Recombinant aavs with improved tropism and specificity

Abstract

The present disclosure provides a modified AAV capsid protein comprising a targeting peptide in variable region VIII (VR VIII) and / or a peptide segment in variable region I (VR 1). The modified AAV capsid protein can form an rAAV, which has a preferred tropism, specificity or biodistribution in vivo or in vitro. The rAAV of the present disclosure can be used for gene therapies targeted at a specific tissue.

Description

Atty Docket No.: 38053-57988 / WO (112WO)RECOMBINANT AAVS WITH IMPROVED TROPISM AND SPECIFICITY 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 502,595, filed May 16, 2023; International Application No. PCT / US2023 / 072424, filed August 17, 2023; U.S. Provisional Application No.63 / 625,790, filed January 26, 2024; and U.S. Provisional Application No.63 / 643,890, filed May 7, 2024, each of which is hereby incorporated by reference in its entirety. 2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted as an XML file on triplicate CD-Rs (labeled as CRF, Copy 1, and Copy 2) and mailed on May 16, 2024, via USPS Priority Mail Express; and is hereby incorporated by reference in its entirety. Said XML file, created on May 16, 2024, is named 57988WO_CRF_sequencelisting.xml, and is 137,114,243 bytes in size. 3. BACKGROUND

[0003] Adeno-associated virus (AAV) 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.

[0004] Although AAV vectors are safer and less inflammatory than other viruses, toxicities have occurred following administration of high doses of rAAVs for gene therapy. Thus, local administration of rAAVs to a target tissue or organ has been used to improve targeting and reduce systemic toxicity. Further, various natural and synthetic AAV variants have been tested to develop an AAV vector with desired tropism and specificity.

[0005] In general, 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. Over the last decade, 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). In some circumstances, the insertion of a novel peptide into a wild type capsid changes the tropism of the variant. For example, insertion of aAtty 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. SUMMARY

[0007] The present disclosure provides a modified AAV capsid protein that can form an rAAV having a preferred tropism and specificity to a therapeutic target. In some embodiments, a modified adeno-associated virus (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 X1X2X3RGDX7X8X9X10, wherein X1, X2, X3, X7, X8, X9and X10are 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. In some cases, 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.

[0008] Overall, 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.

[0009] In one aspect, 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 X1, X2, X3, X7, X8, X9and X10are 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0010] In some embodiments, X7X8X9X10 is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656), or X7X8X9X10 is FNNL and P1P2P3P4P5P6P7P8NDNP12is SSTAGGASNDNA (SEQ ID NO: 47128). In some of these embodiments, X1 is independently selected from S, N, T, A, or Q; X2 is independently selected from G, S, or A; and X3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M. In some of these embodiments, X1is independently selected from T, S, Q, A, D, or N; X2is independently selected from F, W, A, K, Q, N, R, S, Y, M, or T; and X3is independently selected from N, S, Q, T, Y, G, E, F, M, or A. In some of these embodiments, X1is independently selected from N, S, T, A, or Q; X2is independently selected from N, S, Q, T, M, A, Y, or L; and X3is independently selected from I, M, L, T, V, Q, or Y. In some of these embodiments, 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; and X3 is independently selected from H, R, S, A, T, M, G, K, Q, N, or I.

[0011] In some embodiments, 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; X7is F; X8is independently selected from N or Q; X9is N; and X10is independently selected from T or L. In some embodiments, X1is 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; X3is independently selected from N, S, Q, T, Y, G, E, F, M, or A; X7is F; X8is independently selected from N or Q; X9is N; and X10is independently selected from T or L. In some embodiments, 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; X3is independently selected from I, M, L, T, V, Q, or Y; X7is F; X8is independently selected from N or Q; X9is N; and X10is independently selected from T or L. In some embodiments, 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; X7is F; X8is independently selected from N or Q; X9 is N; and X10 is independently selected from T or L.

[0012] In some embodiments, X7X8X9X10 is selected from RSQT, RNVV, RGQI, RGVV, and RSVV and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390). In some of these embodiments, X1is independently selected from S, N, T, A, or Q; X2is independently selected from G, S, or A; and X3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M. In some of these embodiments, X1is independently selected from T, S, Q, A, D, or N; X2is independently selected from F, W, A, K, Q, N, R, S, Y, M, or T; and X3Atty Docket No.: 38053-57988 / WO (112WO) is independently selected from N, S, Q, T, Y, G, E, F, M, or A. In some of these embodiments, 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; and X3is independently selected from I, M, L, T, V, Q, or Y.

[0013] In some embodiments, X1 is independently selected from S, N, T, A, or Q; X2 is independently selected from G, S, or A; X3is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M; X7is R; X8is independently selected from S, G, or N; X9is independently selected from V or Q; and X10 is independently selected from V, T, or I. In some embodiments, X1is independently selected from T, S, Q, A, D, or N; X2is independently selected from F, W, A, K, Q, N, R, S, Y, M, or T; X3is 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. In some embodiments, X1is independently selected from N, S, T, A, or Q; X2is 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; and X10is independently selected from V, T, or I.

[0014] In some embodiments, X7X8X9X10is selected from VRTL, RQGI, ARTL, and RTNL and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390). In some of these embodiments, X1is independently selected from S, N, T, A, or Q; X2is independently selected from G, S, or A; and X3is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M.

[0015] In some embodiments, X1is independently selected from S, N, T, A, or Q; X2is independently selected from G, S, or A; X3is 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.

[0016] In some embodiments, 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 P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390), or X7X8X9X10is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656), or X7X8X9X10is FNNL and P1P2P3P4P5P6P7P8NDNP12is SSTAGGASNDNA (SEQ ID NO:Atty Docket No.: 38053-57988 / WO (112WO) 47128). In some of these embodiments, 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, SGI, SGL, SGM, SGN, SGP, SGQ, SGS, SGT, SGV, SGY, SKD, SMG, SNG, SQG, SRD, SSG, SSN, SSS, TAG, TGA, TGD, TGF, TGG, TGH, TGI, TGL, TGM, TGN, TGP, TGQ, TGS, TGT, TGV, TGY, TKD, TRD, TSG, TSN, TSS, TSV, and TSY.

[0017] In some embodiments, X7X8X9X10is 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 X7X8X9X10is FNNL and P1P2P3P4P5P6P7P8NDNP12 is SSTAGGASNDNA (SEQ ID NO: 47128). In some of these embodiments, 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, QLQ, QLS, QMN, QMS, QMY, QNG, QNN, QNS, QQN, QQY, QRD, QRE, QSP, QSY, QTY, QWN, QWQ, QWS, QWT, QYN, SAA, SAF, SAN, SAS, SFI, SFQ, SFS, SFT, SFY, SGF, SHN, SKE, SLN, SLT, SMN, SMQ, SNN, SQF, SQN, SQS, SRE, SSP, SSY, STG, SWA, SWN, SWQ, SWS, SWT, SYG, SYN, TAF, TAN, TAS, TAY, TFF, TFN, TFQ, TFS, TFT, TFY, TGW, TKE, TLS, TMY, TNG, TNN, TNS, TQF, TQG, TQI, TQN, TQY, TRE, TSF, TSP, TTG, TTY, TVY, TWG, TWL, TWM, TWN, TWQ, TWT, TYG, TYN, TYQ, TYS, and TYT. In some of these embodiments, 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, NHM, NHN, NIM, NIQ, NIT, NIY, NKE, NLM, NLV, NMI, NMM, NMN, NMQ,Atty Docket No.: 38053-57988 / WO (112WO) NMS, NMV, NNA, NNF, NNI, NNL, NNM, NNQ, NNS, NNT, NNV, NNY, NPI, NPM, NPN, NPQ, NPS, NPT, NQH, NQI, NQL, NQM, NQQ, NQT, NQV, NQY, NSA, NSF, NSI, NSL, NSM, NSS, NST, NSV, NSY, NTG, NTH, NTI, NTL, NTM, NTQ, NTS, NTT, NTV, NTY, NVM, NWI, NWM, NWT, NYI, NYM, NYQ, NYS, NYT, NYV, QAL, QDK, QDR, QER, QEV, QGI, QGL, QGY, QHI, QHY, QII, QIL, QLI, QLL, QLM, QLT, QLY, QMI, QML, QMM, QMT, QMV, QNA, QNF, QNI, QNM, QNQ, QNT, QNV, QNW, QNY, QQI, QQL, QQM, QQT, QQV, QSA, QSI, QSL, QSM, QST, QSV, QTI, QTL, QTT, QTV, QVL, QYI, SAH, SAI, SAL, SAM, SAQ, SAT, SAV, SDK, SDR, SEK, SER, SFL, SFM, SFV, SHA, SHF, SHL, SHY, SII, SIM, SIT, SLI, SLV, SMH, SMI, SML, SMM, SMS, SMT, SMV, SNA, SNF, SNI, SNL, SNM, SNQ, SNS, SNT, SNV, SNY, SPA, SPQ, SPT, SQH, SQI, SQL, SQM, SQT, SQV, SQY, SSA, SSI, SSL, SSM, SSQ, SST, SSV, STA, STI, STL, STQ, STT, STV, STY, SVI, SVL, SVM, SVQ, SVT, SWL, SWM, SWV, SYI, SYL, SYM, SYQ, SYS, SYT, SYY, TAI, TAL, TAM, TAQ, TAT, TAV, TDK, TDR, TEK, TER, TFM, TFV, TGR, THF, THY, TIM, TIT, TIV, TLI, TLL, TLM, TMI, TML, TMM, TMT, TMV, TNA, TNF, TNI, TNL, TNM, TNQ, TNT, TNV, TNY, TPQ, TPT, TPV, TQH, TQL, TQM, TQQ, TQT, TQV, TSA, TSI, TSL, TSM, TSQ, TST, TTI, TTL, TTQ, TTT, TTV, TVI, TVL, TVM, and TYL.

[0018] In some embodiments, X7X8X9X10 is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12is SGTTGGSSNDNT (SEQ ID NO: 46656), or X7X8X9X10is FNNL and P1P2P3P4P5P6P7P8NDNP12is SSTAGGASNDNA (SEQ ID NO: 47128). In some of these embodiments, 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, ESR, ETR, EWK, EWR, EYK, EYR, NAH, NDR, NFA, NFH, NFY, NHG, NHQ, NHS, NHT, NHV, NMH, NNH, NRE, NWH, NYA, QAD, QAH, QAM, QDN, QFH, QFM, QHF, QHG, QHM, QHN, QHQ, QHS, QHT, QNH, QQH, QRI, QRY, QSF, QSH, QTM, QWA, QWH, QWI, QYH, QYQ, QYS, QYT, SFA, SFH, SHG, SHI, SHM, SHQ, SHS, SHT, SNH, SNP, SNW, SSH, SWG, SWH, SYA, SYE, SYH, TFH, THG, THM, THN, THQ, THS, THT, THW, TNH, TNP, TPG, TSH, TWA, TWH, and TYM.Atty Docket No.: 38053-57988 / WO (112WO)

[0019] In some embodiments, wherein X7X8X9X10 is selected from LDEL, QSTL, ISRT, LLLS, IHNL, LIGR, FVQR, HVNL, RQGI, KERF, RSVV, ARTL, VRTL, RNVV, RSQT, RGVV, RGQI, and RTNL, and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390). In some of these embodiments, 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, NSK, NSR, NTA, NTK, NTR, NVR, NWK, NWR, NYK, NYR, QAG, QAR, QER, QFK, QFR, QFT, QGK, QGR, QGV, QGW, QHR, QIR, QKA, QKG, QKN, QKP, QKQ, QKR, QKT, QLR, QMG, QMK, QMR, QNG, QNK, QNR, QPR, QQK, QQR, QQS, QRN, QRP, QRV, QSA, QSR, QTR, QTV, QVR, QVV, QYK, SAG, SAR, SDR, SFK, SFR, SGA, SGE, SGF, SGK, SGL, SGM, SGR, SGT, SIR, SKA, SKN, SKP, SKR, SKT, SLR, SMR, SNG, SNK, SNR, SQG, SQR, SRK, SRP, SSG, SSK, SSQ, SSR, STQ, STR, SVR, SWK, SWR, SYK, SYR, TAG, TAR, TFR, TGE, TGF, TGK, TGL, TGM, TGQ, TGR, TIK, TIR, TKE, TKK, TKP, TKQ, TKS, TKT, TKY, TLK, TLR, TMK, TMR, TNK, TNR, TPR, TQK, TQR, TRP, TRQ, TRV, TSG, TSK, TSR, TTK, TTR, TVR, TWK, TWR, and TYK, or X1X2X3 is 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, NFN, NFQ, NFT, NHK, NHR, NHT, NKA, NKK, NKM, NKN, NKQ, NKR, NKS, NKW, NLQ, NLT, NNF, NPG, NPK, NPN, NQN, NQS, NRA, NRF, NRG, NRH, NRI, NRL, NRM, NRR, NRS, NRV, NRW, NRY, NSH, NSN, NSP, NSQ, NSS, NTS, NTY, NVK, NYM, NYN, QAK, QEK, QFG, QGD, QHK, QIK, QIQ, QKF, QKI, QKK, QKL, QKM, QKV, QKW, QKY, QLT, QMM, QMQ, QMS, QMT, QNH, QNT, QNV, QPP, QQP, QQQ, QRA, QRF, QRH, QRI, QRK, QRL, QRM, QRQ, QRR, QRS, QRT, QRW, QRY, QSF, QSI, QSK, QTA, QTI, QTK, QTN, QTT, QWK, QWN, QWR, QYG, QYI, QYN, QYR, QYT, SAN, SER, SFT, SHK, SHR, SIK, SKD, SKF, SKG, SKI, SKK, SKL, SKM, SKQ, SKS, SKV, SKY, SLK, SMG, SMK, SQI, SQN, SQQ, SRA, SRF, SRG, SRH, SRI, SRM, SRN, SRQ, SRR, SRS, SRT, SRV, SRW, SRY, STK, STP, STY, SVK, SWG, TAK, TDR, TFK, THK, THR, TKF, TKG, TKI, TKL, TKM, TKN,Atty Docket No.: 38053-57988 / WO (112WO) TKR, TKV, TKW, TNT, TPS, TQN, TRA, TRE, TRG, TRI, TRK, TRM, TRN, TRR, TRS, TRT, TRY, TSA, TSS, TTY, TVK, TWH, and TYR. In some of these embodiments, 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, DLL, DLM, DLN, DLP, DLQ, DLS, DLT, DLV, DLY, DMD, DME, DMH, DMN, DMP, DMQ, DMS, DMT, DMW, DMY, DNH, DNP, DNW, DPA, DPD, DPE, DPF, DPI, DPL, DPM, DPP, DPQ, DPR, DPT, DPV, DQA, DQD, DQE, DQH, DQT, DQY, DRV, DRW, DSA, DSD, DSE, DSL, DTD, DTF, DTH, DTL, DTM, DTP, DTT, DTW, DVD, DVE, DVF, DVH, DVM, DVN, DVP, DVQ, DVT, DWI, DWL, DWM, DWS, DWY, DYA, DYE, DYF, EAD, EAE, EAH, EAI, EAL, EAS, EAV, EAW, EAY, EDA, EDD, EDE, EDF, EDG, EDK, EDL, EDM, EDN, EDP, EDQ, EDW, EDY, EEA, EED, EEE, EEF, EEL, EEM, EEQ, EEV, EEY, EFA, EFM, EFQ, EFS, EFW, EGD, EHE, EHI, EHV, EID, EIT, ELD, ELN, ELW, EMD, EMF, EMH, EML, EMW, ENI, EPA, EPL, EPM, EPQ, EPW, EQD, EQY, ESF, ESV, ESW, ETA, ETD, ETE, ETL, ETW, EVA, EVE, EVS, EVW, EWE, EWH, EWM, EWN, EWS, EWY, EYF, EYQ, EYT, EYW, NDD, NDY, NEL, NEN, NEY, NFD, NFF, NFP, NFW, NHH, NWW, NYD, QDW, QFW, QPS, QVF, QWS, SDH, SDV, SFI, SIF, SLA, SLG, SMD, SPE, SPW, SWF, SWM, SYD, TDF, TDM, TDW, TDY, TEA, THW, TLW, TWD, and TYI.

[0020] In some embodiments, X7X8X9X10is selected from FNNL, FNNT, and FQNT and P1P2P3P4P5P6P7P8NDNP12is SGTTGGSSNDNT (SEQ ID NO: 46656). In some of these embodiments, 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, NFN, NFQ, NFT, NHK, NHR, NHT, NKA, NKK, NKM, NKN, NKQ, NKR, NKS, NKW, NLQ, NLT, NNF, NPG, NPK, NPN, NQN, NQS, NRA, NRF, NRG, NRH, NRI, NRL, NRM, NRR, NRS, NRV, NRW, NRY, NSH, NSN, NSP, NSQ, NSS, NTS, NTY, NVK, NYM, NYN, QAK, QEK, QFG, QGD, QHK, QIK, QIQ, QKF, QKI, QKK, QKL, QKM, QKV, QKW, QKY, QLT,Atty Docket No.: 38053-57988 / WO (112WO) QMM, QMQ, QMS, QMT, QNH, QNT, QNV, QPP, QQP, QQQ, QRA, QRF, QRH, QRI, QRK, QRL, QRM, QRQ, QRR, QRS, QRT, QRW, QRY, QSF, QSI, QSK, QTA, QTI, QTK, QTN, QTT, QWK, QWN, QWR, QYG, QYI, QYN, QYR, QYT, SAN, SER, SFT, SHK, SHR, SIK, SKD, SKF, SKG, SKI, SKK, SKL, SKM, SKQ, SKS, SKV, SKY, SLK, SMG, SMK, SQI, SQN, SQQ, SRA, SRF, SRG, SRH, SRI, SRM, SRN, SRQ, SRR, SRS, SRT, SRV, SRW, SRY, STK, STP, STY, SVK, SWG, TAK, TDR, TFK, THK, THR, TKF, TKG, TKI, TKL, TKM, TKN, TKR, TKV, TKW, TNT, TPS, TQN, TRA, TRE, TRG, TRI, TRK, TRM, TRN, TRR, TRS, TRT, TRY, TSA, TSS, TTY, TVK, TWH, and TYR; or 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, NSK, NSR, NTA, NTK, NTR, NVR, NWK, NWR, NYK, NYR, QAG, QAR, QER, QFK, QFR, QFT, QGK, QGR, QGV, QGW, QHR, QIR, QKA, QKG, QKN, QKP, QKQ, QKR, QKT, QLR, QMG, QMK, QMR, QNG, QNK, QNR, QPR, QQK, QQR, QQS, QRN, QRP, QRV, QSA, QSR, QTR, QTV, QVR, QVV, QYK, SAG, SAR, SDR, SFK, SFR, SGA, SGE, SGF, SGK, SGL, SGM, SGR, SGT, SIR, SKA, SKN, SKP, SKR, SKT, SLR, SMR, SNG, SNK, SNR, SQG, SQR, SRK, SRP, SSG, SSK, SSQ, SSR, STQ, STR, SVR, SWK, SWR, SYK, SYR, TAG, TAR, TFR, TGE, TGF, TGK, TGL, TGM, TGQ, TGR, TIK, TIR, TKE, TKK, TKP, TKQ, TKS, TKT, TKY, TLK, TLR, TMK, TMR, TNK, TNR, TPR, TQK, TQR, TRP, TRQ, TRV, TSG, TSK, TSR, TTK, TTR, TVR, TWK, TWR, and TYK. ). In some of these embodiments, X1X2X3is 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, NSK, NSR, NTA, NTK, NTR, NVR, NWK, NWR, NYK, NYR, QAG, QAR, QER, QFK, QFR, QFT, QGK, QGR, QGV, QGW, QHR, QIR, QKA, QKG, QKN, QKP, QKQ, QKR, QKT, QLR, QMG, QMK, QMR, QNG, QNK, QNR, QPR, QQK, QQR, QQS, QRN, QRP, QRV, QSA, QSR, QTR, QTV, QVR, QVV, QYK, SAG, SAR, SDR, SFK, SFR, SGA, SGE, SGF, SGK, SGL, SGM, SGR, SGT, SIR, SKA, SKN, SKP, SKR, SKT, SLR, SMR, SNG, SNK, SNR, SQG, SQR, SRK, SRP, SSG, SSK, SSQ, SSR, STQ, STR, SVR, SWK, SWR, SYK, SYR,Atty Docket No.: 38053-57988 / WO (112WO) TAG, TAR, TFR, TGE, TGF, TGK, TGL, TGM, TGQ, TGR, TIK, TIR, TKE, TKK, TKP, TKQ, TKS, TKT, TKY, TLK, TLR, TMK, TMR, TNK, TNR, TPR, TQK, TQR, TRP, TRQ, TRV, TSG, TSK, TSR, TTK, TTR, TVR, TWK, TWR, and TYK, or X1X2X3is 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, DLL, DLM, DLN, DLP, DLQ, DLS, DLT, DLV, DLY, DMD, DME, DMH, DMN, DMP, DMQ, DMS, DMT, DMW, DMY, DNH, DNP, DNW, DPA, DPD, DPE, DPF, DPI, DPL, DPM, DPP, DPQ, DPR, DPT, DPV, DQA, DQD, DQE, DQH, DQT, DQY, DRV, DRW, DSA, DSD, DSE, DSL, DTD, DTF, DTH, DTL, DTM, DTP, DTT, DTW, DVD, DVE, DVF, DVH, DVM, DVN, DVP, DVQ, DVT, DWI, DWL, DWM, DWS, DWY, DYA, DYE, DYF, EAD, EAE, EAH, EAI, EAL, EAS, EAV, EAW, EAY, EDA, EDD, EDE, EDF, EDG, EDK, EDL, EDM, EDN, EDP, EDQ, EDW, EDY, EEA, EED, EEE, EEF, EEL, EEM, EEQ, EEV, EEY, EFA, EFM, EFQ, EFS, EFW, EGD, EHE, EHI, EHV, EID, EIT, ELD, ELN, ELW, EMD, EMF, EMH, EML, EMW, ENI, EPA, EPL, EPM, EPQ, EPW, EQD, EQY, ESF, ESV, ESW, ETA, ETD, ETE, ETL, ETW, EVA, EVE, EVS, EVW, EWE, EWH, EWM, EWN, EWS, EWY, EYF, EYQ, EYT, EYW, NDD, NDY, NEL, NEN, NEY, NFD, NFF, NFP, NFW, NHH, NWW, NYD, QDW, QFW, QPS, QVF, QWS, SDH, SDV, SFI, SIF, SLA, SLG, SMD, SPE, SPW, SWF, SWM, SYD, TDF, TDM, TDW, TDY, TEA, THW, TLW, TWD, and TYI. In some of these embodiments, X1X2X3is 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, DLL, DLM, DLN, DLP, DLQ, DLS, DLT, DLV, DLY, DMD, DME, DMH, DMN, DMP, DMQ, DMS, DMT, DMW, DMY, DNH, DNP, DNW, DPA, DPD, DPE, DPF, DPI, DPL, DPM, DPP, DPQ, DPR, DPT, DPV, DQA, DQD, DQE, DQH, DQT, DQY, DRV, DRW, DSA, DSD, DSE, DSL, DTD, DTF, DTH, DTL, DTM, DTP, DTT, DTW, DVD, DVE, DVF, DVH, DVM, DVN, DVP, DVQ, DVT, DWI, DWL, DWM, DWS, DWY, DYA, DYE, DYF, EAD, EAE, EAH, EAI, EAL, EAS, EAV, EAW, EAY, EDA, EDD, EDE, EDF, EDG, EDK, EDL, EDM, EDN,Atty Docket No.: 38053-57988 / WO (112WO) EDP, EDQ, EDW, EDY, EEA, EED, EEE, EEF, EEL, EEM, EEQ, EEV, EEY, EFA, EFM, EFQ, EFS, EFW, EGD, EHE, EHI, EHV, EID, EIT, ELD, ELN, ELW, EMD, EMF, EMH, EML, EMW, ENI, EPA, EPL, EPM, EPQ, EPW, EQD, EQY, ESF, ESV, ESW, ETA, ETD, ETE, ETL, ETW, EVA, EVE, EVS, EVW, EWE, EWH, EWM, EWN, EWS, EWY, EYF, EYQ, EYT, EYW, NDD, NDY, NEL, NEN, NEY, NFD, NFF, NFP, NFW, NHH, NWW, NYD, QDW, QFW, QPS, QVF, QWS, SDH, SDV, SFI, SIF, SLA, SLG, SMD, SPE, SPW, SWF, SWM, SYD, TDF, TDM, TDW, TDY, TEA, THW, TLW, TWD, and TYI.

