had a quad selected from ARTL, FNNL, FNNT, FQNT, HVNL, RGQI, RGVV, RNVV, RQGI, and RSVV, in combination with a VR1 modification selected from 47VR1, 49VR1, 50VR1 and Mut1.
  • FIGs.78A, 78B, and 78C show the enrichment scores in muscle versus liver for the capsids in the AAV -Lib3 library.
  • the data indicate that liver and muscle tropism of the capsids are decoupled.
  • several capsids are selected. The selection principles include: the capsid has the high enrichment score in muscle; the capsid has lower enrichment score in liver; the capsid has low variability across tissues and animals. The selected capsids are shown in Table 32.
  • FIG.79 shows the enrichment score in muscle tissue for the capsids in the AAV- Lib3 library having the indicated quad sequences and the indicated VR1 sequences.
  • FIG.80 shows that the performance values measured in the AAV -Lib2 library study (x-axis) or AAV -Lib3 library study (y-axis) are highly correlated, validating the experimental data.
  • FIG.81 is a heatmap showing enrichment scores averaged across all muscle tissues for each of the capsids in the AAV -Lib3 library.
  • Hierarchical clustering of the data revealed three quads clusters and four triplets clusters (see FIG.81). Data associated with FIG. 81 can be found in Table 31.
  • the quads cluster1 includes (VR1_quad): 47VR1_FNNL, 47VR1_FNNT, 47VR1_FQNT, and 49VR1_FNNL.
  • the quads cluster2 includes (VR1_quad): Mut1_ RSQT, Mut1_ RNVV, Mut1_ RGQI, Mut1_ RGVV, and Mut1_ RSVV.
  • the quads cluster3 includes (VR1_quad): Mut1_ VRTL, Mut1_ RQGI, Mut1_ ARTL, and Mut1_ RTNL.
  • the quads cluster1 have enhanced muscle tropism when combined with the triplets of triplets clusters 1-4.
  • the quads cluster2 have enhanced muscle tropism when combined with the triplets of triplets clusters 1-3.
  • the quads cluster3 have enhanced muscle tropism when combined with the triplets of triplets cluster 1.
  • the triplets cluster 1 includes: AAG, AAN, AAQ, AAS, AGA, AGG, AGH, AGM, AGN, AGP, AGQ, AGS, AGT, AGV, AKD, ANG, ASA, ASG, ASN, ASP, ASQ, ASS, EAY, EQY, NAG, NGA, NGE, NGF, NGG, NGI, NGL, NGM, NGN, NGP, NGQ, NGS, NGT, NGV, NGW, NGY, NNG, NNN, NQG, NQN, NRD, NSG, NWN, NWS, NYN, QAG, QAQ, QAS, QGA, QGG, QGH, QGM, QGN, QGP, QGQ, QGS, QGT, QGV, QKD, QSG, QSN, QSQ, QSS, SAG, SFG, SFN, SGA, SGE, SGG, SGH, SGI, SGL, SGM, SGN, AGP,
  • the triplets cluster 2 includes: AAA, AAH, AAY, AFG, AFN, AFQ, AFS, AFT, AHN, AKE, AMN, AMQ, ANN, ANS, AQQ, AQS, ARE, ATS, AWM, AWN, AWQ, AWT, AYN, DIR, DKA, DKG, DKI, DKM, DKN, DKQ, DKS, DKT, DRA, DRM, DRQ, DRS, DRT, DRV, DSM, DSS, DSV, DTR, DVR, ENN, ENS, ENT, ERM, NAN, NFN, NFQ, NFS, NFT, NGH, NKD, NMT, NQS, NSN, NSQ, NVN, NWA, NWQ, NWV, QAN, QAT, QAY, QFN, Atty Docket No.: 38053-57988/WO (112WO) QFQ, QFS, QFT, QFT,
  • the triplets cluster3 includes: AAI, AAL, AAM, AAT, AAV, ADL, ADR, AER, AGF, AGI, AGL, AGY, AHI, ALI, ALM, ALV, AMH, AMI, AML, AMM, AMT, ANA, ANF, ANI, ANM, ANQ, ANT, ANV, ANY, AQF, AQI, AQL, AQM, AQT, AQV, ASF, ASI, ASL, ASM, AST, ASV, ASY, ATI, ATL, ATM, ATQ, ATT, ATV, AWI, AYH, AYL, AYQ, DGR, DMR, DQR, DWK, EGR, NAF, NAI, NAL, NAM, NAQ, NAS, NAT, NAV, NAY, NDK, NEK, NER, NFI, NFL, NFM, NHI, NHL, NHM, NHN, NIM,