[0021] In some embodiments, X7X8X9X10 is FNNT and P1P2P3P4P5P6P7P8NDNP12 is SSTAGGATNDNA (SEQ ID NO: 47131), or X7X8X9X10is FNNL and P1P2P3P4P5P6P7P8NDNP12is SSTAGGASNDNA (SEQ ID NO: 47128). In some of these embodiments, 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, DLL, DLM, DLN, DLP, DLQ, DLS, DLT, DLV, DLY, DMD, DME, DMH, DMN, DMP, DMQ, DMS, DMT, DMW, DMY, DNH, DNP, DNW, DPA, DPD, DPE, DPF, DPI, DPL, DPM, DPP, DPQ, DPR, DPT, DPV, DQA, DQD, DQE, DQH, DQT, DQY, DRV, DRW, DSA, DSD, DSE, DSL, DTD, DTF, DTH, DTL, DTM, DTP, DTT, DTW, DVD, DVE, DVF, DVH, DVM, DVN, DVP, DVQ, DVT, DWI, DWL, DWM, DWS, DWY, DYA, DYE, DYF, EAD, EAE, EAH, EAI, EAL, EAS, EAV, EAW, EAY, EDA, EDD, EDE, EDF, EDG, EDK, EDL, EDM, EDN, EDP, EDQ, EDW, EDY, EEA, EED, EEE, EEF, EEL, EEM, EEQ, EEV, EEY, EFA, EFM, EFQ, EFS, EFW, EGD, EHE, EHI, EHV, EID, EIT, ELD, ELN, ELW, EMD, EMF, EMH, EML, EMW, ENI, EPA, EPL, EPM, EPQ, EPW, EQD, EQY, ESF, ESV, ESW, ETA, ETD, ETE, ETL, ETW, EVA, EVE, EVS, EVW, EWE, EWH, EWM, EWN, EWS, EWY, EYF, EYQ, EYT, EYW, NDD, NDY, NEL, NEN, NEY, NFD, NFF, NFP, NFW, NHH, NWW, NYD, QDW, QFW, QPS, QVF, QWS, SDH, SDV, SFI, SIF, SLA, SLG, SMD, SPE, SPW, SWF, SWM, SYD, TDF, TDM, TDW, TDY, TEA, THW, TLW, TWD, and TYI. In some of these embodiments, X1X2X3is 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, ERM, ERP, ERT, ESR, EVR, NAQ, NAT, NEG, NFN, NFQ, NFT, NHK, NHR, NHT, NKA, NKK, NKM, NKN, NKQ, NKR, NKS, NKW, NLQ, NLT, NNF, NPG, NPK, NPN, NQN, NQS, NRA, NRF, NRG, NRH, NRI, NRL, NRM, NRR, NRS, NRV, NRW, NRY, NSH, NSN, NSP, NSQ, NSS, NTS, NTY, NVK, NYM, NYN, QAK, QEK, QFG, QGD, QHK, QIK, QIQ, QKF, QKI, QKK, QKL, QKM, QKV, QKW, QKY, QLT, QMM, QMQ, QMS, QMT, QNH, QNT, QNV, QPP, QQP, QQQ, QRA, QRF, QRH, QRI, QRK, QRL, QRM, QRQ, QRR, QRS, QRT, QRW, QRY, QSF, QSI, QSK, QTA, QTI, QTK, QTN, QTT, QWK, QWN, QWR, QYG, QYI, QYN, QYR, QYT, SAN, SER, SFT, SHK, SHR, SIK, SKD, SKF, SKG, SKI, SKK, SKL, SKM, SKQ, SKS, SKV, SKY, SLK, SMG, SMK, SQI, SQN, SQQ, SRA, SRF, SRG, SRH, SRI, SRM, SRN, SRQ, SRR, SRS, SRT, SRV, SRW, SRY, STK, STP, STY, SVK, SWG, TAK, TDR, TFK, THK, THR, TKF, TKG, TKI, TKL, TKM, TKN, TKR, TKV, TKW, TNT, TPS, TQN, TRA, TRE, TRG, TRI, TRK, TRM, TRN, TRR, TRS, TRT, TRY, TSA, TSS, TTY, TVK, TWH, and TYR; or 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, NSK, NSR, NTA, NTK, NTR, NVR, NWK, NWR, NYK, NYR, QAG, QAR, QER, QFK, QFR, QFT, QGK, QGR, QGV, QGW, QHR, QIR, QKA, QKG, QKN, QKP, QKQ, QKR, QKT, QLR, QMG, QMK, QMR, QNG, QNK, QNR, QPR, QQK, QQR, QQS, QRN, QRP, QRV, QSA, QSR, QTR, QTV, QVR, QVV, QYK, SAG, SAR, SDR, SFK, SFR, SGA, SGE, SGF, SGK, SGL, SGM, SGR, SGT, SIR, SKA, SKN, SKP, SKR, SKT, SLR, SMR, SNG, SNK, SNR, SQG, SQR, SRK, SRP, SSG, SSK, SSQ, SSR, STQ, STR, SVR, SWK, SWR, SYK, SYR, TAG, TAR, TFR, TGE, TGF, TGK, TGL, TGM, TGQ, TGR, TIK, TIR, TKE, TKK, TKP, TKQ, TKS, TKT, TKY, TLK, TLR, TMK, TMR, TNK, TNR, TPR, TQK, TQR, TRP, TRQ, TRV, TSG, TSK, TSR, TTK, TTR, TVR, TWK, TWR, and TYK.

[0022] In some embodiments, X1X2X3RGDX7X8X9X10has a sequence selected from SEQ ID NOs: 98944 to 157056. In some embodiments, 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).Atty Docket No.: 38053-57988 / WO (112WO)

[0023] In some embodiments, X1X2X3RGDX7X8X9X10 is NYQRGDFQNT (SEQ ID NO: 145855) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656). In some embodiments, X1X2X3RGDX7X8X9X10is SYQRGDFQNT (SEQ ID NO: 146577) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656). In some embodiments, X1X2X3RGDX7X8X9X10 is ADRRGDFNNT (SEQ ID NO: 146987) and P1P2P3P4P5P6P7P8NDNP12is SGTTGGSSNDNT (SEQ ID NO: 46656). In some embodiments, X1X2X3RGDX7X8X9X10is NNQRGDFNNT (SEQ ID NO: 148096) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656). In some embodiments, X1X2X3RGDX7X8X9X10is SGQRGDRGQI (SEQ ID NO: 124229) and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390). In some embodiments, X1X2X3RGDX7X8X9X10 is TGYRGDRGQI (SEQ ID NO: 124587) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390). In some embodiments, X1X2X3RGDX7X8X9X10is QGQRGDRGQI (SEQ ID NO: 126394) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390). In some embodiments, X1X2X3RGDX7X8X9X10 is SGVRGDRGVV (SEQ ID NO: 126741) and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390). In some embodiments, X1X2X3RGDX7X8X9X10is TGFRGDARTL (SEQ ID NO: 99313) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390). In some embodiments, X1X2X3RGDX7X8X9X10is QGIRGDRSQT (SEQ ID NO: 136490) and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390). In some embodiments, X1X2X3RGDX7X8X9X10 is SAQRGDRGVV (SEQ ID NO: 48394) and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390).

[0024] In some embodiments, 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. In some embodiments, the AAV capsid protein is selected from VP1, VP2 and VP3. In some embodiments, 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-B; rh.58-B; rh.28-B; rh.51-B; rh.19-B; rh.49-B; rh.52-B; rh.13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu.9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.1-C; hu.18-C; hu.3-C;Atty Docket No.: 38053-57988 / WO (112WO) hu.25-C; hu.15-C; hu.16-C; hu.11-C; hu.10-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu.17-E; hu.6-E; hu.66-E; rh.38- E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; Anc113; Anc126; Anc127; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc110; and Anc80DI. In some embodiments, 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. In some embodiments, the targeting peptide is positioned between 565 and 595 within VR VIII of the modified AAV capsid protein. In some embodiments, (i) 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; (ii) 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; (iii) 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, and N588 of the reference capsid protein; (iv) 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, thereby replacing residues D582, Q583, and S584 of the reference capsid protein; (v) the reference AAV capsid protein is a capsid protein of AAV5 or a modification thereof and the targeting peptide is between Q574 and T578 ofAtty Docket No.: 38053-57988 / WO (112WO) the reference AAV capsid protein, thereby replacing residues S575, S576, and S577 of the reference capsid protein; (vi) 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, thereby replacing residues S586, S587, and S588 of the reference capsid protein; (vii) 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, thereby replacing residues A587, A588, and N589 of the reference capsid protein; (viii) the reference AAV capsid protein is a capsid protein of AAV8 or a modification thereof and the targeting peptide is between Q587 and A591 of the modified AAV capsid protein thereby replacing residues Q588, Q589, and N590 of the reference capsid protein; (ix) 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 thereby replacing residues S586, A587, and Q588 of the reference capsid protein; (x) 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 thereby replacing residues Q588, Q589, and N590 of the reference capsid protein; (xi) 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 thereby replacing residues N565, Q566, and N567 of the reference capsid protein; (xii) 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 thereby replacing residues N590, Q591, and N592 of the reference capsid protein; (xiii) 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 thereby replacing residues S586, S587, and N588 of the reference capsid protein;Atty Docket No.: 38053-57988 / WO (112WO) (xiv) 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 thereby replacing residues S586, A587, and N588; (xv) 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 thereby replacing residues S586, S587, and N588 of the reference capsid protein; (xvi) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein; (xvii) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein; (xviii) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein; (xix) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein; or (xx) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein.

[0025] In some embodiments, variable region I (VR I) corresponds to amino acid residues between about position 259 to about position 275 of the modified capsid protein. In some embodiments, the peptide segment is at a position between S261 and Y274 of an AAV9 capsid protein (SEQ ID NO: 61). In some embodiments, the peptide segment is at a position between S260 and Y273 of an Anc80 capsid protein (SEQ ID NO: 132). In someAtty Docket No.: 38053-57988 / WO (112WO) embodiments, the peptide segment is at a position between S260 and Y273 of an Anc80L65 capsid protein (SEQ ID NO: 142).

[0026] In another aspect, provided herein is a polynucleotide encoding the modified AAV capsid protein.

[0027] In another aspect, provided herein is a vector comprising the polynucleotide. In some embodiments, the vector further comprises a promoter operably linked to the polynucleotide.

[0028] In another aspect, provided herein is a host cell comprising the modified AAV capsid protein, the polynucleotide, or the vector.

[0029] In another aspect, provided herein is a recombinant AAV virion (rAAV) comprising the modified AAV capsid protein. In some embodiments, the rAAV virion further comprises an exogenous polynucleotide. In some embodiments, 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. In some embodiments, 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.

[0031] In some embodiments, the exogenous polynucleotide further comprises a regulatory sequence. In some embodiments, the regulatory sequence comprises expression regulatory elements (EREs). In some embodiments, the EREs comprise a CAG promoter. In some embodiments, 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.

[0032] In another aspect, provided herein is a pharmaceutical composition comprising the modified AAV capsid protein or the AAV virion.

[0033] In another aspect, provided herein is 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0034] In some embodiments, the disease is a muscular disease and / or the condition is muscle degeneration. In some embodiments, the muscle is a striated muscle, preferably heart or a skeletal muscle or diaphragm. In some embodiments, 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.

[0035] In another aspect, provided herein is a modified adeno-associated virus (AAV) capsid protein for use in treating and / or preventing a muscular disease and / or muscle degeneration.

[0036] In another aspect, provided herein is 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.

[0037] In another aspect, provided herein is 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.

[0038] In another aspect, provided herein is a method of transferring an exogenous polynucleotide into a muscle cell, comprising the step of administering the rAAV virion to a subject. In some embodiments, 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. In some embodiments, the muscle:liver infection ratio ranges from 1 to 100. In some embodiments, the muscle:liver infection ration ranges from 1 to 10. In some embodiments, the muscle:liver infection ratio ranges from 2 to 8. In some embodiments, the administration results in expression of the exogenous polynucleotide in the muscle cell, at a muscle:liver expression ratio of greater than 10. In some embodiments, the muscle:liver expression ratio ranges from 10 to 100. In some embodiments, the muscle:liver expression ratio ranges from 20 to 80. In some embodiments, 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.Atty Docket No.: 38053-57988 / WO (112WO) In some embodiments, the muscle cell is selected from triceps surae, biceps, heart and quadricep.

[0040] In another aspect, provided herein is 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. In some embodiments, the target cell is heart and X7X8X9X10is FVQR, IHNL, KERF, LDEL, LIGR, QSTL, RTNL, or VRTL; the target cell is muscle and X7X8X9X10is ARTL, FNNL, FNNT, FQNT, HVNL, RGQI, RGVV, RNVV, RQGI, or RSVV; or the target cell is skeletal muscle and X7X8X9X10is ISRT or RSQT.

[0041] In some embodiments, the method comprises administering 1e12 vg / kg to 1e14 vg / kg dose of rAAV to the subject. In some embodiments, 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.

[0044] 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.

[0045] FIGs.3A-3D provide the sequence alignment of VP1 sequences of ancestral AAVs using AAV2 as a reference. The location of the liver toggle sites, residue 168 (FIG.3A), and residue 266 (FIG.3B), and the insertion site of a targeting peptide (FIG.3C), are indicated. One or more representative member sequences for each of the Anc80, Anc81, Anc82, Anc83, Anc84, Anc94, Ac110, Anc113, Anc 126 and Anc127 libraries were used for the alignment. 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. Anti-GFP immunohistochemistry was performed on liver with vehicle (FIG.4A), AAV9 (FIG.4B), AAVmut1(FIG.4C), AAVdeco1(FIG.4D), or AAVmut1-deco1(FIG.4E); and skeletal muscle (quadriceps) tissue cross-sections of mice injected with vehicle (FIG.4F), AAV9 (FIG.4G), AAVmut1(FIG.4H), AAVdeco1(FIG.4I), or AAVmut1-deco1(FIG.4J).

[0047] 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).

[0048] FIGs.6A-6E show eGFP 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 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.6A), heart (FIG.6B), triceps surae (FIG.6C), quadriceps (FIG.6D), or diaphragm (FIG.6E).

[0049] FIGs.7A-7D show eGFP vector genome (DNA) and eGFP expression (mRNA) in liver and quad tissues of C57BL / 6 mice treated with vehicle, AAVMut1and AAVMut1-deco1AAV 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.

[0050] FIG.8 shows eGFP mRNA expression in various tissues of BalbC mice treated with vehicle, AAVmut1and AAVmut1-deco1AAV 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).

[0051] FIGs.9A and 9B show exemplary IHC tissue analysis obtained from of Run 1 samples from NHPs. 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 anAtty Docket No.: 38053-57988 / WO (112WO) AAVmut1_deco1vector treated NHP; exemplary IHC quadriceps tissue is shown in FIG.9B, obtained from AAV9 vector treated NHP on left and AAVmut1_deco1vector treated NHP on the right.

[0052] 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 AAVmut1_deco1vector.

[0053] 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 AAVmut1_deco1vector.

[0054] 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 AAVmut1_deco1vector.

[0055] 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 AAVmut1_deco1vector.

[0056] FIG.14 shows the % GFP positive cells in various cardiac tissues (average from Runs 1 and 2) obtained from NHPs administered vehicle, AAV9 or AAVmut1_deco1vector.

[0057] 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 AAVmut1_deco1vector.

[0058] 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 AAVmut1_deco1vectors.

[0059] 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 AAVmut1_deco1vectors. 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0060] 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 AAVmut1deco1on the right of each plot) and indicating whether the sample was taken from the left or right side of the organ / animal.

[0061] FIGs.19A-19D show the results of mRNA transcript analysis measured by eGFP copies of eGFP over RPP30 mRNA. FIG.19A (liver), FIG.19B (quadriceps), FIG.19C (biceps), and FIG.19D (heart), are illustrated where the x-axis represents AAV vectors (wild type AAV9 on the left and AAVmut1deco1on the right) and indicating whether the sample was taken from the left or right side of the organ / animal.

[0062] FIG.20 shows human MTM1 protein expression in RD cells. The expression level of human MTM protein was determined by automated JESS-ProteinSimple instrument. Each bar represents by peak area values of JESS, either before (blue) or after (orange) being normalized to total protein load. Data were obtained from one run using the 1:4 dilution as described in the western protocol.

[0063] FIG.21 provides a study design to assess distribution patterns of Anc80 variants in non-human primates, as described in Example 8.

[0064] 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).

[0065] 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.

[0066] 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. On the x-axis, “0” represents amino acid residue “G”, and “1.0” represents amino acid “A”.Atty Docket No.: 38053-57988 / WO (112WO)

[0067] 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”.

[0068] 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”. The upper right (UR) quadrant had high biodistribution of 3 Anc80 variants in the quadricep muscle with liver toggle on and off. The lower left (LR) quadrant had low biodistribution of 56 Anc80 variants in the quadricep muscle with liver toggle on. The lower right (LL) quadrant had higher distribution of 891 Anc80 variants in the quadricep muscle with liver toggle on “liver on” and off.

[0070] 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”. The upper right (UR) quadrant had high biodistribution of 125 Anc80 variants with liver toggle on and off. The lower left (LL) quadrant had low biodistribution of 271 Anc80 variants in the quadricep muscle with liver toggle on and off. The lower right (LR) quadrant had lower distribution of 566 Anc80 variants in the quadricep muscle with liver toggle on “liver on”.

[0071] 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. To use the limited data from two primates to evaluate the low doses of each variant in the ANc80 library, the average fold- enrichment of each variant was used rather than rely soley on enrichments from each animal.

[0072] 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 69Atty 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. The lower left (LL) quadrant had low biodistribution of 687 Anc80 variants in the quadricep muscle with liver toggle on and off. The lower right (LR) quadrant had lower distribution of 257 Anc80 variants in the quadricep muscle with liver toggle on “liver on”.

[0073] 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). “0” represents the color green, while 1 represents the color red. Variants having different amino acids at the variation positions are color coded. Variants were sorted and ranked in decreasing order by liver on fold-change of tissue enrichment in quadricep muscle. As shown, position P3 contains amino acid G in red for all 100 Anc80 variants.

[0074] 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.

[0075] 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”. As shown in the bottom left scatter plot, 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).

[0076] 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”, and Y3 denotes amino acid residue “D”, in X1X2X3RGDX7X8X9X10 described in the present disclosure. Y4 is denoted as “X7” of X1X2X3RGDX7X8X9X10, Y5 is denoted as “X8” of X1X2X3RGDX7X8X9X10, Y6 is denoted as X9” of X1X2X3RGDX7X8X9X10, and Y7 is denoted as “X10” of X1X2X3RGDX7X8X9X10. Example amino acid modifications of Y4, Y5, Y6, and Y7 regions of the targeting peptide include those described herein.Atty Docket No.: 38053-57988 / WO (112WO)

[0077] 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 X1X2X3RGDX7X8X9X10), 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. The various colors represent the amino acid at the particular position.

[0078] 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. The cumulative density of variants is shown at specific tissue enrichment scores (Log2(fold)). The lines are colored by the probability of amino acids at a particular position.

[0079] 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.

[0080] FIG.37D shows a sequence motif enriched in muscle tropic capsids.

[0081] FIG.37E shows a network comparison of the targeting peptides described herein to literature myopeptides.

[0082] 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 scatterAtty Docket No.: 38053-57988 / WO (112WO) plot show that SEQ ID NO: 238 (“ATLVT013XX38181”) outperformed wild-type AAV9, AAV9deco1(AAV9 capsid with the deco1 peptide), and AAV9myo3E(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.

[0083] 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.

[0084] 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).

[0085] 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.

[0086] FIG.41, 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”). Additionally, the present inventors created additional targeting peptides 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: 44880 and the 4 amino acids after the amino acids “RGD” in the targeting peptide having an amino acid sequence of SEQ ID NO: 44880.Atty Docket No.: 38053-57988 / WO (112WO)

[0087] 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.

[0088] 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. Surprisingly, as shown in the network plot, 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. Additionally, MYODV6 and MYODV8 each have a higher overall log2 fold change of tissue enrichment in each muscle tissue compared to the targeting peptide having an amino acid sequence of SEQ ID NO: 44880.

[0089] FIG.43 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 H1 (RGDLIGR (SEQ ID NO: 1422)) targeting peptide located in VR VIII between amino acids at positions 588 and 589 or an H2 (RGDQSTL (SEQ ID NO: 3052)) targeting peptide located in VR VIII between amino acids at positions 588 and 589. Muscle tissues analyzed included biceps femoris, diaphragm, tibialis anterior, triceps brachii, and ventricle wall of the heart. Higher tissue enrichment score values represent greater tropism.

[0090] 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. 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.

[0091] FIG.45 shows a plot for median percent GFP+cells for liver and the indicated muscle tissues. Capsids tested included AAV9 and AAV9 comprising an M3 (“DV6”Atty Docket No.: 38053-57988 / WO (112WO) 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); 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); and a mut1 VR I substitution, or 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) with a wild type VR I. Higher percent GFP+cells represent greater tropism.

[0092] 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. Capsids tested included 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); 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) and a mut1 VR I substitution, or 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) with a wild type VR I.

[0094] 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0095] 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-Lib1capsid library.

[0096] FIG.49B provides a sequence alignment of variable region I in certain AAV variants using AAV2 variable region I as a reference for numbering.

[0097] FIG.50 shows vector genome (DNA) in liver of C57BL / 6 mice treated with the AAV-Lib1Library. Log2Fold DNA levels are presented for each of the 3,456 capsid variants (variant ID on x-axis) in addition to AAV9 and AAVmut1. Log2Fold DNA levels of AAV9 and AAVmut1are identified specifically in FIG.50.

[0098] 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-Lib1library: alanine (A) and glycine (G).

[0099] 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-Lib1that 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-Lib1that include a glycine (G) at P6(amino acid position 266).

[0100] 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-Lib1that include an alanine (A) at P6(amino acid position 266) and the amount of vector DNA in the liver (Log2(Fold)) for the sum of all capsids in AAV-Lib1that include a glycine (G) at P6(amino acid position 266).

[0101] 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. In addition, FIG.52A provides an example formula for calculating an enrichment score for the modified capsid proteins in the AAV-Lib1library.

[0102] 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-Lib1library that include either an alanine (A), a glutamate (E), a glutamine (Q), and a threonine (T) at P3(amino acid position 263).Atty Docket No.: 38053-57988 / WO (112WO)

[0103] 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-Lib1library that include either an alanine (A) or a glycine (G) at P5(amino acid position 265).

[0104] 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-Lib1library that include either an alanine (A) or a glycine (G) at P6(amino acid position 266).

[0105] FIG.53A is an enrichment plot showing the amount of vector DNA in the liver for the sum of all the capsids the AAV-Lib1library that include either an alanine (A) or a glycine (G) at P6 (amino acid position 266).

[0106] FIG.53B is an enrichment plot showing the amount of vector DNA in the liver for the sum of all the capsids the AAV-Lib1library having a P5P6combination of AA, AG, GA, or GG.

[0107] 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-Lib1library having a P3, P5, and P6combination 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).

[0108] FIG.54 shows output of a network analysis of data from the AAV-Lib1library. Each circle indicates a capsid variant and a line connecting the capsids indicates sequence similarity.

[0109] FIG.55 is an illustration of the structure of an AAV VP1 protein with positions P3, P5, P6, and P8in VRI highlighted.

[0110] 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-Lib1library that include an alanine (A), a glutamate (E), a glutamine (Q), or a threonine (T) at P3(amino acid position 263).

[0111] 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-Lib1library that include an alanine (A), a glutamate (E), a glutamine (Q), or a threonine (T) at P3(amino acid position 263).Atty Docket No.: 38053-57988 / WO (112WO)

[0112] 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-Lib1library that include either an alanine (A) or a glycine (G) at P5(amino acid position 265).

[0113] 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-Lib1library that include either an alanine (A) or a glycine (G) at P5(amino acid position 265).

[0114] 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-Lib1library that include either an alanine (A) or a glycine (G) at P6(amino acid position 266).

[0115] 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-Lib1library that include either an alanine (A) or a glycine (G) at P6 (amino acid position 266).

[0116] 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 AAVmut1and AAV9 controls are indicated by arrows and corresponding text.

[0117] 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 P3, P5, and P6. Positions of AAVmut1and AAV9 controls are indicated by arrows and corresponding text.

[0118] 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 P3, P5, and P6, respectively. Positions of AAVmut1and AAV9 controls are indicated by arrows and corresponding text.

[0119] 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 AAVmut1and AAV9 controls are indicated by arrows and corresponding text.

[0120] 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 ofAtty 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 AAVmut1and AAV9 controls are indicated by arrows and corresponding text.

[0121] 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 P3, P5, and P6, respectively. Each dot represent a capsid having TGG at P3, P5, and P6. 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)) are identified and shaded accordingly. Particular subpopulations having NGTT, NSTT, SGAA, and SGAT at positions P1, P2, P4, and P12 are identified. Positions of AAVmut1and AAV9 controls are indicated by arrows and corresponding text.

[0122] 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 P3, P5, and P6. A subpopulation having a serine (S), a glycine (G), a threonine, and a histidine (H) at positions at positions P1, P2, P4, and P12is identified. Positions of AAVmut1and AAV9 controls are indicated by arrows and corresponding text.

[0123] FIG.58F shows average enrichment in liver RNA (x-axis) plotted against enrichment in heart RNA (y-axis) for all AAV-Lib1capsids having a threonine (T), a glycine (G), and a glycine (G) combination of amino acid residues at P3, P5, and P6, respectively. Positions of AAVmut1and AAV9 controls are indicated by corresponding text.

[0124] 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. The 24 selected capsids correspond to groups: 1 NSTSGGP7P8NDNH (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: SGTTGGP7P8NDNT (SEQ ID NO: 44932); and group 7: SSTAGGP7P8NDNA (SEQ ID NO: 44933). Positions of AAVmut1and AAV9 controls are indicated by corresponding text.

[0125] 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), aAtty 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.

[0126] FIG.59 shows vector genome (DNA) in liver of NHP treated with the AAV- Lib1 Library. Log2Fold DNA levels are presented for each of the 3,456 capsid variants (variant ID on x-axis) in addition to AAV9 and AAVmut1. Log2Fold DNA levels of AAV9 and AAVmut1 are identified specifically in FIG.59.

[0127] 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).

[0128] FIG.61 is a plot showing liver enrichment for rAAV from AAV-Lib1in mice (y-axis) versus liver enrichment for rAAV from AAV-Lib1in NHP (x-axis). Correlation is indicated by 0.72 R^2-value. Capsids plotted on FIG.61 include capsids having a peptide segment with a P3TP5GP6G motif.

[0129] 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 X1, X2, and X3where 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 X7, X8, X9, and X10, where the color indicates the type of amino acid residue.

[0130] FIGs.63A-63B show an enrichment plots (Avg_logFC versus Inverse CV (Coefficient of Variation)) for capsids in the AAV-Lib2library. FIG.63A is an enrichment plot showing capsid performance in muscle (i.e., all muscle tissue) for all 50,500 capsids in the AAV-Lib2library. FIG.63B shows enrichment plots for capsids from the AAV-Lib2library having a quad selected from: FNNL, FNNT, and RGQI.

[0131] 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-Lib2library having the indicated quad sequences.Atty Docket No.: 38053-57988 / WO (112WO)

[0132] FIG.65 shows a heatmap of tissue enrichment scores in muscle tissues for each of the capsids in the AAV-Lib2library. AAV-Lib2library includes about 50,500 capsids having one of the 20 quad and one of XX triplet sequences. Data for each capsid appears as a cell in the heatmap where the row indicates the quad and the column indicates the triplet. Data is grouped using hierarchical clustering. Clustering identified Group I, Group II, and Group III.

[0133] FIG.66A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV-Lib2library 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.

[0134] FIG.66B shows a motif plot of the targeting peptides for the top 100 capsids in the AAV-Lib2library having a quad selected from: RGQI, RSVV, and RGVV (Group 1). Data used to generate the motif plot is averaged across all muscle tissue.

[0135] FIG.66C shows a plot of unique variant count for the indicated triplets for the top 100 capsids in the AAV-Lib2library having a quad selected from: RGQI, RSVV, and RGVV (Group 1).

[0136] FIG.67A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV-Lib2library 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.

[0137] FIG.67B shows a motif plot of the targeting peptides for the top 100 capsids in the AAV-Lib2library 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.

[0138] FIG.67C shows a plot of unique variant count for the indicated triplets for the top 100 capsids in the AAV-Lib2library having a quad selected from: HGVL, YSSV, YSTM, and YTSV (Group 2).

[0139] FIG.68A shows enrichment scores averaged across all muscle tissue for each of the capsids in the AAV-Lib2library 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.

[0140] FIG.68B shows a motif plot of the targeting peptides for the top 100 capsids in the AAV-Lib2library 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)

[0141] FIG.68C shows a plot of unique variant count for the indicated triplets for the top 100 capsids in the AAV-Lib2library having a quad selected from: FNNT, FNNL, FQNT, and YNSL (Group 3).

[0142] 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-LIB2library having the indicated quad sequences.

[0143] FIG.70A shows enrichment scores for liver tissue for each of the capsids in the AAV-Lib2library 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.

[0144] FIG.70B shows a motif plot of the targeting peptides for the top 100 capsids in the AAV-Lib2library (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.

[0145] 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-Lib2library. The top 10 performing capsids are identified by the darker shaded circles.

[0146] FIG.71B shows a table of enrichment scores for the top 10 capsids, where the top 10 capsids include the indicated targeting peptide.

[0147] 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.

[0148] 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.

[0149] FIG.72B shows a table of the amino acid residues at each position or the targeting peptides described in FIG.72A.

[0150] 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.

[0151] 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.73AAtty Docket No.: 38053-57988 / WO (112WO)

[0152] FIG.74A shows enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV-Lib2library. The 10 capsids are highlighted.

[0153] FIG.74B shows enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV-Lib2library. The 10 capsids are highlighted.

[0154] FIG.75A shows enrichment scores in skeletal muscle versus enrichment scores heart for each of the capsids in the AAV-Lib2library.

[0155] FIG.75B shows enrichment scores in skeletal muscle versus enrichment scores in heart for each of the capsids in the AAV-Lib2library having the quad “LIGR”.

[0156] FIG.75C shows enrichment scores in skeletal muscle versus enrichment scores in heart for each of the capsids in the AAV-Lib2library having the quad (“QSTL”).

[0157] FIG.75D shows enrichment scores in skeletal muscle versus enrichment scores in heart for each of the capsids in the AAV-Lib2library having the quad (“RGVV”).

[0158] FIG.75E shows enrichment scores for heart over enrichment scores in skeletal muscle for each of the capsids in the AAV-Lib2library having the indicated quads.

[0159] 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-Lib3library 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.

[0160] FIG.77 shows the scaled enrichment scores of capsids having a targeting peptide with the indicated VR1 and VR8 modifications. As non-limiting examples, 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 aAtty 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.

[0161] FIGs.78A, 78B, and 78C show the enrichment scores in muscle versus liver for the capsids in the AAV-Lib3library. FIG.78A shows the overall enrichment scores in muscle versus liver for the all capsids in the AAV-Lib3library. As non-limiting examples, 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 ofAtty 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.

[0162] FIG.79 shows the box plot of enrichment scores in muscle tissue for the capsids in the AAV-Lib3library having the indicated quad sequences and the indicated VR1 sequences.

[0163] FIG.80 shows that the performance of selected quads with different VR1 backgrounds, measured in the AAV-Lib2library study (x-axis) or AAV-Lib3library study (y- axis).

[0164] FIG.81 shows a heatmap of tissue enrichment scores in muscle tissues for each of the capsids in the AAV-Lib3library. 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.

[0165] 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.

[0166] 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.

[0167] FIG 84 show motif plots that identify the enriched motifs of Quads cluster 3 combined with Triplets cluster1.

[0168] 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0169] FIG.86 shows a heatmap of tissue enrichment scores in liver tissues for each of the capsids in the AAV-Lib3library. 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.

[0170] 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.

[0171] FIGs.88A and 88B show the heart versus skeletal muscle enrichment scores of the capsids in the AAV-Lib3library. FIG.88A shows the overall enrichment scores in skeletal muscle versus heart muscle for the all capsids in the AAV-Lib3library. FIG.88B shows the enrichment scores for heart muscle over skeletal muscle for the capsids in the AAV-Lib3library having the indicated quads.

[0172] 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.

[0173] 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).

[0174] 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.

[0175] 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.

[0176] 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 showingAtty 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] FIG.98 shows the immunohistochemistry (IHC) staining in different heart region sections of NHPs received M1 or AAV9 expressing eGFP at low dose level.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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

[0186] “AAV” is adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.

[0187] The term “AAV capsid protein” or simply “capsid protein” refers to a VP1, VP2, or VP3 capsid protein. In some embodiments, 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; rh.13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu.9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.1-C; hu.18-C; hu.3-C; hu.25-C; hu.15-C; hu.16-C; hu.11-C; hu.10-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu.17-E; hu.6-E; hu.66-E; rh.38- E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; Anc113; Anc126; Anc127; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc110; and Anc80DI.

[0188] The term “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.

[0189] The term “targeting peptide” as used herein refers to a 10 amino acid sequence (X1X2X3RGDX7X8X9X10) 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0190] The term “peptide segment” as used herein 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 P1P2P3P4P5P6P7P8P9P10P11P12within VR I where “Pn” refers to a position in the peptide segment.

[0191] The term “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. As used herein, the term "amino acid" comprises naturally occurring L- and D- amino acids and artificial, i.e. non-naturally occurring, α-amino acids. Preferably, the amino acid is a naturally occurring amino acid. In preferred embodiments, the amino acid is a naturally occurring L-α-amino acid.

[0192] For the avoidance of doubt, as used herein, 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.

[0193] 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. The term “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. The term “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.

[0194] The term “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. The term “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.

[0195] The term “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.Atty Docket No.: 38053-57988 / WO (112WO)

[0196] The term “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.

[0197] The term “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. In some instances, 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.

[0198] 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.

[0199] 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).

[0200] The term “inducible” 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 substantially only when an inducer which corresponds to the promoter is present in the cell.

[0201] As used herein, the term “internalizing moiety” refers to a moiety capable of interacting with a target tissue or a cell type to effect delivery of the attached molecule intoAtty Docket No.: 38053-57988 / WO (112WO) the cell (i.e., penetrate desired cell; transport across a cellular membrane). In certain embodiments, an MTM1 polypeptide encoded by the rAAV of the disclosure can be a fusion protein comprising an internalizing moiety. In some embodiments, the internalizing moiety selectively, although not necessarily exclusively, targets and penetrates muscle cells. In certain embodiments, the internalizing moiety has limited cross-reactivity, and thus preferentially targets a particular cell or tissue type. In certain embodiments, 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. In certain embodiments, the internalizing moiety mediates transit across cellular membranes via an ENT2 transporter. Exemplary internalizing moieties are disclosed in U.S. Patent No.9447394 B2, the contents of which are incorporated by reference herein.

[0202] The term “inverted terminal repeat” (or “ITR”) refers to a polynucleotide sequence found at the ends of AAV genomes that form a hairpin, which contributes to the genome’s ability to self-prime (allowing for primase-independent synthesis of the complementary second DNA strand) and provides for encapsidation of the genome into an AAV particle. An ITR can be a wild-type ITR or a variant thereof.

[0203] The terms “liver-toggle mutant”, “liver-toggle mutant of a reference AAV capsid protein” and the like, as used herein, 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”). 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. In some embodiments, 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. In other embodiments, 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. In yet further embodiments, 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).

[0204] The term “modification” when in conjunction with an amino acid residue, amino acid residues, or a modified sequence, refers to insertion(s), deletion(s), and / or substitution(s).

[0205] The term “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.

[0206] The term “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.

[0207] The terms “operably linked” and “operatively linked” refer 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. For example, 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.

[0208] The term “parenteral” administration of a composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0209] The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.Atty Docket No.: 38053-57988 / WO (112WO)

[0210] The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, excipients, stabilizers and adjuvants. For examples of carriers, excipients, stabilizers and adjuvants, see Remington: The Science and Practice of Pharmacy, 22nd Revised Ed., Pharmaceutical Press, 2012.

[0211] The abbreviation “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.

[0212] The term “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. In general, 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. Thus, 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 “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.

[0213] The term “tissue-specific” promoter or ERE as used herein 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.

[0214] The terms “treatment”, “treating”, and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. The effect may beAtty 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” as used herein 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).

[0215] The terms “vector”, “AAV vector” and “rAAV vector” refer to an rAAV that comprises a heterologous polynucleotide, e.g., a transgene.

[0216] The terms “variable region” or “VR” as used herein refer 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.

[0217] Unless otherwise defined, 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.Atty Docket No.: 38053-57988 / WO (112WO) 6.2. Modified AAV capsid proteins

[0218] 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 X10are independently selected from any amino acid residue, wherein the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8NDNP12and P1,P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue. In some embodiments, 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.