  • the triplets cluster4 includes: ADK, AFA, AFH, AFM, AGK, AHF, AHG, AHM, AHQ, AHS, AHT, AHY, AKY, ANH, ANP, AQH, ARI, ASH, ATA, ATG, AWA, AWP, AWS, AYS, AYT, DAR, DHA, DHS, DHT, DIS, DNK, DRG, DRH, DSR, DWR, DYN, DYR, DYS, EAR, EFR, EHH, EHK, EHN, EHR, EIR, EKA, EKE, EKG, EKH, EKI, EKL, EKM, EKN, EKQ, EKS, EKT, EKV, ELR, EMK, EMR, ENK, ENR, EQR, ERA, ERH, ERI, ERL, ERN
  • FIGs.82A-D show the motif plots that identify the enriched motifs in the AAV- Lib3 library having a VR1_quad selected from: 47VR1_FNNL, 47VR1_FNNT, 47VR1_FQNT, and 49VR1_FNNL (quads cluster 1).
  • the quads cluster 1 combined with triplets cluster 1 includes a targeting peptide having a sequence of X1X2X3RGDX7X8X9X10, wherein X 1 is independently selected from a serine (S), an asparagine (N), a threonine (T), an alanine (A), or a glutamine (Q); X 2 is independently selected from a glycine (G), a serine (S), or an alanine (A); X 3 is independently selected from a glycine (G), an asparagine (N), a serine (S), a glutamine (Q), an aspartic acid (D), a valine (V), a tyrosine (Y), a proline (P), an alanine (A), a threonine (T), or a methionine (M); X7 is a phenylalanine (F); X8 is independently selected from an aspara
  • the quads cluster 1 combined with triplets cluster 2 includes a targeting peptide having a sequence of X1X2X3RGDX7X8X9X10, wherein X 1 is independently selected from a threonine (T), a serine (S), a glutamine (Q), an alanine (A), an aspartic acid (D), or an asparagine (N); X 2 is independently selected from a phenylalanine (F), a tryptophan (W), an alanine (A), a lysine (K), a glutamine (Q), an asparagine (N), an arginine (R), a serine (S), a tyrosine (Y), a methionine (M), or a threonine (T); X 3 is independently selected from an asparagine (N), a serine (S), a glutamine (Q), a threonine (T), a t
  • the quads cluster 1 combined with triplets cluster 3 includes a targeting peptide having a sequence of X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 , wherein X 1 is independently selected from an asparagine (N), a serine (S), a threonine (T), an alanine (A), or a glutamine (Q); X2 is independently selected from an asparagine (N), a serine (S), a glutamine (Q), a threonine (T), a methionine (M), an alanine (A), a tyrosine (Y), or a leucine (L); X3 is independently selected from an isoleucine (I), a methionine (M), a leucine (L), a threonine (T), a valine (V), a glutamine (Q), or a tyrosine (Y); X 7 is a
  • the quads cluster 1 combined with triplets cluster 4 includes a targeting peptide having a sequence of X1X2X3RGDX7X8X9X10, wherein X1 is independently selected from a glutamic acid (E), a glutamine (Q), an alanine (A), a serine (S), a threonine (T), an asparagine (N), or an aspartic acid (D); X2 is independently selected from a histidine (H), a tyrosine (Y), a tryptophan (W), an arginine (R), a lysine (K), a phenylalanine (F), or an asparagine (N); X3 is independently selected from a histidine (H), an arginine (R), a serine (S), an alanine (A), a threonine (T), a methionine (M), a glycine (G),
  • FIGs.83A-C show the motif plot that identifies the enriched motifs in the AAV- Lib3 library having a VR1_quad selected from: Mut1_ RSQT, Mut1_ RNVV, Mut1_ RGQI, Mut1_ RGVV, and Mut1_ RSVV (quads cluster 2). As shown in FIG.