[0219] One aspect of the present disclosure provides 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 X10are independently selected from any amino acid residue, wherein the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8NDNP12and P1,P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue.

[0220] In some embodiments, the modified AAV capsid protein does not comprise RGDLLLS (SEQ ID NO: 1).

[0221] In some embodiments, the modified AAV capsid protein has a peptide segment that does not comprise an alanine (A) at P6 and a threonine (T) at P8

[0222] In some embodiments, the modified AAV capsid does not comprise RGDLLLS (SEQ ID NO: 1), and the peptide segment does not comprise an alanine (A) at P6and a threonine (T) at P8.

[0223] In some embodiments, 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)

[0224] In some embodiments, 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.

[0225] In some embodiments, the capsid protein further comprises one or more modifications outside of the VR I and VR VIII of the reference AAV capsid protein.

[0226] In some embodiments, 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).

[0227] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0228] In some embodiments, 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. In some embodiments, 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. In some embodiments, the one or more modifications outside of the VR VIII is a modification in the VR I region of the reference AAV capsid protein. In some embodiments, the one or more modifications results in a liver-off phenotype. In some embodiments, the one or more modifications outside of the VR VIII is a liver toggle mutation. 6.2.1. Targeting peptide

[0229] In some embodiments, the targeting peptide has a sequence X1X2X3RGDX7X8X9X10 , wherein X1, X2, X3, X7, X8, X9 and X10 are independently selectedAtty Docket No.: 38053-57988 / WO (112WO) from any amino acid residue. In some embodiments, 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. In some embodiments, X7, X8, X9and X10are independently selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, and V.

[0230] In some embodiments, 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. In some embodiments, 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.

[0231] In some embodiments, X1, X2, and X3are independently selected from any amino acid residue.

[0232] In yet other embodiments, X1, X2, and X3 have not been modified from the amino acids at corresponding positions of the reference AAV capsid protein. In this case, X1, X2, and X3are identical to the amino acids at corresponding positions of the reference AAV capsid protein.

[0233] In yet other embodiments, X1, and X2have not been modified from the amino acids at corresponding positions of the reference AAV capsid protein. In yet other embodiments, X1 has not been modified from the amino acids at corresponding position of the reference AAV capsid protein. In yet other embodiments, X2 has not been modified from the amino acids at corresponding position of the reference AAV capsid protein. In some embodiments, X1, X2, and / or X3 are the natural amino acid residues of the reference AAV capsid protein.

[0234] X1, X2, and X3can be introduced by amino acid substitutions, insertions, mutations, and / or deletions of the amino acids at the corresponding sites in the reference AAV capsid. For example, in some embodiments, any one of the corresponding sites X1, X2, and X3in the reference AAV capsid can be deleted or substituted. In some embodiments, theAtty Docket No.: 38053-57988 / WO (112WO) corresponding site X1 in the reference AAV capsid is deleted or substituted. In some embodiments, the corresponding site X2 in the reference AAV capsid is deleted or substituted. In some embodiments, the corresponding site X3in 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 X1and X3in 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.

[0235] In some embodiments, X1is selected from S, E, A, D, N, Q, or T. In some embodiments, X1 is D or E. In some embodiments, 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, X1is E.

[0236] 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, X2is selected from K, E, D, A, S, F, N, V or L. In some embodiments, X2is selected from K, E or D. In some embodiments, X2is selected from N, A or Y. In some embodiments, X2 is selected from N, Y or S.

[0237] In some embodiments, X3is 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, X2is N. In some embodiments, X2is A. In some embodiments, X3 is selected from R or Q. In some embodiments, X3 is R. In some embodiments, X3is Q. In some embodiments, X2is N; and X3is R. In some embodiments, X1is E; X2is N; and X3is R. In some embodiments, X1is S; X2is N; and X3is R. In some embodiments, X3 is selected from Y, V or F. In some embodiments, X3 is selected from N, A, Y or S. In some embodiments, X3 is selected from I, Q, R, V, T or M.

[0238] In some embodiments, X2X3is NR. In some embodiments, X1X2X3is ENR. In some embodiments, X1X2X3 is SNR.

[0239] In some embodiments, 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, DMV, DMI, EPH, QEG, DIN, NEI, EYY, DIV, SEG, DVG, DYQ, EGF, NDI, EGY, DVF, DVH, DGF, DIY, DSF, DGW, EHY, DRE, TEH, DTS, NEN, NEM, NEH, TEN, DSN, DVT, DQS, DKD, DTH, DVV, DQK, NET, DKP, TEV, NDS, QET, EVL, SET, SDT, AEQ, QEF, SEY, SEF, SPF, EGQ, ETH, TDQ, QEA, QDQ, AEN, ESS, NDT, EFM, EFI, EHM, DFM, QDT, SME, DYT, EHV, ENV, EAV, EAI, ESI, DAT, ENQ, EAM, ADN, EFQ, SDS, TDH, SDH, DAS, TWE, SSF, DRD, EFL, TDF, QDA, EMH, SGE, AEW, DAH, TET, TDM, TNE, SAE, NSE, SFE, QDI, DSA, ADV, SEI, AEI, QDV, ADT, DNM, DNQ, ADL, TDL, SDL, SDM, TDV, DNI, DHY, DTY, DAA, DSY, QLY, DVM, DAY, DMT, DQT, DAQ, DTV, DSH, QDF, DST, DNL, DSI, DFV, DNY, DAF, DKI, DKF, DTM, DSL, NDV, TDI, DSV, DAV, DKV, DAM, DNV, DKM, DKL, DKW, DSM, ENI, SDI, DKT, QGE, NNE, QYW, AGM, ESH, QDH, QEH, DYH, ENS, DKQ, TGE, DSS, EST, DSQ, DNS, EFV, SWE, DKN, DKS, SEH, EAL, EAS, ADQ, SDQ, ADI, SDV, EAH, ASE, EAF, EYV, DKA, SNE, AGE, EGV, EQF, ETF, EVY, EQY, QEY, QEN, SAD, AEF, EMF, SDF, ADF, ADY, AVF, TLY, SIY, SLY, AIY, DYV, AEY, ENF, EMY, QDY, ETY, EAY, TVY, SVY, QVF, QVY, TMY, DKY, ALY, AVY, TMW, EQW, SPW, SIW, DTQ, TFF, AMF, EYT, DRV, SPY, SIF, SLF, ENY, EWY, EYI, EFY, TLF, TVF, TYY, TFY, SVF, SYW, TAW, SAW, TIY, NIM, NFM, NII, NIV, SII, SIV, SEM, TTI, NSY, SSY, SNY, SEV, AEH, ALT, SGQ, QTY, SYY, AMY, QMY, SGF, SGY, ALF, TTF, SFN, QAF, ASF, SAF, TAF, TAY, TGL, SAY, QAY, ADH, ASI, SSV, SSI, TGV, SGV, DPH, TEG, DLY, ESE, TEF, NPE, DHA, DLI, DWG, EWM, EET, EIL, TEL, DYL, EMW, EVW, EPV, EDV, SLD, EGH, QRY, TDG, SQE, ELH, EYW, EMM, EFF, QEI, EQG, DVS, ESP, EIQ, EPM, EPQ, SPE, EPN, NQD, ELQ, TDT, ELT, EPI, ETM, ETG, EHI, EMI, EIT, ETV, EVV, EVM, TPY, QEW, EIW, EPT, EIH, DFY, DQL, NDA, DIW, TDY, ELF, DVY, ESG, EPG, EIG, ELG, EVG, DHI, EIS, DYM, DFT, DFH, EQS, TPF, EEF, DWN, SDN, EYL, AET, DMN, QSP, DNA, DHM, DHT, EQI, ESN, EYF, EHF, NAD, AEG, AES, TEM, SEL, DYS, EVH, EAN, EGL, TFE, ETL, TYE, SHE, ELM, TYD, SYE, ESA, EFT, EAT, AMD, AND, DMS, QMD, SLQ, EYS, QDN, EYQ, EKD, QYD, QFD, DER, AFD, EAA, EFS, DHV, QSE, TSD, SFD, DGY, TWD, QWD, EHL, QGD, QSD, AWD, AGD, QIY, EMQ, EAG, ERD, DDR, DAL, QDL, DNT, EGN, ENH, NGE, DYN, DNH, DNF, ESQ, ESM, ESL, SSD, ENL, EMV, QDS, TSE, SWD, ASD, ENT, SSE, EGM, EGS, EGT, EGI, AIF, NIN, NIQ, NYW, EPW, EGG, TIW, QIW, TTW, EKW, NLY, SVM, TPT, AAW, AIH, TIH, QGT, QFF, QYF, QFY, EHH, QLH, TYF, STY, DRL, EYM, SFF, QLF, QIF, APF, TYW, QII, QPF, SPI, TIL, SVL, NVI, SIM, SLL, QEL, AEM, AEL, QEV, QDM, NDM, SYL, ASL, TPL, ANI, NMI, TIM, AEV, TMI, SWI, SFI, SNW, TNF, NAF, ASP, QSQ, APY, SFM,Atty Docket No.: 38053-57988 / WO (112WO) TAN, QAW, SGS, SGH, TGF, QSN, ERY, QMN, TGQ, NGM, NGV, AMN, ALN, QFV, AAF, TFV, TSY, EDK, ASN, SAV, QSV, QGV, QST, ASM, TAI, QSM, QSL, QSI, SSL, AGV, TGT, AGI, QGM, TGM, SGM, SGI, THD, QVE, ALE, QLE, EWP, AFP, AEE, ADE, NIE, TYP, QIE, TFP, SYP, ELW, DVW, NEW, EPA, EVA, TLE, AWE, NME, QME, ATE, AYE, DQM, TPD, DFG, QPE, EDI, EWL, ELL, QPD, EWF, ALD, SVD, QVD, ADA, APD, AHE, EQL, DTA, AFE, DAW, QAE, TME, STE, THE, QID, AAE, EIA, EML, SDA, SIE, TQE, TVD, NWE, NQE, EYG, QYP, EIV, EII, QFP, TID, EQN, EVS, TYG, EAQ, ELV, QLD, TLD, QQD, TIN, TLN, SPN, TPN, NFD, NEY, DSW, NMD, DGA, DYA, DIH, DNP, DQW, DQV, EFA, EPS, DFN, DWV, ELI, DTF, EQT, DHW, ESF, DEK, DMH, EWS, EHA, TTD, EWI, QLQ, SQD, QHD, NSD, EMA, TND, ATD, DGS, TAD, TMD, QES, AHD, QAD, DGH, DGL, NTE, ENG, SMD, NDQ, EMS, ENA, EMT, EYN, DDK, EWA, EWQ, EFH, QWE, DGT, DGM, EWV, EWH, EWT, QND, DGV, ESY, AAD, SND, ANE, TIG, QIG, AVD, SFP, AWP, QDW, SLW, TPA, AII, TIV, SWP, AFY, APN, AVN, TVN, TIS, TIQ, TLQ, AIQ, QVN, NFY, QFG, EMN, NVF, SYF, SFW, TPQ, AIL, TWI, QWN, TWN, QWQ, AQF, ASA, TWV, EWN, TMF, NGI, ATN, SAS, TMQ, TMN, AYN, TQF, SMF, QMQ, SMQ, QAN, AAY, SFQ, TYQ, TFQ, TWQ, AVI, and SGL.

[0240] In some embodiments, X1X2X3is 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, SVY, SWD, SWE, TDF, TDV, TDY, TEF, TEY, TIY, TLY, and TWQ.

[0241] In some embodiments, 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, AMF, STY, TNW, ANW, AWM, TSF, DRL, DRV, SPY, NVF, SIF, QLF, SLF, QIF, SYF, APF, AGF, SVF, SAW, TIY, DHK, DAK, EGK, DYK, QNL, QPL, SPV, NPM, STL, NIL, AGA, NTV, SQA, QQA, NNF, NWL, NMA, NNA, NHN, NFH, ELR, NYM, ERA, NFS, SRD, NMM, NNL, NNQ, NNS, QDR, SDK, NNI, AQH, ANA, QNH, QWG, AWA, QWA, EQK, SHS, QFA, SFA,Atty Docket No.: 38053-57988 / WO (112WO) TFA, AYG, EKQ, ERN, EMR, EFR, AYM, EAR, EMK, EYK, EWK, QPI, QPF, NPL, SPI, SPL, NPI, SIV, SLL, SSA, AEM, TML, TLL, SML, TYL, QDM, NSN, AQL, NAT, SQQ, QTI, NAH, SQL, STH, NTT, AEK, ANV, QGL, AGL, QMV, STM, NQM, STI, TQL, SMM, SKE, SQT, SYL, ASL, SLV, NAM, TTV, TPI, TPL, ATL, DNK, DSR, ANL, SHL, SNL, TNL, QNI, QAL, ANI, QNM, QYM, SQI, SNI, QNV, SHV, SHM, QMM, ANM, AHM, SQM, NAL, AQT, EKL, NNM, TNM, SNM, QQI, TQV, TQI, THM, SQV, AAV, THL, AQI, NNV, TNN, TNI, SNN, TQM, NAI, TNV, SWT, NMV, NQI, NQL, NMI, NSS, SHF, QNF, SWL, AQM, SYV, TFM, SYM, TYM, ATI, TTI, SLI, ALI, AYI, QLV, TMI, SMI, QFI, NMY, NAY, NMN, NMF, ANF, NWT, QWM, TFT, SWM, TWM, QNW, STF, AFM, NSY, SSY, SNY, THY, TNY, QNY, THF, NSV, ATY, SKD, TNF, TWT, QWT, SFS, TFS, DRQ, NYN, DFR, EKG, ENN, AWN, QWQ, TQH, ALV, QHQ, DVR, STS, SQS, AAA, QGQ, QSQ, QAQ, SIH, APY, QGN, AGQ, ASQ, AAQ, SQN, NQN, ERM, ALH, NYH, TGH, ATH, QTH, TQT, QQH, AQF, QMH, EKN, ERV, ERS, AFT, EKF, EKT, QSA, NFT, QYH, NYS, TSA, QYY, AYY, AHY, QFN, AWQ, NWQ, QAW, NMS, NWN, QMF, AMH, TSN, TAH, TMH, QFH, NWS, SWN, QRD, QKD, AKE, TSS, AYH, TSH, AHQ, ADK, ADR, ASA, ASS, SNS, QSS, SAH, SMH, QAH, AAH, AHN, QNS, QAS, ANS, ANN, SWA, SWS, TWS, ERI, EKS, QWS, AYS, AFS, SYS, QFS, QYN, QFT, AYT, TNS, TYS, SFT, QNQ, QYS, SYT, ANQ, SWV, SNQ, ANY, TNQ, ATF, SSQ, SSN, TWA, SGQ, SSS, TWY, SMY, TGN, TGY, QWY, SQF, TGF, QTY, TTY, AMY, QMY, ERY, TGQ, SGY, TMF, ALN, ARD, ATN, NAS, SAS, AQN, SYG, SYN, TFN, TMN, AYN, SGN, QNN, DKG, AAN, SMN, SAN, TTF, TQF, SFN, QAF, QFV, AAF, ASF, SAF, SMF, ASY, AFN, TAF, TSY, TAY, QMQ, SMQ, TYV, TGL, QAY, AAY, ADH, EDK, ASN, QYQ, SFQ, TYQ, TWQ, SWQ, AYQ, SYQ, AMV, TMV, TAV, AQV, TYT, TAT, QGI, QQV, SAQ, NAV, SAV, ASV, QSV, QAV, QAI, QAM, TAM, DTR, DQR, DMR, NNT, NYT, NMT, NST, ATT, SMV, SNT, QNT, AAT, SAM, SAL, QGV, QST, QYT, ASM, TSM, AMT, TST, ANH, SNV, TNT, ANT, SST, AST, TAI, DAR, SSM, TAL, NSM, TSL, SNF, TSI, TSV, TDK, SAT, SAI, QSL, QSI, ASI, SSL, SSV, SSI, AGI, QGM, TGM, SGM, SGL, SGV, ARL, NWE, SFL, TPY, DRP, DTK, DMK, SEY, SME, EAI, AHD, ADN, EFQ, EMH, SGE, DNQ, DNI, QLY, DGT, EGN, DYN, EWV, ESM, EAL, ASD, DKA, ADF, ADY, SLY, AEY, ETY, EAY, TGA, AVM, TPT, SVI, QSH, TTQ, QTN, TLH, AWY, SLT, AWV, QQY, NFV, SFY, TYY, SHA, TEK, QFK, NNY, EPR, NIT, NHI, NTY, SHY, NFM, NFN, NDK, NER, SER, NDR, QDK, SDR, QNA, TNA, THT, SNH, AYA, NFA, AFA, AHA, QHA, EYR, ENR, EHK, QVL, SVL, NIV, NVI, ADM, SAA, SQH, NQV, ALL, ATM, QYA, STT, DGR, NHT, SHT, QHT, TTL, NHM, QHL, DNR, QHM, QFM, QMI,Atty Docket No.: 38053-57988 / WO (112WO) AHV, THI, AHT, TNH, NHV, THV, SHI, ATV, TWI, NYV, TYI, DYR, SFI, NQF, NWM, NSL, NSP, TQY, DRS, ASP, TKD, QWI, QWN, TWN, QWV, THN, TTM, TQQ, NMQ, TYA, STQ, STN, AGN, EKH, ERT, TFH, SFH, AFH, ENK, NYQ, SFM, QGS, AHF, QLT, QHY, AQY, TRD, ERH, QWH, TYH, TRE, TAQ, AER, TDR, ASH, EKI, AFV, TYN, ALT, NGN, SYY, EWN, NGM, NAN, TAS, TFV, SFV, QAN, SGT, SAY, AFQ, TFQ, QFQ, EFK, SYH, QHV, AMM, THQ, AAI, AAM, NSQ, QAT, TSQ, SHQ, QSM, AGV, AGT, TGT, TGI, AVI, TGV, SGI, ESP, SDA, TPN, EAW, NEQ, DSN, DIK, DNA, QES, SLQ, EKD, SDH, SSF, TET, QDI, DSA, DNM, DKT, DGM, SQY, TLT, QEH, DKQ, TGE, DNS, SDV, SNE, EGT, EGV, AAP, AHG, TWK, TQA, TVT, APQ, SVT, NGT, SSH, NLV, AGY, QFF, QYF, NWV, EKA, EKY, SMG, NAG, ASG, TFI, ELK, SEK, QTL, NML, DHS, ETR, NQQ, QWL, ANP, AHH, NEK, NNN, SNA, EWR, ESR, SHN, SFG, SYA, APV, QPV, SPT, TPQ, QVI, TPV, AVL, SEM, NDM, API, QLM, QTM, QQL, QHI, NFL, AYV, NKE, TIM, QYV, SWI, DRN, AWI, SMA, NRE, QTS, QVV, QQT, QMA, QQS, QAA, ATQ, ERL, TGS, QQQ, AQQ, QHF, TAN, QSY, QSN, NGQ, TWH, TMS, QMS, TQN, TMT, QTV, THS, TTT, SMT, QMT, SMR, ADA, AAE, EPY, EYL, NWI, TSP, DGQ, NDH, QFD, EHL, ERD, DAF, ENI, ENH, QQW, AGM, ENL, EAF, EYV, ENT, SSE, AGE, QEY, DRH, NIQ, SVV, SVM, QPT, TVL, QTQ, SVQ, TIQ, NLQ, SIQ, AWL, EHH, NGY, NHF, NSA, NAA, NTH, QLI, NWH, QRE, AML, EVR, SNP, NHS, THH, NQS, TTS, AHS, EKV, NFQ, APL, QPM, NPV, SIL, TII, STV, QEL, TFL, SIT, NAQ, TMM, AAL, QQM, AMI, QFL, AHI, SYI, SSP, NVV, TTN, TAA, TQS, TMA, ALA, QQN, NSH, TTH, SRE, ERQ, QHS, SWH, SEV, AVT, ALQ, DKH, AMQ, ARE, TWV, NGL, NGV, TMQ, TLV, EER and QTT.

[0242] In some embodiments, X1X2X3is 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, TAT, TDR, TFM, THV, TNF, TNH, TNI, TNM, TNQ, TNV, TSY, TWA, and TYM.

[0243] In some embodiments, X1X2X3is 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, ADR, AHN, QNS, ANN, SWS, EKS, AFS, QFS, TNQ, TGY, NGL, ARD, AKD, AAF, SMF, AFN, SGT, TGL, ASN, SFQ, AFQ, TFQ, QFQ, EFK, SWQ, AYQ, SYQ, DQR, DMR, ASM, ANT, SHQ, TSL, SNF, QSL, ASI, SGL, SGI, NDA, DHQ, DFK, DTK, DNA, SDS, TDH, DGL, QDV, SDM, DKT, DGM, SQY, DKS, SSD, EMV, TSE, EYV, SSE, EGM, SDF, TTG, QLG, NNP, AGS, QSF, TLM, NGA, AWY, NKD, SWY, TFF, NSG, NGY, QEK, SEK, TEK, AQA, NVL, NHI, TER, NML, NMA, NNA, NHN, ANP, AMS, NER, SER, DKK, NMM, QDK, AHS, TNA, THT, SNH, QNH, ESK, SHS, AYM, SSA, NAT, QLM, QQM, SQI, SNI, QNV, SHV, SHM, NNV, TNN, AHT, TNH, NHV, TQM, THV, SHI, NYL, NMV, NFL, AYV, NQI, NQL, NMI, NSS, SHF, SYM, NYV, AYI, SWI, QFI, ANF, TFT, TWM, QNW, NSY, THY, TNY, NYN, AWN, SMA, NSH, TGH, SFH, SWH, SFM, NFT, QYH, QGS, TGS, TSA, QQQ, QYY, AMQ, TAN, AWH, NMS, TSN, TAH, TMH, QWH, NWS, SWN, QRD, TYH, AKE, TAQ, AER, AYH, ASA, SNS, QSS, SAH, SMH, ASH, QAS, ANS, SYS, QYN, TYN, TNS, SFT, ANQ, SGS, SGH, SSS, TWY, QTY, SYY, NGM, TMS, SYG, TQN, SYN, TFN, TMN, AYN, SGN, QNN, SMN, SAN, QAF, ASF, ASY, TAS, TAF, TFV, SMQ, SFV, TMT, QAN, SAY, EDK, AMV, QHV, THS, TAT, QGI, QQV, NAV, SAV, ASV, QSV, QAV, AAI, AAM, QAM, TAM, DTR, NNT, NYT, NMT, ATT, QTT, TTT, QAT, SMV, SNT, QNT, AAT, SAM, SAL, QGV, QST, QYT, TSM, ANH, SNV, TSQ, TNT, SST, AST, TAI, TSI, TSV, TDK, SAT, QSM, SAI, QSI, SSV, SSI, AGT, TGT, AGI, QGM, TGM, TGV, SGM, SGV, SQE, EHT, EYL, DSG, SFE, QND, TQA, AWV, NFV, TSG, SSG, DRG, ANW, TFI, TSF, QNL, STL, TWL, NNF, NND, NYM, NFI, ETR, SNP, EIR, NFS, NNH, NNL, AHL, NNQ, NDR, NNN, QDR, SDK, SNA, AFG, NYA, QHA, QFA, SHN, NFQ, SYA, TFA, EMR, EFR, EAR, NQM, TTL, TQL, SMM, SKE, SYL, ASL, SLV, TTV, AAL, NHL, NHM, QYL, QHL, ATL, DNR, DSR, ANL, SHL, SNL, TNL, QAL, AMI, ANI, QNM, QYM, QHM, QFM, QMM, QMI, ANM, AHM, SQM, AQT, EKL, NNM, TNM, SNM, QQI, TQV, TQI, THI, QHI, THL, SNN, TNV, SWL, AQM, SYV, TFM, TYM, TWI, AFI, ATI, DYR, QLV, TMI, SYI, SFI, NWM, NWT, NSL, STF, SNW, AFM, SSY, NSV, TNF, NAF, TFS, QWV, ERT, TQT, AFT, NYQ, ERL, NRD, TRD, AWQ, TDR, TSH, ASS, AYT, TYS, QYS, SYT, SWV, SNQ, SMS, NAS, SAS, QFV, SAF, TWQ, TAV, AMM, TYT, QAI, TST, DAR, SSM, TAL, NES, DWK, DLR, DIR, SDT, EAN, ETH, TYD, SYE, EAM, TND, AYD, TSP, DRT, NDH, SSF, EFH, DNQ, DSH, DNT, SDI, DKQ, EWT, EAL, EAS, ESL, ASD, EAH, ASE, EAF, ENT, SIY, QDY, APG, AIG, SSW, NPG, ERP, TVI, QSH, SLS, EDR, AWL, SLH, EQH, EHH, NWV, EKY, STY, DRI, EYH, DRV, SPY, NNY, DWR, ERA, QRE, EVR,Atty Docket No.: 38053-57988 / WO (112WO) SRD, AWA, SFA, QTI, SHT, THM, AQI, AHI, SWT, ATV, TTI, SMI, QYV, NMY, SWM, QNY, TWT, QWT, TQY, NRE, QTS, QHQ, DVR, AAA, TYA, EKH, ALH, AWT, ATH, SRE, TFH, QMH, ERV, ERS, QHY, ALQ, QSY, ERH, TRE, TSS, QAH, AAH, SWA, TWS, QWS, AYS, AFV, QNQ, ANY, ALT, SMY, TWV, TTY, AMY, QMY, ERY, TWH, TSY, TAY, TYV, QAY, AAY, QYQ, TYQ, SYH, AQV, EER, THQ, and SMT.

[0244] In some embodiments, X1X2X3is 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, SMH, SNA, SNE, SNF, SNI, SNQ, SNT, SRE, SST, SWQ, SWT, SYG, SYY, TFQ, THL, THQ, THV, TMI, TNL, TNS, TSH, TSQ, and TWQ.

[0245] In some embodiments, the targeting peptide comprises a 7-mer peptide (RGDX7X8X9X10) having an amino acid sequence selected from SEQ ID NOs.: 238-44858. In this case, X1X2X3can be the amino acids at the corresponding positions of the reference AAV capsid. For example, X1X2X3can be the naturally occurring amino acids at the corresponding positions of the reference AAV capsid. In some embodiments, the targeting peptide comprises a 7-mer peptide (RGDX7X8X9X10) 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.

[0246] In some embodiments, the modified AAV capsid protein comprises targeting peptide including a 7-mer peptide (RGDX7X8X9X10) having an amino acid sequence selected from SEQ ID NOs.: 238-247. In this case, X1X2X3 can be the amino acids at the corresponding positions of the reference AAV capsid. In some embodiments, 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. In some embodiments, 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. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs.: 238-737. In some embodiments, the 7-mer has aAtty 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.

[0247] In some embodiments, 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.

[0248] In some embodiments, the targeting peptide comprises a 7-mer peptide (RGD X7 X8 X9 X10) having an amino acid sequence of SEQ ID NO: 238.

[0249] In some embodiments, the targeting peptide comprises an amino acid sequence selected from SEQ ID NOs.:.44859- 44883, 44911, 44912, 44913, and 44918- 44919. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 peptideAtty 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. In some embodiments, 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. In certain embodiments, 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.

[0250] 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. In certain embodiments, 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. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44864. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44865. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44866. In some embodiments, the targeting peptide comprises an amino acid sequence of SEQ ID NO: 44867. In some embodiments, 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. In some embodiments, 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.

[0251] 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, X7is Y or H. In some embodiments, X7is F or Y.

[0252] In some embodiments, X8is 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, X8is T, G or S. In some embodiments, X8is N or Q. In some embodiments, X9is selected from S or V.

[0253] 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. In some embodiments, X9is selected from N or S.

[0254] In some embodiments, X10 is selected from I, V, S, L, M, R, T, and Q. In some embodiments, X10 is I or V. In some embodiments, X10 is I. X10 is V. In some embodiments, X10is V, L or M. In some embodiments, X10is T or L.

[0255] In some embodiments, the targeting peptide comprises a sequence of RGDRX8X9X10. In some embodiments, X8 is S or G. In some embodiments, X8 is S. In some embodiments, X8is G. In some embodiments, X9is selected from any amino acid residue. In some embodiments, X9is selected from V, S, N, G, and Q. In some embodiments, X9is V. In some embodiments, X9 is S. In some embodiments, X9 is N. In some embodiments, X9 is G. In some embodiments, X9is Q. In some embodiments, X10is selected from I, V, S, L and Q. In some embodiments, X10is I. In some embodiments, X10is V. In some embodiments, X10is S. In some embodiments, X10 is L. In some embodiments, X10 is Q. In some embodiments, X10 is I or V.

[0256] In some embodiments, 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), X1X2X3RGDRQGI (SEQ ID NO: 98929), X1X2X3RGDFQNT (SEQ ID NO: 98934), X1X2X3RGDHGVL (SEQ ID NO: 98938), X1X2X3RGDYTSV (SEQ ID NO: 98941), X1X2X3RGDYTSM (SEQ ID NO: 98942), X1X2X3RGDLTVT (SEQ ID NO: 98935), X1X2X3RGDFNNT (SEQ ID NO: 98943), X1X2X3RGDYSSV (SEQ ID NO: 98937),Atty Docket No.: 38053-57988 / WO (112WO) X1X2X3RGDHVNL (SEQ ID NO: 98924), X1X2X3RGDQSTL (SEQ ID NO: 98926), X1X2X3RGDLIGR (SEQ ID NO: 98925), X1X2X3RGDFNNL (SEQ ID NO: 98933), X1X2X3RGDLLLS (SEQ ID NO: 98932), X1X2X3RGDYNSL (SEQ ID NO: 98940), X1X2X3RGDRDYL (SEQ ID NO: 98939), or X1X2X3RGDYVGL (SEQ ID NO: 98936).

[0257] In some embodiments, the targeting peptide has a sequence of X1X2X3RGDRGVV (SEQ ID NO: 98928), X1X2X3RGDRSVV (SEQ ID NO: 98931) or X1X2X3RGDRGQI (SEQ ID NO: 98927).

[0258] In some embodiments, the targeting peptide has a sequence of X1X2X3RGDYTSV (SEQ ID NO: 98941), X1X2X3RGDYTSM (SEQ ID NO: 98942), X1X2X3RGDRGVV (SEQ ID NO: 98928), X1X2X3RGDRSVV (SEQ ID NO: 98931), X1X2X3RGDYSSV (SEQ ID NO: 98937), or X1X2X3RGDHGVL (SEQ ID NO: 98938).

[0259] In some embodiments, the targeting peptide has a sequence of X1X2X3RGDFQNT (SEQ ID NO: 98934), X1X2X3RGDHGVL (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 X1X2X3RGDYTSV (SEQ ID NO: 98941).

[0260] In some embodiments, X1X2X3is selected from the group consisting of EFK, AAY, DQK, QVY, DKL, DNV, ENF, EWK, QNV, and TFM.

[0261] In certain embodiments, the modified sequence comprises a 7-mer peptide selected from: RGDRSX9I, RGDRGX9I, RGDRSX9V, or RGDRGX9V.

[0262] In some embodiments, the targeting peptide has an amino acid sequence selected from X1X2X3RGDRGQI (SEQ ID NO: 98927), X1X2X3RGDRSVV (SEQ ID NO: 98931) or X1X2X3RGDRGVV (SEQ ID NO: 98928).

[0263] In some embodiments, 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, SVY, SWD, SWE, TDF, TDV, TDY, TEF, TEY, TIY, TLY, and TWQ.Atty Docket No.: 38053-57988 / WO (112WO)

[0264] In some embodiments, X1X2X3 is selected from the group consisting of: ETI, DQN, DLL, EKW, DNN, EYS, or TVF.

[0265] In some embodiments, X7is selected from Y and H; X8is selected from T, G and S; X9 is selected from S and V; X10 is selected from V, L and M

[0266] In some embodiments, the targeting peptide has an amino acid sequence selected from: X1X2X3RGDHGVL (SEQ ID NO: 98938), X1X2X3RGDYSSV (SEQ ID NO: 98937), X1X2X3RGDYTSM (SEQ ID NO: 98942) or X1X2X3RGDYTSV (SEQ ID NO: 98941).

[0267] In some embodiments, X1X2X3is 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, TAT, TDR, TFM, THV, TNF, TNH, TNI, TNM, TNQ, TNV, TSY, TWA, and TYM.

[0268] In some embodiments, X1X2X3is selected from TVF, APM, AQI, TTS, NQF, NFL or SMN.

[0269] In some embodiments, X7 is selected from F and Y; X8 is selected from N and Q; X9is selected from N and S; X10is selected from T and L.

[0270] In some embodiments, the targeting peptide has an amino acid sequence selected from: X1X2X3RGDFNNT (SEQ ID NO: 98943), X1X2X3RGDFNNL (SEQ ID NO: 98933), X1X2X3RGDFQNT (SEQ ID NO: 98934) or X1X2X3RGDYNSL (SEQ ID NO: 98940).

[0271] In some embodiments, 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, SMH, SNA, SNE, SNF, SNI, SNQ, SNT, SRE, SST, SWQ, SWT, SYG, SYY, TFQ, THL, THQ, THV, TMI, TNL, TNS, TSH, TSQ, and TWQ.Atty Docket No.: 38053-57988 / WO (112WO)

[0272] In some embodiments, X1X2X3 is selected from the group consisting of: TVF, APM, AQI, TTS, NQF, NFL or SMN.

[0273] In certain embodiments, 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), RGDRGQX10(SEQ ID NO: 157062), RGDRGX9V(SEQ ID NO: 157063), RGDRGX9I(SEQ ID NO: 157064), RGDRGX9S(SEQ ID NO: 157065), RGDRGX9L(SEQ ID NO: 157066), RGDRGX9Q(SEQ ID NO: 157067), RGDHX8X9L(SEQ ID NO: 157068), RGDRX8X9I(SEQ ID NO: 157069), RGDRX8X9V(SEQ ID NO: 157070), RGDRX8X9L(SEQ ID NO: 157071), RGDYX8X9L(SEQ ID NO: 157072), RGDYX8X9V(SEQ ID NO: 157073), RGDYX8X9M(SEQ ID NO: 157074), and RGDLX8X9T (SEQ ID NO: 157075).

[0274] In certain embodiments, the targeting peptide comprises a sequence of H1 (RGDLIGR (SEQ ID NO: 1422)) or a sequence of H2 (RGDQSTL (SEQ ID NO: 3052)). In some embodiments, 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. In such embodiments, 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.

[0275] In certain embodiments, 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)). In some embodiments, 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. In such embodiments, 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.

[0276] In certain embodiments, the modified sequence does not comprise a 7-mer peptide selected from: RGDRMVF, RGDRTVI, SRGDRPM, and ISLRGDR. In some embodiments, 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, theAtty 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. In some embodiments, 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 X1X2LRGDR, where X1and X2are independently any amino acid residue.

[0277] In some embodiments, the targeting peptide has a sequence selected from: X1X2X3RGDHVNL (SEQ ID NO: 98924); X1X2X3RGDLIGR (SEQ ID NO: 98925); X1X2X3RGDQSTL (SEQ ID NO: 98926); X1X2X3RGDRGQI (SEQ ID NO: 98927); X1X2X3RGDRGVV (SEQ ID NO: 98928); X1X2X3RGDRQGI (SEQ ID NO: 98929); X1X2X3RGDRSQT (SEQ ID NO: 98930); X1X2X3RGDRSVV (SEQ ID NO: 98931); X1X2X3RGDLLLS (SEQ ID NO: 98932); X1X2X3RGDFNNL (SEQ ID NO: 98933); X1X2X3RGDFQNT (SEQ ID NO: 98934); X1X2X3RGDLTVT (SEQ ID NO: 98935); X1X2X3RGDYVGL (SEQ ID NO: 98936); X1X2X3RGDYSSV (SEQ ID NO: 98937); X1X2X3RGDHGVL (SEQ ID NO: 98938); X1X2X3RGDRDYL (SEQ ID NO: 98939); X1X2X3RGDYNSL (SEQ ID NO: 98940); X1X2X3RGDYTSV (SEQ ID NO: 98941); X1X2X3RGDYTSM (SEQ ID NO: 98942); and X1X2X3RGDFNNT (SEQ ID NO: 98943). In some embodiments, X1is selected from S, E, A, D, N, Q, or T. In some embodiments, X1is selected from S or E. In some embodiments, X1is S. In some embodiments, X1is 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, X2is selected from N or A. In some embodiments, X2is N. In some embodiments, X2is A. In some embodiments, X3is 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, X3 is selected from R or Q. In some embodiments, X3 is R. In some embodiments, X3 is Q. In some embodiments, X2 is N; and X3is R. In some embodiments, X1is E; X2is N; and X3is R. In some embodiments, X1is S; X2 is N; and X3 is R.

[0278] In some embodiments, 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 AAVAtty 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.

[0279] In some embodiments, 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. In some embodiments, 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.

[0280] In some embodiments, 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.

[0281] Sequences of exemplary targeting peptides that can be used various embodiments of the present disclosure are provided in SEQ ID Nos: 1-53, 153-157, 160-162, 238-44858, 3881, 12092, 14601, 15342, 21498, 31396, 44859- 44883, 44910, 44911, 44912, 44913, 44918, 44919, and 48391-157057.

[0282] 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 ENRRGDYNSL (SEQ ID NO: 44859). 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 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% sequenceAtty Docket No.: 38053-57988 / WO (112WO) identity to the amino acid sequence of ENRRGDYNNT (SEQ ID NO: 44862). 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 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). 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 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). 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 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). 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 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). 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 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 ofAtty Docket No.: 38053-57988 / WO (112WO) SNRRGDFQNT (SEQ ID NO: 44876). 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 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). 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 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). 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 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). 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: 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. In some embodiments, 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)

[0283] In some embodiments, in the targeting peptide, 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, X7is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y. In some embodiments, X8 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y. In some embodiments, X9 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y. In some embodiments, X10is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y.

[0284] In some embodiments, X7is selected from L, Q, D, H, M, P, and K. In some embodiments, X7 is L. In some embodiments, X8 is selected from G, V, S, D, M, and N. In some embodiments, X8is G. In some embodiments, X9is selected from V, M, P, S, and D. In some embodiments, X9is V. In some embodiments, X10is selected from S, N, L, H, and M. In some embodiments, X10 is S.

[0285] In some embodiments, X7 is L. In further embodiments, X8 is G. In further embodiments, X9is L. In further embodiments, X10is S.

[0286] In some embodiments, X7 is A. In further embodiments, X8 is V. In further embodiments, X9 is G. In further embodiments, X10 is V.

[0287] In some embodiments, X7is L. In further embodiments, X8is L. In further embodiments, X9is L. In further embodiments, X10is S.

[0288] In some embodiments, X7 is L; X8 is selected from G, L and V; X9 is selected from L and G; and / or X10is selected from S, V and L. In a further embodiment, X7is L, X8is G or L, and / or X10is S. In certain embodiments, X8and X9is G or L.

[0289] In certain embodiments, X7, X8, and X9 are independently selected from L, V, and A; at least two of X7, X8, and X9are independently L. In some embodiments, X7, X8, and X9are L. In certain embodiments, X8is L.

[0290] In some embodiments, the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X7X8X9X10 is a quad selected for enhanced targeting to muscle. In some embodiments, 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).

[0291] In some embodiments, the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X7X8X9X10is a quad selected for enhanced targeting toAtty Docket No.: 38053-57988 / WO (112WO) skeletal muscle. In some embodiments, the quad is FNNL, YNSL, RQGI, FQNT, YVGL, YSSV, YTSM, RSVV, YTSV, RDYL, or FNNT.

[0292] In some embodiments, the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X7X8X9X10 is a quad selected for enhanced targeting to cardiac muscle. In some embodiments, the quad is LIGR, QSTL, or RGVV.

[0293] In some embodiments, the targeting peptide has a sequence of X1X2X3RGDX7X8X9X10, wherein X7X8X9X10is a quad selected for enhanced targeting to liver. In some embodiments, the quad is RGVV, RSVV, RGQI, RSQT, RQGI, FQNT, HGVL, YTSV, YTSM, LTVT, FNNT, YSSV, or HVNL

[0294] In certain embodiments, 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. In certain embodiments, 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).

[0295] In some embodiments, the targeting peptide comprises a sequence of RGDLLLS (SEQ ID NO: 1).

[0296] In some embodiments, the targeting peptide comprises a sequence selected from: SEQ ID NOs.: 238-44858. In some embodiments, the modified AAV capsid protein comprises a targeting peptide comprising an amino acid sequence selected from SEQ ID NOs.: 238-247.

[0297] In some embodiments, the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-337. In some embodiments, the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-437. In some embodiments, the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-537. In some embodiments, the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-637. In some embodiments, the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238- 737. In some embodiments, the targeting peptide comprises a sequence selected from SEQ ID NOs.: 238-837. In some embodiments, the targeting peptide comprises a sequence selectedAtty 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.