  • the quads cluster 2 combined with triplets cluster 1 includes a targeting peptide having a sequence of X1X2X3RGDX7X8X9X10, wherein X1 is independently selected from a serine (S), an asparagine (N), a threonine (T), an alanine (A), or a glutamine (Q); X 2 is independently selected from a glycine (G), a serine (S), or an alanine (A); X 3 is independently selected from a glycine (G), an asparagine (N), a serine (S), a glutamine (Q), an aspartic acid (D), a valine (V), a tyrosine (Y), a proline (P), an alanine (A), a threonine (T), or a methionine (M); X 7 is an arginine (R); X 8 is independently selected from a serine (S), a arginine
  • the quads cluster 2 combined with triplets cluster 2 includes a targeting peptide having a sequence of Atty Docket No.: 38053-57988/WO (112WO) X 1 X 2 X 3 RGDX 7 X 8 X 9 X 10 , wherein X 1 is independently selected from a threonine (T), a serine (S), a glutamine (Q), an alanine (A), an aspartic acid (D), or an asparagine (N); X 2 is independently selected from a phenylalanine (F), a tryptophan (W), an alanine (A), a lysine (K), a glutamine (Q), an asparagine (N), an arginine (R), a serine (S), a tyrosine (Y), a methionine (M), or a threonine (T); X3 is independently selected from an asparagine (N), a
  • the quads cluster 2 combined with triplets cluster 3 includes a targeting peptide having a sequence of X1X2X3RGDX7X8X9X10, wherein X1 is independently selected from an asparagine (N), a serine (S), a threonine (T), an alanine (A), or a glutamine (Q); X2 is independently selected from an asparagine (N), a serine (S), a glutamine (Q), a threonine (T), a methionine (M), an alanine (A), a tyrosine (Y), or a leucine (L); X3 is independently selected from an isoleucine (I), a methionine (M), a leucine (L), a threonine (T), a valine (V), a glutamine (Q), or a tyrosine (Y); X 7 is an arginine (R);
  • FIG.84 show the motif plot that identifies the enriched motifs in the AAV -Lib3 library having a VR1_quad selected from: Mut1_ VRTL, Mut1_ RQGI, Mut1_ ARTL, and Mut1_ RTNL (quads cluster 3).
  • the quads cluster 3 combined with triplets cluster 1 includes a targeting peptide having a sequence of X1X2X3RGDX7X8X9X10, wherein X1 is independently selected from a serine (S), an asparagine (N), a threonine (T), an alanine (A), or a glutamine (Q); X 2 is independently selected from a glycine (G), a serine (S), or an alanine (A); X 3 is independently selected from a glycine (G), an asparagine (N), a serine (S), a glutamine (Q), an aspartic acid (D), a valine (V), a tyrosine (Y), a proline (P), an alanine (A), a threonine (T), or a methionine (M); X7 is independently selected from an arginine (R), a valine (V), or an a
  • FIG.85A shows that the liver de-targeting effect of the modified AAV capsid is mainly modulated by the VR1 sequence and liver de-targeting effect of the VR1 mutant sequences rank as: Mut1>47VR1>50VR1>49VR1.
  • the quad sequence does not significantly impact the liver de-targeting effect of the VR1 sequence (FIG. 85B).
  • FIG.86 is a heatmap showing the liver enrichment score for each of the capsids in the AAV -Lib3 library.
  • Cluster1 triplets have higher liver enrichment for 49VR1 and 50VR1 but have low liver enrichment for Mut1 and 47VR1.
  • Cluster2 triplets have higher liver enrichment for 47VR1, 49VR1 and 50VR1.
  • the position 1 (P1) of the triplet is not an aspartic acid (D) or a glutamic acid (E).