[0298] In some embodiments, the targeting peptide is the targeting peptide disclosed in US2017 / 0166926, incorporated by reference in its entirety herein.

[0299] The targeting peptide can have any of the sequences selected from SEQ ID NOs: 2-51 and 53 provided herein. In some embodiments, the targeting peptide is the 7-mer peptide TLAVPFK (SEQ ID NO: 53).

[0300] 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

[0301] A modified AAV capsid protein of the present disclosure comprises a targeting peptide within VR VIII of the reference AAV capsid protein (FIG.1).

[0302] Preferably, 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.

[0303] 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. In some embodiments, the site of the targeting peptide corresponds to amino acid position 588 of the AAV9 VP1 capsid protein. In other words, X1of the targeting peptide is at a site corresponding to amino acid position 580 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is at a site corresponding to amino acid position 581 of the AAV9 VP1 capsid protein. In other words, X1of the targeting peptide is at a site corresponding to amino acid position 582 of the AAV9 VP1 capsid protein. In other words, X1of the targeting peptide is at a site corresponding to amino acid position 583 of the AAV9 VP1 capsid protein. In other words, X1of the targeting peptide is at a site corresponding to amino acid position 584 of the AAV9 VP1 capsid protein. In other words, X1of 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 theAtty 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. In other words, X1of 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, X1of the targeting peptide is at a site corresponding to amino acid position 586 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 AAV9 VP1 capsid protein. In other words, X1of 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, X1of the targeting peptide is at a site corresponding to amino acid position 591 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is at a site corresponding to amino acid position 592 of the AAV9 VP1 capsid protein.

[0304] 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. Table 1 Exemplary insertion sites Insertion sites 1 Insertion sites 2 Insertion sites 3 AAV1 D590 STD- S588 SSS- Q585 Q-SSS-T P591 PAT T589 TDP T589 AAV2 R588 GNR- N587 RGN- Q584 Q-RGN-R Q589 QAA R588 RQA R588 AAV3b S586 LQS- N588 SSN- Q585 Q-SSN-T S587 SNT T589 TAP T589 AAV4 S584 DQS- S586 SNS- G581 G-DQS-N N585 NSN N587 NLP N585 AAV5 S575 NQS- T577 SST- Q574 Q-SST-T S576 STT T578 TAP T578 AAV6 D590 STD- S588 SSS- Q585 Q-SSS-T P591 PAT T589 TDP T589 AAV7 N589 AAN- Q586 Q-AAN-T T590 TAA T590 AAV8 N590 QQN- Q587 Q-QQN-T T591 TAP T591Atty Docket No.: 38053-57988 / WO (112WO) AAV9 Q588 SAQ- Q585 Q-SAQ-A A589 AQA A589 AAVrh10 N590 QQN- Q587 Q-QQN-A A591 AAP A591 AAVpo.1 N567 NQN- N569 NSN- N564 N-NQN-S S568 SNT T570 THP S568 AAV12 N592 NQN- T594 NAT- N589 N-NQN-A A593 ATT T595 TAP A593 Anc80 N588 QSXN-T Q585 Q-SXN-T T589 T589 Anc80L65 N588 QSAN-T Q585 Q-SAN-T T589 T589 Anc80-55 N588 QSSN-T Q585 Q-SSN-T T589 T589 Anc80-129 N588 QSAN-T Q585 Q-SAN-T T589 T589 Anc80-156 N588 QSAN-T Q585 Q-SAN-T T589 T589 Anc80-751 N588 QSAN-T Q585 Q-SAN-T T589 T589 Anc80-1029 N588 QSAN-T Q585 Q-SAN-T T589 T589 Anc80-1712 N588 QSAN-T Q585 Q-SAN-T T589 T589

[0305] 3 exemplary insertion sites are indicated in Table 1. For insertion sites 1 and 2, the targeting peptide is inserted between 2 amino acid positions, where the insertion site is denoted as “-”. For example, for AAV1 capsid protein insertion site 1, the targeting peptide is inserted between positions D590 and P591. In some embodiments, 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. For example, for 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. For example, for AAV1 capsid protein insertion site 3, 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”.

[0306] In some embodiments, 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.

[0307] In some embodiments, the targeting peptide is at between 570 and 610 within the VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptideAtty 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.

[0308] In some embodiments, 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. In some embodiments, 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.

[0309] 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. In some embodiments, 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.

[0310] 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. In some embodiments, 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.

[0311] 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. In some embodiments, the reference AAV capsid protein is a capsid protein of AAV4 or a modification thereof and the targeting peptide is betweenAtty Docket No.: 38053-57988 / WO (112WO) positions 581 and 586 or between positions 583 and 590 of the modified AAV capsid protein. In some embodiments, 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.

[0312] In some embodiments, 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.

[0313] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0314] In some embodiments, 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.

[0315] In some embodiments, 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. In some embodiments, 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. In some embodiments, the reference AAV capsidAtty 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.

[0316] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0317] In some embodiments, 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.

[0318] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0319] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0320] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0321] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0322] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0323] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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)

[0324] 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 T589 and A590 or between N587 and T588 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 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.

[0325] 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. 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 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.

[0326] 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. 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 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.

[0327] 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. 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 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. Various embodiments of modified AAV capsid proteins

[0328] The present disclosure provides a modified AAV capsid protein 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. In some embodiments, the modified AAV capsid protein comprises a targeting peptide selected from SEQ ID NOs.: 238-44858 introduced into the VR VIII. In some embodiments, 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.

[0329] In some embodiments, the modified AAV capsid protein is an AAV9 capsid protein containing a targeting peptide, RGDLLLS (SEQ ID NO: 1), inserted into the VR VIII. In one embodiment, the modified AAV capsid protein has a sequence of SEQ ID NO: 158. In some embodiments, the modified AAV capsid protein has the amino acids 138 to 736 of SEQ ID NO: 158. In some embodiments, 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. In some embodiments, 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. 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 an amino acid sequence selected from: SEQ ID NOs.: 44900-44909.Atty Docket No.: 38053-57988 / WO (112WO)

[0330] 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.

[0331] In another aspect, 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.

[0332] In one embodiment, 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. In one embodiment, the modified AAV capsid protein has a sequence of SEQ ID NO: 159. In some embodiments, the modified AAV capsid protein has the amino acids 138 to 736 of SEQ ID NO: 159. In some embodiments, the modified AAV capsid protein has the amino acids 203 to 736 of SEQ ID NO: 159.

[0333] 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: 159.

[0334] 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 preferred embodiments, an AAV comprising the modified AAV capsid protein has increased targeting to a target cell, tissue or organ when administered to a subject. In some embodiments, an AAV comprising the modified AAV capsid protein has decreased distribution outside of a target cell, tissue or organ when administered to a subject. In some embodiments, tropism of a modified AAV capsid protein can be measured using an enrichment score. A non-limiting example of an enrichment score is based on the combination of amino acid residues present in the modified sequence within VR VIII. An exemplary enrichment formula is provided below:

[0335] ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ℎ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ^^ ^^ ^^2 ^^ ^^ ^^ ^^ ^^ℎ ^^ ^^ ^^ ^^ = ^^ ^^ ^^2 ^^ ^^Tissue− ^^ ^^ ^^2 ^^ ^^TestArticleAtty Docket No.: 38053-57988 / WO (112WO)

[0336] The formula for the scaled log2 fold change is provided below:

[0338] In some embodiments, the modified AAV capsid protein comprises (i) a reference AAV capsid protein, and (ii) a 7-mer peptide having the sequence RGDLLLS (SEQ ID NO: 1) inserted into a site within VR VIII of the reference AAV capsid protein. 6.2.4. Reference AAV capsid proteins

[0339] 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.

[0340] 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. Accordingly, 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.

[0341] In some embodiments, 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. No: AF028705.1), AAV4 (Genbank Ace. No: AAC58045.1), AAV5 (Genbank Ace. No: AAD13756.1), AAV6 (Genbank Ace. No: AF028704.1), AAV7 (Genbank Ace. No: AAN03855.1), AAV 8 (Genbank Ace. No: AAN03857.1), AAV10 (Genbank Ace. No: AAT46337.1), AAVrh10 (Genbank Ace. No: AY243015.1), AAV11 (Genbank Ace. No: AAT46339.1), AAV12 (Genbank Ace. No: ABI16639.1), or AAV13 (Genbank Ace. No: ABZ10812.1), AAVpol (Genbank Ace. No: FJ688147.1). In certain embodiments, the AAV capsid protein is the capsid protein of AAV9 (Genbank Ace. No: AAS99264.1).Atty Docket No.: 38053-57988 / WO (112WO)

[0342] 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: 63 (AAV hu.68), SEQ ID NO: 64 (AAV LK03), SEQ ID NO: 65 (AAV hu.1 (AAS99260)), SEQ ID NO: 66 (AAV hu.2 (AAS99270)), SEQ ID NO: 67 (AAV hu.3 (AAS99280)), SEQ ID NO: 68 (AAV hu.4 (AAS99287)), SEQ ID NO: 69 (AAV hu.6 (AAS99306)), SEQ ID NO: 70 (AAV hu.7 (AAS99313)), SEQ ID NO: 71 (AAV hu.9 (AAS99314)), SEQ ID NO: 72 (AAV hu.10 (AAS99261)), SEQ ID NO: 73 (AAV hu.11 (AAS99262)), SEQ ID NO: 74 (AAV hu.15 (AAS99265)), SEQ ID NO: 75 (AAV hu.16 (AAS99266)), SEQ ID NO: 76 (AAV hu.17 (AAS99267)), SEQ ID NO: 77 (AAV hu.18 (AAS99268)), SEQ ID NO: 78 (AAV hu.20 (AAS99271)), SEQ ID NO: 79 (AAV hu.21 (AAS99272)), SEQ ID NO: 80 (AAV hu.22 (AAS99273)), SEQ ID NO: 81 (AAV hu.23 (AAS99274)), SEQ ID NO: 82 (AAV hu.25 (AAS99276)), SEQ ID NO: 83 (AAV hu.27 (AAS99277)), SEQ ID NO: 84 (AAV hu.28 (AAS99278)), SEQ ID NO: 85 (AAV hu.29 (AAS99279)), SEQ ID NO: 86 (AAV hu.31 (AAS99281)), SEQ ID NO: 87 (AAV hu.32 (AAS99282)), SEQ ID NO: 88 (AAV hu.34 (AAS99283)), SEQ ID NO: 89 (AAVhu.37 (AAS99285)), SEQ ID NO: 90 (AAV hu.39 (AAS99286)), SEQ ID NO: 91 (AAV hu.41 (AAS99289)), SEQ ID NO: 92 (AAV hu.42 (AAS99290)), SEQ ID NO: 93 (AAV hu.43 (AAS99291)), SEQ ID NO: 94 (AAV hu.44 (AAS99292)), SEQ ID NO: 95 (AAV hu.45 (AAS99293)), SEQ ID NO: 96 (AAV hu.46 (AAS99294)), SEQ ID NO: 97 (AAV hu.47 (AAS99295)), SEQ ID NO: 98 (AAV hu.48 (AAS99296)), SEQ ID NO: 99 (AAV hu.51 (AAS99298)), SEQ ID NO: 100 (AAV hu.52 (AAS99299)), SEQ ID NO: 101 (AAV hu.53 (AAS99300)), SEQ ID NO: 102 (AAV hu.54 (AAS99301)), SEQ ID NO: 103 (AAV hu.55 (AAS99302)), SEQ ID NO: 104 (AAV hu.56 (AAS99303)), SEQ ID NO: 105 (AAV hu.57 (AAS99304)), SEQ ID NO: 106 (AAV hu.60 (AAS99307)), SEQ ID NO: 107 (AAV hu.61 (AAS99308)), SEQ ID NO: 108 (AAV hu.63 (AAS99309)), SEQ ID NO: 109 (AAV hu.66 (AAS99311)), SEQ ID NO: 110 (AAV hu.67 (AAS99312)), SEQ ID NO: 111 (AAV rh.10 (AAO88201)), SEQ ID NO: 112 (AAV rh.13 (AAO88199)), SEQ ID NO: 113 (AAV rh.19 (AAO88194)), SEQ ID NO: 114 (AAV rh.22 (AAO88192)), SEQ ID NO: 115 (AAV rh.23 (AAO88191)), SEQ ID NO: 116 (AAV rh.24 (AAO88190)), SEQ ID NO: 117 (AAV rh.35 (AAO88186)), SEQ ID NO: 118 (AAV rh.43 (AAS99245)), SEQ ID NO: 119 (AAV rh.48 (AAS99246)), SEQ ID NO: 120 (AAV rh.49Atty Docket No.: 38053-57988 / WO (112WO) (AAS99247)), SEQ ID NO: 121 (AAV rh.50 (AAS99248)), SEQ ID NO: 122 (AAV rh.51 (AAS99249)), SEQ ID NO: 123 (AAV rh.52 (AAS99250)), SEQ ID NO: 124 (AAV rh.53 (AAS99251)), SEQ ID NO: 125 (AAV rh.54 (AAS99252)), SEQ ID NO: 126 (AAV rh.55 (AAS99253)), SEQ ID NO: 127 (AAV rh.57 (AAS99254)), SEQ ID NO: 128 (AAV rh.58 (AAS99255)), SEQ ID NO: 129 (AAV rh.62 (AAS99258)), SEQ ID NO: 130 (AAV rh.64 (AAS99259)), SEQ ID NO: 131 (AAV rh.56 (JA400164)), SEQ ID NO: 143 (Anc80L1), SEQ ID NO: 144 (Anc80L27), SEQ ID NO: 145 (Anc80L33), SEQ ID NO: 146 (Anc80L36), SEQ ID NO: 147 (Anc80L44), SEQ ID NO: 148 (Anc80L59), SEQ ID NO: 149 (Anc80L60), SEQ ID NO: 150 (Anc80L62), SEQ ID NO: 151 (Anc82DI), SEQ ID NO: 152 (AAV rh.74); 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). The reference AAV capsid protein can be a VP2 or VP3 protein having a part of one of the sequences. For example, VP2 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 and VP3 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 protein.

[0343] 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. For example, VP2 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 and VP3 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1 protein. When a SEQ ID NO for a library sequence is used in this disclosure, it refers to a sequence of any one member of the library.

[0344] In some embodiments, the reference AAV capsid protein is a liver-toggle mutant described in WO2019 / 217911, which is incorporated by reference in its entirety herein.

[0345] In some embodiments, 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.58-B; rh.28- B; rh.51-B; rh.19-B; rh.49-B; rh.52-B; rh.13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27- B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu.9-C; hu.54-C; hu.53-C; hu.60- C; hu.55-C; hu.2-C; hu.1-C; hu.18-C; hu.3-C; hu.25-C; hu.15-C; hu.16-C; hu.11-C; hu.10-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; hu.42-E; rh.57-E; rh.40-E; rh74; hu.67-E; hu.17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc80-55, Anc80-129, Anc80-156, Anc80-751, Anc80-1029, Anc80-1712; Anc110; and Anc80DI. In some embodiments, 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.

[0346] In some embodiments, the reference AAV capsid protein is a protein having a sequence selected from SEQ ID Nos: 54-131 and 143-152. In some embodiments, 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.

[0347] 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0348] In some embodiments, the reference AAV capsid protein is a liver toggle mutant of a capsid protein of Anc80L65.Atty Docket No.: 38053-57988 / WO (112WO)

[0349] In some embodiments, 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.

[0350] In some embodiments, 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

[0351] 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. In some embodiments, the modified AAV capsid protein comprises a peptide segment of 12 amino acids (P1P2P3P4P5P6P7P8P9P10P11P12) within VR I which is different from the reference AAV capsid protein. In some embodiments, 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. In some embodiments, 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.

[0352] In some embodiments, 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 (P1P2P3P4P5P6P7P8P9P10P11P12) 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. In some embodiments, the peptide segment comprises 11 amino acids (P2P3P4P5P6P7P8P9P10P11P12) 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 inAtty Docket No.: 38053-57988 / WO (112WO) a reference AAV capsid protein (e.g., any of the reference AAV capsid proteins described herein).

[0353] In some embodiments, 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.

[0354] In some embodiments, 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.

[0355] In some embodiments, 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 P1P2P3P4P5P6P7P8NDNP12and P1, P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue.

[0356] In some embodiments, 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 P1P2P3P4P5P6P7P8NDNP12and P1,P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue.

[0357] In some embodiments, the modified AAV capsid protein can include a peptide segment having the amino acid sequence of P1P2P3P4P5P6P7P8NDNP12where: (i) P1 is independently selected from an asparagine (N), a serine (S), or a threonine (T), (ii) P2is 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) P4is 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) P7is independently selected from an alanine (A) or a serine (S), (viii) P8is independently selected from a serine (S) or a threonine (T), andAtty Docket No.: 38053-57988 / WO (112WO) (ix) P12 is independently selected from a histidine (H), a threonine (T), or an alanine (A).

[0358] In some embodiments, 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 P1P2P3P4P5P6P7P8P9P10P11P12, wherein positions P9, P10, and P11, or a combination thereof, are not modified compared to a reference AAV capsid protein.

[0359] In some embodiments, 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.

[0360] In another aspect, 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.

[0361] In another aspect, 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 P1P2P3P4GGP7P8NDNP12(SEQ ID NO: 44921), wherein P1, P2, P3, P4, P7, P8,andP12are independently selected from any amino acid residue.

[0362] In some embodiments, the modified AAV capsid protein can comprise a peptide segment having the amino acid sequence of P1P2P3P4GGP7P8NDNP12(SEQ ID NO: 44921) where: (i) P1 is independently selected from an asparagine (N) or a serine (S) ; (ii) P2is independently selected from a serine (S) or a glycine (G); (iii) P3is 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) P12is independently selected from a histidine (H), a threonine (T), or an alanine (A).

[0363] In some embodiments, the modified AAV capsid protein can comprise a peptide segment having a sequence of P1P2TP4GGP7P8NDNP12(SEQ ID NO: 44922), wherein P1, P2, P4, P7, P8, and P12are independently selected from any amino acid residue.

[0364] In some embodiments, the modified AAV capsid protein can comprise a peptide segment has a sequence of P1P2QP4GGP7P8NDNP12(SEQ ID NO: 44923), wherein P1, P2, P4, P7, P8, and P12are independently selected from any amino acid residue.

[0365] In some embodiments, the modified AAV capsid protein can comprise a peptide segment having a sequence of P1P2TP4GGP7TNDNP12 (SEQ ID NO: 44924), wherein P1, P2, P4, P7, and P12are independently selected from any amino acid residue.

[0366] In some embodiments, the modified AAV capsid protein can comprise a peptide segment has a sequence of NP2TP4GGP7P8NDNP12 (SEQ ID NO: 44925), wherein P2, P4, P7, P8, and P12are independently selected from any amino acid residue.

[0367] In some embodiments, the modified AAV capsid protein can comprise a peptide segment has a sequence of SP2TP4GGP7P8NDNP12 (SEQ ID NO: 44926), wherein P2, P4, P7, P8, and P12are independently selected from any amino acid residue.

[0368] In some embodiments, 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 P1P2P3P4P5P6P7P8P9P10P11P12, wherein positions P5, P6, or a combination thereof, are not modified compared to a reference AAV capsid protein. In some embodiments, 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 P5, P6, P9, P10, and P11, or a combination thereof, are not modified compared to a reference AAV capsid protein.Atty Docket No.: 38053-57988 / WO (112WO)

[0369] In another aspect, 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. In some embodiments, P7is independently selected from an alanine (A) or a serine (S) and P8is independently selected from a serine (S) or a threonine (T). In some embodiments, 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 NSTSGGP7P8NDNH (SEQ ID NO: 44927), wherein P7and P8are independently selected from any amino acid residue. In some embodiments, 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). In some embodiments, 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).

[0370] In another aspect, 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 NSTTGGP7P8NDNH (SEQ ID NO: 44928), wherein P7and P8are independently selected from any amino acid residue. In some embodiments, P7is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T). In some embodiments, 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. In some embodiments, P7 is independently selected from an alanine (A) or a serine (S) and P8is independently selected from a serine (S) or a threonine (T). In some embodiments, 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).

[0371] In another aspect, 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 SGQTGGP7P8NDNH (SEQ ID NO: 44929), wherein P7and P8are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from an alanine (A) or a serine (S) and P8is independently selected from a serine (S) or a threonine (T). In some embodiments, 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 P8are independently selected from any amino acid residue. In some embodiments, P7is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T). In some embodiments, 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).

[0372] In another aspect, 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. In some embodiments, P7 is independently selected from an alanine (A) or a serine (S) and P8is independently selected from a serine (S) or a threonine (T). In some embodiments, 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 SGTAGGP7P8NDNT (SEQ ID NO: 44930), wherein P7and P8are independently selected from any amino acid residue. In some embodiments, P7is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T). In some embodiments, the peptide segment has a sequence of: (i) SGTAGGASNDNT (SEQ ID NO: 46554), (ii) SGTAGGSSNDNT (SEQ IDAtty Docket No.: 38053-57988 / WO (112WO) NO: 46560), or a corresponding sequence having one or more modifications (e.g., insertion, deletion, and / or substitution). In some embodiments, the peptide segment does not comprise SGTAGGATNDNT (SEQ ID NO: 46557) or SGTAGGSTNDNT (SEQ ID NO: 46563).

[0373] In another aspect, 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. In some embodiments, P7is independently selected from an alanine (A) or a serine (S) and P8is independently selected from a serine (S) or a threonine (T). In some embodiments, 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 SGTSGGP7P8NDNA (SEQ ID NO: 44931), wherein P7and P8are independently selected from any amino acid residue. In some embodiments, 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). In some embodiments, 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).

[0374] In another aspect, 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. In some embodiments, P7is independently selected from an alanine (A) or a serine (S) and P8 is independently selected from a serine (S) or a threonine (T). In some embodiments, 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 SGTTGGP7P8NDNT (SEQ ID NO: 44932), wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7is independently selected from an alanine (A) or a serine (S) and P8is independently selected from a serine (S) or a threonine (T). In someAtty Docket No.: 38053-57988 / WO (112WO) embodiments, 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). In some embodiments, the peptide segment is SGTTGGSSNDNT (SEQ ID NO: 46656).

[0375] In another aspect, 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 SSTAGGP7P8NDNA (SEQ ID NO: 44933), wherein P7and P8are independently selected from any amino acid residue. In some embodiments, 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). In some embodiments, 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. In some embodiments, P7is independently selected from an alanine (A) or a serine (S) and P8is independently selected from a serine (S) or a threonine (T). In some embodiments, 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). In some embodiments, the peptide segment is SSTAGGASNDNA (SEQ ID NO: 47128). In some embodiments, the peptide segment is SSTAGGATNDNA (SEQ ID NO: 47131).

[0376] In some embodiments, the peptide segment is NSTSGASTNDNA (SEQ ID NO: 48390).

[0377] In some embodiments, the peptide segment is selected a peptide segment as shown in Table 20.

[0378] 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 whenAtty Docket No.: 38053-57988 / WO (112WO) administered to a subject. In some embodiments, an AAV comprising the modified AAV capsid protein has decreased distribution outside of a target cell, tissue or organ when administered to a subject.

[0379] In some embodiments, tropism of a modified AAV capsid protein can be measured using an enrichment score. A non-limiting example of 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 P1P2P3P4P5P6P7P8P9P10P11P12. FIG.52A provides an exemplary enrichment score formula, which is reproduced below.^^ ^^ ^^ ^^ ^^ℎ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^=^^^ + ^^^ ^^^ ^^ + ^^ଶ ^^^ ^^ + ^^ଷ ^^ଶ ^^ + ^^ସ ^^ଷ ^^ + ^^ହ ^^ଷ ^^ + ^^ହ ^^ଷ ^^ + . . . + ^^^ଷ ^^^ଶ ^^ + ^^^ସ ^^^ଶ ^^where 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, and “T”, “S”, “E”, “Q”, “H”, and “T” refer to the amino acid residue at the X amino acid position. 6.2.6. Variable Region I (VR I)

[0380] In some embodiments, 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)).

[0381] In some embodiments, 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

[0382] In some embodiments, the peptide segment is at a position between S261 and Y274 of an AAV9 capsid protein (SEQ ID NO: 61).

[0383] In some embodiments, the peptide segment is at a position c between S260 and Y273 of an Anc80 capsid protein (SEQ ID NO: 132).

[0384] In some embodiments, 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)

[0385] 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

[0386] 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.

[0387] In some embodiments, 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. In some embodiments, the one or more amino acid substitutions is at a variable region, VR I, of the reference AAV capsid protein (FIG.1).

[0388] 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.

[0389] In some embodiments, 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.

[0390] In some embodiments, 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. In some embodiments, 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.

[0391] In some embodiments, 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. In some embodiments, 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.

[0392] 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.

[0393] In some embodiments, 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.

[0394] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0395] In some embodiments, a reference AAV capsid is an AAV capsid protein disclosed in WO2019 / 217911, which is incorporated by reference in its entirety herein.

[0396] In particular, an AAV capsid protein that is described therein to generate a “liver off” (“liver de-targeting”) AAV can be used in embodiments herein. In other embodiments, an AAV capsid protein that is described therein to generate a “liver on” (“liver targeting”) AAV can be used herein.

[0397] In some embodiments, two amino acid positions corresponding to position 266 and position 168 of Anc80 VP1 protein are used as liver-toggle positions. In some embodiments, 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. In some embodiments, 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.

[0398] In some embodiments, more than one toggle region residues are introduced to enhance the liver-off or the liver-on phenotypes. In some embodiments, a double mutant AAV9 G267A S269T is used.

[0399] In some embodiments, a reference AAV capsid is Anc80L65 capsid protein with a G266A mutation. In some embodiments, 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. In some embodiments, 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.

[0400] 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.

[0401] In some embodiments, 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. In some embodiments, the reference AAV capsid protein does not include an alanine (A) at an amino acid position corresponding to position 267 in AAV9 VP1. In some embodiments, the one or more modifications to a reference AAV capsid protein to comprise a peptide segment within variable region I (VR I) do not include modification to an alanine (A) at an amino acid position corresponding to position 267 in AAV9 VP1. In some embodiments, the reference AAV capsid protein comprises a glycine (G) at an amino acid position corresponding to position 267 in AAV9 VP1.

[0402] In some embodiments, 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. In some embodiments, 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. Combinations of Targeting Peptides and Peptide Segments

[0403] In some embodiments, 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.

[0404] In some embodiments, 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, 47137, and 48390.

[0405] In some embodiments, 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).

[0406] In some embodiments, 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).

[0407] In some embodiments, 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).

[0408] In some embodiments, 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).

[0409] In some embodiments, 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).

[0410] In some embodiments, 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).

[0411] In some embodiments, 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).Atty Docket No.: 38053-57988 / WO (112WO)

[0412] In some embodiments, 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.

[0413] In some embodiments, 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).

[0414] In some embodiments, 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).

[0415] In some embodiments, 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).

[0416] In some embodiments, 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).

[0417] In some embodiments, 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).Atty Docket No.: 38053-57988 / WO (112WO) 6.3. Polynucleotides Encoding Modified AAV Capsid Proteins; Vectors; Host Cells

[0418] In another aspect, the present disclosure provides a polynucleotide encoding a modified AAV capsid protein described herein. In some embodiments, the polynucleotide is codon optimized for expression in a bacterial or mammalian cell.

[0419] In some embodiments, the polynucleotide is inserted into an expression vector. In some embodiments, 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. In some embodiments, the vector is used to generate an AAV virion comprising the modified AAV capsid protein. In some embodiments, the vector further comprises an AAV rep protein or a fragment thereof. In some embodiments, the reference capsid protein for the modified AAV capsid protein and the rep protein are originated from an AAV of the same clade. In some embodiments, the reference capsid protein for the modified AAV capsid protein and the rep protein are originated from an AAV of different clades.

[0420] In some embodiments, 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. In some embodiments, the host cell is a mammalian cell or a yeast cell.

[0421] In some embodiments, the host cell further comprises another polynucleotide encoding an AAV protein. In some embodiments, 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.

[0422] In some embodiments, 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. In some embodiments, any one or more of the required components (e.g., a recombinant nucleic acid vector, rep sequences, cap sequences, and / or helper functions) 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. In some embodiments, such a stable host cell contains the required component(s) under the control of an inducibleAtty Docket No.: 38053-57988 / WO (112WO) promoter. In some embodiments, the required component(s) is under the control of a constitutive promoter. 6.4. Recombinant nucleic acid vector containing an expressible polynucleotide

[0423] In one aspect, the present disclosure provides a recombinant nucleic acid vector containing an expressible polynucleotide. In some embodiments, the recombinant nucleic acid vector is encapsulated in the modified AAV capsid proteins disclosed herein. In preferred embodiments, the expressible polynucleotide comprises a transgene (in cis or trans configuration with other viral sequences).

[0424] 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). In some embodiments, the modified rAAV comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA.

[0425] The transgene can be selected depending, at least in part, on the particular disease or deficiency being treated. Simply by way of example, 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 amyloidosis, Marfan’s syndrome, fatal familial insomnia, Creutzfeldt-Jakob disease, sickle-cell disease, Huntington’s disease, fronto-temporal lobar degeneration, Usher syndrome, lactose intolerance, lipid storageAtty Docket No.: 38053-57988 / WO (112WO) disorders (e.g., Niemann-Pick disease, type C), Batten disease, choroideremia, glycogen storage disease type II (Pompe disease), ataxia telangiectasia (Louis-Bar syndrome), congenital hypothyroidism, severe combined immunodeficiency (SCID), and / or amyotrophic lateral sclerosis (ALS). 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). For example, 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). By way of example, 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. MTM1 transgene

[0426] In certain embodiment, 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.

[0427] 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. PCT / US2022 / 015842, which is herein incorporated by reference in its entirety

[0428] 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). Thus, in certain embodiments, the MTM1 polypeptides comprise amino acid residues 29-486 of SEQ ID NO:165 or the amino acid sequence of SEQ ID NO:164.

[0429] In some embodiments, 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. In some embodiments, 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)

[0430] In other embodiments, 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. In some embodiments, the polypeptide portion is an internalizing moiety.

[0431] In some embodiments, 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). In certain embodiments, 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).

[0432] In other embodiments, 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). In certain embodiments, 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).

[0433] In some embodiments, the MTM1 coding sequence may further comprise a nucleotide sequence that encodes a linker and / or an internalizing moiety. In some embodiments, the internalizing moiety is an antibody or an antigen-binding fragment thereof. 6.4.2. Regulatory Sequences

[0434] 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 isAtty 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. For example, the regulatory sequence induces expression of a transgene in a specific target such as heart, liver, skeletal muscle, cardiac muscle, or other muscle.

[0435] Other suitable features of the rAAV include 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). 6.4.2.1 CAG Promoters

[0436] 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. Without being bound by theory, it is believed 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.

[0437] In some embodiments, 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.

[0438] In some embodiments, 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)

[0439] In some embodiments, 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.

[0440] An exemplary CAG ERE is used in the rAVE expression cassette (GeneDetect.com).

[0441] In some embodiments, 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. In some embodiments, 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.

[0442] Further modifications of the CAG promoter can be used in the rAAV of the disclosure. For example, 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). Thus, 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. 6.4.2.2 Other Promoters

[0443] 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).

[0444] 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.

[0445] In some embodiments, the promoter is a skeletal-muscle specific promoter. In some embodiments, the promoter is a cardiac-specific promoter.Atty Docket No.: 38053-57988 / WO (112WO)

[0446] Examples of 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 β-actin promoter, a phosphoglycerol kinase (PGK) promoter, and a EF1α promoter.

[0447] Examples of tissue-specific promoters include, without limitation a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, an α-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter.

[0448] Examples of 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. 6.5. Modified AAV Virions

[0449] The present disclosure further provides a modified recombinant AAV (rAAV) virion comprising a modified AAV capsid protein described herein. In some embodiments, the modified rAAV comprises a modified AAV capsid protein and a recombinant nucleic acid vector.

[0450] In some embodiments, 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. In some embodiments, 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.

[0451] In some embodiments, 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. In some embodiments, 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. In typical embodiments, the corresponding reference AAV capsidAtty 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.

[0452] In some embodiments, the target is brain, muscle, spinal cord, eye, liver, muscle, or other organ. In some embodiments, the off-target tissue is brain, muscle, spinal cord, eye, liver, muscle, or other organ. In one embodiment, the target is muscle.

[0453] In some embodiments, 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. Methods of Producing rAAV

[0454] The rAAV of the disclosure comprise a recombinant nucleic acid vector containing an expressible polynucleotide. In some embodiments, 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.

[0455] In some embodiments, 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).

[0456] In order to replicate and package the vector, 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.

[0457] 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.Atty Docket No.: 38053-57988 / WO (112WO)

[0458] In certain embodiments of this invention, the AAV vector and complementary packaging gene(s), if any, are provided in the form of bacterial plasmids, AAV particles, or any combination thereof. In other embodiments, 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.

[0459] A variety of different genetically altered cells can thus be used in the context of this invention. By way of illustration, 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). Alternatively, 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.

[0460] Thus, 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) can be introduced into a packaging host cell using one or more viral vectors.

[0461] Once assembled, rAAV particles can be purified, if desired, using routine methods. As used herein, “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. 6.7. Pharmaceutical Composition Comprising Modified rAAV

[0462] In one aspect, the present disclosure provides a 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 AAVAtty Docket No.: 38053-57988 / WO (112WO) capsid protein as described herein and a recombinant nucleic acid vector containing an expressible polynucleotide.

[0463] In particular embodiments, the present disclosure provides a pharmaceutical composition comprising 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.

[0464] The pharmaceutical composition can be used to deliver the recombinant nucleic acid vector to a target within a mammalian subject. When the pharmaceutical composition is administered, 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. In some embodiments, 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.

[0465] 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.

[0466] 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.

[0467] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, 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).

[0468] 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. As used herein, 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.

[0469] 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.

[0470] 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.

[0471] In some embodiments, 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

[0472] 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. Suitable carriers include saline, which may be formulated with a variety of buffering solutions (e.g., phosphate bufferedAtty Docket No.: 38053-57988 / WO (112WO) saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, and water. A modified rAAV typically is 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 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. Routes of administration can be combined, if desired. 6.7.2. Dosages

[0473] 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. For example, 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 101to about 1 x 1016genome copies (GCs) / ml of viruses (e.g., a solution containing concentrations of from about 1 x 103to about 1 x 1014GCs / ml). In some embodiments, the total dose of the rAAV administered to a subject is less than 3 x 1014GCs, e.g., 1 x 1014GCs or less, 5 x 1013GCs or less, 1 x 1013GCs or less, 5 x 1012GCs or less, or 1 x 1012GCs or less.

[0474] In another embodiment, 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 101to 1 x 1012genome copies (GCs) of viruses (e.g., about 1 x 103to 1 x 109GCs).

[0475] In some embodiments, 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.

[0476] 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 / orAtty 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

[0477] Targeting of modified rAAVs can be tested in an experimental animal by measuring rAAV infection or expression of an expressible polynucleotide. In some embodiments, 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.

[0478] Targeting of modified rAAVs can be measured after systemic or local administration of rAAVs. In some embodiments, targeting of modified rAAVs is measured after intravenous infusion of rAAVs. 6.7.3.1 RNA data - Muscle:Liver Infection Ratio

[0479] In some embodiments, 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. In a particular embodiment, the transcripts are measured by RT-ddPCR. In some embodiments, 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.

[0480] In some embodiments, a muscle:liver infection ratio (RNA) 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).

[0481] In some embodiments, 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.

[0482] In certain embodiments, when the (transgene transcripts / housekeeping transcripts) in the liver is zero or below detection limits, the muscle:liver infection ratio is reported as >10,000 by convention.Atty Docket No.: 38053-57988 / WO (112WO)

[0483] In some embodiments, 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. In some embodiments, the muscle is triceps surae, biceps, heart or quadricep.

[0484] In some embodiments, 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. In some embodiments, the muscle is triceps surae, bicep, heart or quadricep. 6.7.3.2 DNA data - Muscle:Liver Infection Ratio

[0485] In some embodiments, 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. In some embodiments, 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.

[0486] In some embodiments, a muscle:liver infection ratio (DNA) 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). ൬^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ DNA genomes^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ( ^^ ^^ ^^) = ℎ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^^^ ^^ ^^ ^^ ^^ ^^: ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^൬^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ DNA genomesℎ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^ ^^ ^^ ^^ ^^ ^^ ^^ ^^

[0487] In some embodiments, modified rAAV of the present disclosure provides a (transgene genomes / housekeeping genomes) ratio in liver of less than 1, 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.

[0488] In certain embodiments, when the (transgene genomes / housekeeping genomes) in the liver is zero or below detection limits, the muscle:liver infection ratio is reported as >10,000 by convention.

[0489] In some embodiments, 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. In some embodiments, the muscle is triceps surae, biceps, heart or quadricep.

[0490] In some embodiments, 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. In some embodiments, the muscle is triceps surae, biceps, heart, or quadricep. In some embodiments, 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. 6.7.3.3 IHC data Muscle:Liver Infection Ratio

[0491] In some embodiments, 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). In a particular embodiment, the transgene expression is measured by immunohistochemistry. In some embodiments, 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.

[0492] In some embodiments, a muscle:liver infection ratio (IHC) 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).

[0493] In some embodiments, 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.

[0494] In certain embodiments, when the (transgene %GFP / housekeeping %GFP) in the liver is zero or below detection limits, the muscle:liver infection ratio is reported as >10,000 by convention.

[0495] In some embodiments, 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. In some embodiments, the muscle is triceps surae, biceps, heart or quadricep.

[0496] In some embodiments, 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. In some embodiments, the muscle is triceps surae, bicep, heart or quadricep. 6.8. Method of use

[0497] A modified rAAV as described herein can be used in research and / or therapeutic applications. In some embodiments, a modified rAAV is for genetically modifying a cell in vitro or in vivo. In some embodiments, 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.

[0498] In some embodiments, a modified rAAV of the present disclosure is used for treatment of a muscle disease. In some embodiments, the disease is a muscular disease and / or the condition is muscle degeneration. In some embodiments, 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 myofascialAtty Docket No.: 38053-57988 / WO (112WO) pain syndrome. In some embodiments, the modified rAAV is used to deliver the rAAV to a striated muscle, preferably heart or a skeletal muscle or diaphragm.

[0499] In some embodiments, 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. Merely to illustrate, “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.

[0500] 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.

[0501] 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. In particular embodiments, 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.

[0502] 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. Routes of administration can be combined, if desired.

[0503] The dose of a viral vector administered to a subject will depend primarily on factors such as the age, weight, and health (e.g., disease progression) of the subject. For example, 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 101to about 1 x 1016genome copies (GCs) / ml of virusesAtty Docket No.: 38053-57988 / WO (112WO) (e.g., a solution containing concentrations of from about 1 x 103to about 1 x 1014GCs / ml). In some embodiments, the total dose of the rAAV administered to a subject is less than 3 x 1014GCs, e.g., 1 x 1014GCs or less, 5 x 1013GCs or less, 1 x 1013GCs or less, 5 x 1012GCs or less, or 1 x 1012GCs or less.

[0504] Transduction and / or expression of the transgene can be monitored at various time points following administration by DNA, RNA, or protein assays.

[0505] Accordingly, 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.

[0506] In some embodiments, the modified rAAV capsid of the present disclosure is used to deliver a transgene to a target tissue. In some embodiments 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.