  • Cluster3 triplets have lower liver enrichment for 49VR1 and 50VR1.
  • Cluster 3 is enriched for aspartic acid (D) and glutamic acid (E) at P1 of the triplet.
  • the Cluster1 triplets include: AAP, AAR, AER, AFK, AFR, AGA, AGR, AIR, AKP, AKQ, AKR, ALR, AMK, AMR, ANK, ANR, AQG, AQK, AQR, ARK, ARP, ARR, ARS, ASG, ASP, ASR, ATR, AVR, AWK, AWR, DSG, EKP, NAG, NAK, NAR, NER, NFK, NFR, NGA, NGD, NGH, NGI, NGK, NGL, NGN, NGQ, NGR, NGS, NGT, NGV, NGY, NIK, NIR, NKI, NKP, NKT, NKV, NLK, NLR, NMK, NMR, NNK, NNR, NPR, NQG,
  • the Cluster2 triplets include: ADK, AEK, AGL, AGM, AHG, AHK, AIK, AKA, AKD, AKE, AKF, AKH, AKI, AKK, AKL, AKM, AKN, AKS, AKT, AKV, AKY, ALK, ANG, ANN, ANT, APK, AQI, AQT, ARA, ARE, ARF, ARG, ARI, ARN, ARQ, ART, ARV, ARY, ASA, ASK, ASN, ATG, ATK, ATY, AWN, AYH, AYK, AYP, AYR, DKK, DKR, DNR, DRR, DWR, DYR, EGR, EIR, EKK, EKM, EKR, ENK, EQK, ERK, ERM, ERP, ERT, ESR, EVR,
  • the Cluster3 triplets include: ADL, ADN, ADS, ADT, AEA, AED, AEE, AHE, AHL, AIW, ALE, APE, APL, ARW, AWA, AYE, DAD, DAE, DAI, DAL, DAM, DAT, DAV, DDD, DDE, DDF, DDH, DDL, DDM, DDP, DDQ, DDT, DDV, DDY, DED, DEM, DEN, DEP, DEW, DFA, DFD, DFF, DFI, DFM, DFN, DFP, DFV, DFW, DGD, DGI, DHD, DHE, DHL, DHW, DHY, DIE, DII, DIL, DIM, DIN, DIP, DIS, DIT, DIV, DKA, DKD, DKE, DKH, DKQ, DKW, DLA, DLF, DLG, DLH, DLI, DLL, DLM, DLN
  • FIGs.87A-C show the motif plot that identifies the enriched motifs in the AAV- Lib3 library of the liver triplet clusters.
  • the liver triplet Cluster 1 includes a triplet peptide having a sequence of X 1 X 2 X 3 , wherein X 1 is independently selected from an asparagine (N), a threonine (T), a glutamine (Q), a serine (S), or an alanine (A); X 2 is independently selected from a glycine (G), a lysine (K), a serine (S), an arginine (R), a glutamine (Q), an asparagine (N), an alanine (A), a threonine (T), a phenylalanine (F), a methionine (M), or a tryptophan (W); and X3 is independently selected from an arginine (R), a lys
  • the liver triplet Cluster 2 includes a triplet peptide having a sequence of X1X2X3, wherein X1 is independently selected from a glutamine (Q), an alanine (A), an asparagine (N), a serine (S), or a threonine (T); X2 is independently selected from an arginine (R), a lysine (K), a threonine (T), a serine (S), or a tyrosine (Y); and X 3 is independently selected from a lysine (K), an arginine (R), an asparagine (N), a threonine (T), a glycine (G), a methionine (M), an isoleucine (I), a serine (S), or a glutamine (Q).
  • X1 is independently selected from a glutamine (Q), an alanine (A), an asparagine (N), a serine (S), or
  • the liver triplet Cluster 3 includes a triplet peptide having a sequence of X 1 X 2 X 3 , wherein X1 is independently selected from an aspartic acid (D), a glutamic acid (E), an alanine (A), a serine (S), an asparagine (N), or a threonine (T); X2 is independently selected from an aspartic acid (D), a proline (P), a leucine (L), a glutamic acid (E), a phenylalanine (F), a tryptophan (W), a methionine (M), an alanine (A), a valine (V), a threonine (T), an isoleucine (I), a histidine (H), or a tyrosine (Y); and X 3 is independently selected from an aspartic acid (D), a tryptophan (W), a glutamic acid (E), a
  • Heart-specific targeting peptides [00737] The AAV -Lib3 library data was also analyzed to assess whether certain capsids had increased tropism for a particular subset of muscle tissue, for example, increased tropism in the heart over skeletal muscle or increased tropism in skeletal muscle over the heart. [00738] As shown in FIG. 88A, the heart and skeletal muscle enrichment scores were correlated, especially at the high end. Some capsids favored heart muscle over skeletal muscle. However, these capsids did not have the highest enrichment score in heart.
  • FIG.88B shows the enrichment scores for heart muscle over skeletal muscle for the capsids in the AAV -Lib3 library having the indicated quads.
  • analysis of the data in FIG. 88B revealed that targeting peptides having quads selected from: FVQR, KERF, LDEL, LIGR, QSTL, and VRTL were the most “heart-specific” quads. 8.27.4.
  • Conclusion [00740] Overall, this data shows that a subset of the modified AAV capsid comprising various targeting peptides tested in NHP have increased muscle tropism. The liver de-targeting effect of the modified AAV capsid is mainly modulated by the variants of the VR1 sequence.
  • IHC immunohistochemistry
  • RT-ddPCR Bio-Rad One-Step reverse transcription droplet digital PCR
  • M1 and M3 also enhanced the transcription of GFP DNA in heart tissues by 6.3- 21.7 times and 3.7-69.3 times respectively compared to the GFP transcription with wild type AAV9 at various doses (FIGs.93A, 93B and 93C). 8.29.
  • M1 (AAV9-38181-mut1) and M3 have superior cardiotropism in NHPs
  • M1 and M3 showed improved GFP expression compared to AAV9 in NHPs (FIG. 99).
  • the increase in GFP expression was observed at different NHP skeletal muscle regions including quadriceps, tibialis anterior, and biceps femoris (FIG.100). 8.31.
  • M1 AAV9-38181-mut1
  • Cynomolgus macaques were dosed with wild type AAV9 or modified AAV9 expressing CAG.eGFP by IV-injection at 1e14vg/kg.
  • the modified AAV9 comprises an M3 targeting peptide, an M1 targeting peptide with wild type VRI, or an M1 targeting peptide with mut1 VRI substitution.
  • the modified AAV9 comprising a targeting peptide with the amino acid sequence of SEQ ID NO: 44880 was used as a control.
  • Each treatment group contained 3 animals.
  • Whole blood was collected prior to dosing at days -28 and 1, and post-doing on days 8, 15, 22, 28/29. Whole blood was collected in K2EDTA tubes, aliquoted and stored at - 80°C until DNA isolation was performed.
  • DNA from whole blood was isolated using Qiagen DSP DNA Blood mini kits following the Qiagen protocol and stored at -20°C.
  • FIGs.102A and 102B show the levels of liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST) for wild type AAV9 and AAV9 an M1 Atty Docket No.: 38053-57988/WO (112WO) targeting peptide with mut1 VRI substitution (M1). The data show that M1 did not cause liver enzyme elevation in NHP serum. 9.

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Abstract

La présente invention concerne une protéine de capside d'AAV modifiée comprenant un peptide de ciblage dans la région variable VIII (VR VIII) et/ou un segment de peptide dans la région variable I (VR 1). La protéine de capside d'AAV modifiée peut former un rAAV, qui a un tropisme, une spécificité ou une biodistribution préférés in vivo in vivo ou in vitro. Le rAAV de la présente invention peut être utilisé pour des thérapies géniques ciblées au niveau d'un tissu spécifique.
PCT/US2024/029714 2023-05-16 2024-05-16 Aav de recombinaison à tropisme et spécificité améliorés WO2024238807A2 (fr)

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