[0507] In some embodiments, the target tissue is heart. In some embodiments, 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. 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 5. The modified AAV capsid protein of embodiment 1 or 2, wherein the targeting peptide does not comprise RGDLLLS (SEQ ID NO: 1); and 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 7. The modified AAV capsid protein of embodiment 6, wherein the modified AAV capsid protein comprises one or more modifications comprising an amino acid insertion, deletion, substitution, or a combination thereof as compared to a reference AAV capsid protein to introduce the targeting peptide within VR VIII. 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 10. The modified AAV capsid protein of any one of embodiments 1-9, wherein the AAV capsid protein is selected from VP1, VP2 and VP3. 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; 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; rh.13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C;Atty Docket No.: 38053-57988 / WO (112WO) rh.56-C; hu.9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.1-C; hu.18-C; hu.3-C; hu.25-C; hu.15-C; hu.16-C; hu.11-C; hu.10-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu.17-E; hu.6-E; hu.66-E; rh.38- E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; Anc113; Anc126; Anc127; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc110; and Anc80DI. 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. The modified AAV capsid protein of any one of embodiments 1-18, wherein X7, X8, X9 and X10 are independently selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, and Y. 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 21. The modified AAV capsid protein of embodiment 20, wherein X1, X2, and X3are amino acids identical to the amino acids at corresponding positions of the reference AAV capsid protein. Embodiment 22. The modified AAV capsid protein of embodiment 20, wherein X1is 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 24. The modified AAV capsid protein of embodiment 20, wherein X3 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 27. The modified AAV capsid protein of embodiment 20, wherein X2 and X3 are amino acids that are identical to the amino acids at corresponding positions of the reference AAV capsid protein.Atty Docket No.: 38053-57988 / WO (112WO) Embodiment 28. The modified AAV capsid protein of embodiment 20, wherein X1, X2, and X3 are amino acids that are not 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 30. The modified AAV capsid protein of embodiment 20, wherein X2is 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 X3is 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 X1and X3are amino acids that are not identical to the amino acids at corresponding positions of the reference AAV capsid protein. Embodiment 33. The modified AAV capsid protein of embodiment 20, wherein X1and X2are 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 X2and X3are 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. Embodiment 36. 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.Atty Docket No.: 38053-57988 / WO (112WO) 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 X1is 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 X1is S, A or E. Embodiment 41. The modified AAV capsid protein of any one of embodiments 1-36, wherein X2is 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 X2is 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 N, A or Y. Embodiment 45. The modified AAV capsid protein of embodiment 41, wherein X2is selected from N, Y or S. Embodiment 46. The modified AAV capsid protein of any one of embodiments 1-45, wherein X3is 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 X3is selected from Y, V or F. Embodiment 48. The modified AAV capsid protein of embodiment 46, wherein X3is selected from N, A, Y or S. Embodiment 49. The modified AAV capsid protein of embodiment 46, wherein X3 is selected from I, Q, R, V, T or M.Atty Docket No.: 38053-57988 / WO (112WO) Embodiment 50. The modified AAV capsid protein of any one of embodiments 1-49, wherein X2X3 is NR. Embodiment 51. The modified AAV capsid protein of any one of embodiments 1-50, wherein X1X2X3 is ENR. Embodiment 52. The modified AAV capsid protein of any one of embodiments 1-50, wherein X1X2X3is SNR. 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, DTW, DTI, ELY, TEY, TEI, DAI, DQY, DMY, EWG, DMV, DMI, EPH, QEG, DIN, NEI, EYY, DIV, SEG, DVG, DYQ, EGF, NDI, EGY, DVF, DVH, DGF, DIY, DSF, DGW, EHY, DRE, TEH, DTS, NEN, NEM, NEH, TEN, DSN, DVT, DQS, DKD, DTH, DVV, DQK, NET, DKP, TEV, NDS, QET, EVL, SET, SDT, AEQ, QEF, SEY, SEF, SPF, EGQ, ETH, TDQ, QEA, QDQ, AEN, ESS, NDT, EFM, EFI, EHM, DFM, QDT, SME, DYT, EHV, ENV, EAV, EAI, ESI, DAT, ENQ, EAM, ADN, EFQ, SDS, TDH, SDH, DAS, TWE, SSF, DRD, EFL, TDF, QDA, EMH, SGE, AEW, DAH, TET, TDM, TNE, SAE, NSE, SFE, QDI, DSA, ADV, SEI, AEI, QDV, ADT, DNM, DNQ, ADL, TDL, SDL, SDM, TDV, DNI, DHY, DTY, DAA, DSY, QLY, DVM, DAY, DMT, DQT, DAQ, DTV, DSH, QDF, DST, DNL, DSI, DFV, DNY, DAF, DKI, DKF, DTM, DSL, NDV, TDI, DSV, DAV, DKV, DAM, DNV, DKM, DKL, DKW, DSM, ENI, SDI...

Claims

Atty Docket No.: 38053-57988 / WO (112WO) WHAT IS CLAIMED IS:

1. 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 X1, X2, X3, X7, X8, X9and X10are independently selected from any amino acid residue, and wherein the peptide segment has an amino acid sequence of P1P2P3P4P5P6P7P8NDNP12and P1, P2, P3, P4, P5, P6, P7, P8, and P12 are independently selected from any amino acid residue.

2. The modified AAV capsid protein of claim 1, a. wherein X7X8X9X10is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12is SGTTGGSSNDNT (SEQ ID NO: 46656), or b. wherein X7X8X9X10is FNNL and P1P2P3P4P5P6P7P8NDNP12is SSTAGGASNDNA (SEQ ID NO: 47128).

3. The modified AAV capsid protein of claim 2, wherein X1is independently selected from S, N, T, A, or Q; X2is independently selected from G, S, or A; and X3is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M.

4. The modified AAV capsid protein of claim 2, wherein X1is independently selected from T, S, Q, A, D, or N; X2is independently selected from F, W, A, K, Q, N, R, S, Y, M, or T; and X3is independently selected from N, S, Q, T, Y, G, E, F, M, or A.

5. The modified AAV capsid protein of claim 2, wherein X1is independently selected from N, S, T, A, or Q; X2is independently selected from N, S, Q, T, M, A, Y, or L; and X3is independently selected from I, M, L, T, V, Q, or Y.

6. The modified AAV capsid protein of claim 2, wherein X1is independently selected from E, Q, A, S, T, N, or D; X2is independently selected from H, Y, W, R, K, F, or N; and X3is independently selected from H, R, S, A, T, M, G, K, Q, N, or I.Atty Docket No.: 38053-57988 / WO (112WO) 7. The modified AAV capsid protein of claim 1, wherein X1is independently selected from S, N, T, A, or Q; X2is independently selected from G, S, or A; X3is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M; X7 is F; X8 is independently selected from N or Q; X9 is N; and X10 is independently selected from T or L.

8. The modified AAV capsid protein of claim 1, wherein X1is 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; X3 is independently selected from N, S, Q, T, Y, G, E, F, M, or A; X7 is F; X8 is independently selected from N or Q; X9 is N; and X10 is independently selected from T or L.

9. The modified AAV capsid protein of claim 1, wherein 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; X3 is independently selected from I, M, L, T, V, Q, or Y; X7is F; X8is independently selected from N or Q; X9is N; and X10is independently selected from T or L.

10. The modified AAV capsid protein of claim 1, wherein X1is independently selected from E, Q, A, S, T, N, or D; X2is independently selected from H, Y, W, R, K, F, or N; X3is independently selected from H, R, S, A, T, M, G, K, Q, N, or I; X7is F; X8is independently selected from N or Q; X9is N; and X10is independently selected from T or L.

11. The modified AAV capsid protein of claim 1, wherein X7X8X9X10is selected from RSQT, RNVV, RGQI, RGVV, and RSVV and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390).

12. The modified AAV capsid protein of claim 11, wherein X1is independently selected from S, N, T, A, or Q; X2 is independently selected from G, S, or A; and X3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M.

13. The modified AAV capsid protein of claim 11, wherein X1is 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; and X3 is independently selected from N, S, Q, T, Y, G, E, F, M, or A.Atty Docket No.: 38053-57988 / WO (112WO) 14. The modified AAV capsid protein of claim 11, wherein X1is independently selected from N, S, T, A, or Q; X2is independently selected from N, S, Q, T, M, A, Y, or L; and X3 is independently selected from I, M, L, T, V, Q, or Y.

15. The modified AAV capsid protein of claim 1, wherein X1is independently selected from S, N, T, A, or Q; X2is independently selected from G, S, or A; X3is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M; 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.

16. The modified AAV capsid protein of claim 1, wherein X1 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; X3 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; X9is independently selected from V or Q; and X10is independently selected from V, T, or I.

17. The modified AAV capsid protein of claim 1, wherein X1is independently selected from N, S, T, A, or Q; X2is independently selected from N, S, Q, T, M, A, Y, or L; X3is independently selected from I, M, L, T, V, Q, or Y; X7is R; X8is independently selected from S, G, or N; X9is independently selected from V or Q; and X10is independently selected from V, T, or I.

18. The modified AAV capsid protein of claim 1, wherein X7X8X9X10is selected from VRTL, RQGI, ARTL, and RTNL and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390).

19. The modified AAV capsid protein of claim 18, wherein X1is independently selected from S, N, T, A, or Q; X2 is independently selected from G, S, or A; and X3 is independently selected from G, N, S, Q, D, V, Y, P, A, T, or M.

20. The modified AAV capsid protein of claim 1, wherein X1is 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; X7 is independently selected from R, V,Atty Docket No.: 38053-57988 / WO (112WO) or A; X8is independently selected from R, T, or Q; X9is independently selected from T, G, or N; and X10is independently selected from L or I.

21. The modified AAV capsid protein of claim 1, a. wherein X7X8X9X10is selected from VRTL, RQGI, ARTL, and RTNL and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390), or b. wherein X7X8X9X10 is selected from RSQT, RNVV, RGQI, RGVV, and RSVV and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390), or c. wherein X7X8X9X10 is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12is SGTTGGSSNDNT (SEQ ID NO: 46656), or d. wherein X7X8X9X10 is FNNL and P1P2P3P4P5P6P7P8NDNP12 is SSTAGGASNDNA (SEQ ID NO: 47128).

22. The modified AAV capsid protein of claim 21, wherein 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, SGI, SGL, SGM, SGN, SGP, SGQ, SGS, SGT, SGV, SGY, SKD, SMG, SNG, SQG, SRD, SSG, SSN, SSS, TAG, TGA, TGD, TGF, TGG, TGH, TGI, TGL, TGM, TGN, TGP, TGQ, TGS, TGT, TGV, TGY, TKD, TRD, TSG, TSN, TSS, TSV, and TSY.

23. The modified AAV capsid protein of claim 1, a. wherein X7X8X9X10 is selected from RSQT, RNVV, RGQI, RGVV, and RSVV and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390), or b. wherein X7X8X9X10 is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656), orAtty Docket No.: 38053-57988 / WO (112WO) c. wherein X7X8X9X10is FNNL and P1P2P3P4P5P6P7P8NDNP12is SSTAGGASNDNA (SEQ ID NO: 47128).

24. The modified AAV capsid protein of claim 23, wherein X1X2X3is 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, QLQ, QLS, QMN, QMS, QMY, QNG, QNN, QNS, QQN, QQY, QRD, QRE, QSP, QSY, QTY, QWN, QWQ, QWS, QWT, QYN, SAA, SAF, SAN, SAS, SFI, SFQ, SFS, SFT, SFY, SGF, SHN, SKE, SLN, SLT, SMN, SMQ, SNN, SQF, SQN, SQS, SRE, SSP, SSY, STG, SWA, SWN, SWQ, SWS, SWT, SYG, SYN, TAF, TAN, TAS, TAY, TFF, TFN, TFQ, TFS, TFT, TFY, TGW, TKE, TLS, TMY, TNG, TNN, TNS, TQF, TQG, TQI, TQN, TQY, TRE, TSF, TSP, TTG, TTY, TVY, TWG, TWL, TWM, TWN, TWQ, TWT, TYG, TYN, TYQ, TYS, and TYT.

25. The modified AAV capsid protein of claim 23, wherein 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, NHM, NHN, NIM, NIQ, NIT, NIY, NKE, NLM, NLV, NMI, NMM, NMN, NMQ, NMS, NMV, NNA, NNF, NNI, NNL, NNM, NNQ, NNS, NNT, NNV, NNY, NPI, NPM, NPN, NPQ, NPS, NPT, NQH, NQI, NQL, NQM, NQQ, NQT, NQV, NQY, NSA, NSF, NSI, NSL, NSM, NSS, NST, NSV, NSY, NTG, NTH, NTI, NTL, NTM, NTQ, NTS, NTT, NTV, NTY, NVM, NWI, NWM, NWT, NYI, NYM, NYQ, NYS, NYT, NYV, QAL, QDK, QDR, QER, QEV, QGI, QGL, QGY, QHI, QHY, QII, QIL, QLI, QLL, QLM, QLT, QLY, QMI, QML, QMM, QMT, QMV, QNA, QNF, QNI, QNM, QNQ, QNT, QNV, QNW, QNY, QQI, QQL, QQM, QQT, QQV, QSA, QSI, QSL, QSM, QST, QSV, QTI, QTL, QTT, QTV,Atty Docket No.: 38053-57988 / WO (112WO) QVL, QYI, SAH, SAI, SAL, SAM, SAQ, SAT, SAV, SDK, SDR, SEK, SER, SFL, SFM, SFV, SHA, SHF, SHL, SHY, SII, SIM, SIT, SLI, SLV, SMH, SMI, SML, SMM, SMS, SMT, SMV, SNA, SNF, SNI, SNL, SNM, SNQ, SNS, SNT, SNV, SNY, SPA, SPQ, SPT, SQH, SQI, SQL, SQM, SQT, SQV, SQY, SSA, SSI, SSL, SSM, SSQ, SST, SSV, STA, STI, STL, STQ, STT, STV, STY, SVI, SVL, SVM, SVQ, SVT, SWL, SWM, SWV, SYI, SYL, SYM, SYQ, SYS, SYT, SYY, TAI, TAL, TAM, TAQ, TAT, TAV, TDK, TDR, TEK, TER, TFM, TFV, TGR, THF, THY, TIM, TIT, TIV, TLI, TLL, TLM, TMI, TML, TMM, TMT, TMV, TNA, TNF, TNI, TNL, TNM, TNQ, TNT, TNV, TNY, TPQ, TPT, TPV, TQH, TQL, TQM, TQQ, TQT, TQV, TSA, TSI, TSL, TSM, TSQ, TST, TTI, TTL, TTQ, TTT, TTV, TVI, TVL, TVM, and TYL.

26. The modified AAV capsid protein of claim 1, a. wherein X7X8X9X10is selected from FNNL, FNNT, and FQNT, and P1P2P3P4P5P6P7P8NDNP12is SGTTGGSSNDNT (SEQ ID NO: 46656), or b. wherein X7X8X9X10is FNNL and P1P2P3P4P5P6P7P8NDNP12is SSTAGGASNDNA (SEQ ID NO: 47128).

27. The modified AAV capsid protein of claim 26, wherein 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, ESR, ETR, EWK, EWR, EYK, EYR, NAH, NDR, NFA, NFH, NFY, NHG, NHQ, NHS, NHT, NHV, NMH, NNH, NRE, NWH, NYA, QAD, QAH, QAM, QDN, QFH, QFM, QHF, QHG, QHM, QHN, QHQ, QHS, QHT, QNH, QQH, QRI, QRY, QSF, QSH, QTM, QWA, QWH, QWI, QYH, QYQ, QYS, QYT, SFA, SFH, SHG, SHI, SHM, SHQ, SHS, SHT, SNH, SNP, SNW, SSH, SWG, SWH, SYA, SYE, SYH, TFH, THG, THM, THN, THQ, THS, THT, THW, TNH, TNP, TPG, TSH, TWA, TWH, and TYM.Atty Docket No.: 38053-57988 / WO (112WO) 28. The modified AAV capsid protein of claim 1, wherein X7X8X9X10is selected from LDEL, QSTL, ISRT, LLLS, IHNL, LIGR, FVQR, HVNL, RQGI, KERF, RSVV, ARTL, VRTL, RNVV, RSQT, RGVV, RGQI, and RTNL, and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390).

29. The modified AAV capsid protein of claim 28, a. wherein X1X2X3is 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, NSK, NSR, NTA, NTK, NTR, NVR, NWK, NWR, NYK, NYR, QAG, QAR, QER, QFK, QFR, QFT, QGK, QGR, QGV, QGW, QHR, QIR, QKA, QKG, QKN, QKP, QKQ, QKR, QKT, QLR, QMG, QMK, QMR, QNG, QNK, QNR, QPR, QQK, QQR, QQS, QRN, QRP, QRV, QSA, QSR, QTR, QTV, QVR, QVV, QYK, SAG, SAR, SDR, SFK, SFR, SGA, SGE, SGF, SGK, SGL, SGM, SGR, SGT, SIR, SKA, SKN, SKP, SKR, SKT, SLR, SMR, SNG, SNK, SNR, SQG, SQR, SRK, SRP, SSG, SSK, SSQ, SSR, STQ, STR, SVR, SWK, SWR, SYK, SYR, TAG, TAR, TFR, TGE, TGF, TGK, TGL, TGM, TGQ, TGR, TIK, TIR, TKE, TKK, TKP, TKQ, TKS, TKT, TKY, TLK, TLR, TMK, TMR, TNK, TNR, TPR, TQK, TQR, TRP, TRQ, TRV, TSG, TSK, TSR, TTK, TTR, TVR, TWK, TWR, and TYK, or b. X1X2X3is 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, NFN, NFQ, NFT, NHK, NHR, NHT, NKA, NKK,Atty Docket No.: 38053-57988 / WO (112WO) NKM, NKN, NKQ, NKR, NKS, NKW, NLQ, NLT, NNF, NPG, NPK, NPN, NQN, NQS, NRA, NRF, NRG, NRH, NRI, NRL, NRM, NRR, NRS, NRV, NRW, NRY, NSH, NSN, NSP, NSQ, NSS, NTS, NTY, NVK, NYM, NYN, QAK, QEK, QFG, QGD, QHK, QIK, QIQ, QKF, QKI, QKK, QKL, QKM, QKV, QKW, QKY, QLT, QMM, QMQ, QMS, QMT, QNH, QNT, QNV, QPP, QQP, QQQ, QRA, QRF, QRH, QRI, QRK, QRL, QRM, QRQ, QRR, QRS, QRT, QRW, QRY, QSF, QSI, QSK, QTA, QTI, QTK, QTN, QTT, QWK, QWN, QWR, QYG, QYI, QYN, QYR, QYT, SAN, SER, SFT, SHK, SHR, SIK, SKD, SKF, SKG, SKI, SKK, SKL, SKM, SKQ, SKS, SKV, SKY, SLK, SMG, SMK, SQI, SQN, SQQ, SRA, SRF, SRG, SRH, SRI, SRM, SRN, SRQ, SRR, SRS, SRT, SRV, SRW, SRY, STK, STP, STY, SVK, SWG, TAK, TDR, TFK, THK, THR, TKF, TKG, TKI, TKL, TKM, TKN, TKR, TKV, TKW, TNT, TPS, TQN, TRA, TRE, TRG, TRI, TRK, TRM, TRN, TRR, TRS, TRT, TRY, TSA, TSS, TTY, TVK, TWH, and TYR.

30. The modified AAV capsid protein of claim 28, wherein 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, DLL, DLM, DLN, DLP, DLQ, DLS, DLT, DLV, DLY, DMD, DME, DMH, DMN, DMP, DMQ, DMS, DMT, DMW, DMY, DNH, DNP, DNW, DPA, DPD, DPE, DPF, DPI, DPL, DPM, DPP, DPQ, DPR, DPT, DPV, DQA, DQD, DQE, DQH, DQT, DQY, DRV, DRW, DSA, DSD, DSE, DSL, DTD, DTF, DTH, DTL, DTM, DTP, DTT, DTW, DVD, DVE, DVF, DVH, DVM, DVN, DVP, DVQ, DVT, DWI, DWL, DWM, DWS, DWY, DYA, DYE, DYF, EAD, EAE, EAH, EAI, EAL, EAS, EAV, EAW, EAY, EDA, EDD, EDE, EDF, EDG, EDK, EDL, EDM, EDN, EDP, EDQ, EDW, EDY, EEA, EED, EEE, EEF, EEL, EEM, EEQ, EEV, EEY, EFA, EFM, EFQ, EFS, EFW, EGD, EHE, EHI, EHV, EID, EIT, ELD, ELN, ELW, EMD, EMF, EMH, EML, EMW, ENI, EPA, EPL, EPM, EPQ, EPW, EQD, EQY, ESF, ESV, ESW, ETA, ETD, ETE, ETL, ETW, EVA, EVE, EVS, EVW, EWE, EWH, EWM,Atty Docket No.: 38053-57988 / WO (112WO) EWN, EWS, EWY, EYF, EYQ, EYT, EYW, NDD, NDY, NEL, NEN, NEY, NFD, NFF, NFP, NFW, NHH, NWW, NYD, QDW, QFW, QPS, QVF, QWS, SDH, SDV, SFI, SIF, SLA, SLG, SMD, SPE, SPW, SWF, SWM, SYD, TDF, TDM, TDW, TDY, TEA, THW, TLW, TWD, and TYI.

31. The modified AAV capsid protein of claim 1, wherein X7X8X9X10is selected from FNNL, FNNT, and FQNT and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656).

32. The modified AAV capsid protein of claim 31, wherein X1X2X3 is not selected from a. 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, NFN, NFQ, NFT, NHK, NHR, NHT, NKA, NKK, NKM, NKN, NKQ, NKR, NKS, NKW, NLQ, NLT, NNF, NPG, NPK, NPN, NQN, NQS, NRA, NRF, NRG, NRH, NRI, NRL, NRM, NRR, NRS, NRV, NRW, NRY, NSH, NSN, NSP, NSQ, NSS, NTS, NTY, NVK, NYM, NYN, QAK, QEK, QFG, QGD, QHK, QIK, QIQ, QKF, QKI, QKK, QKL, QKM, QKV, QKW, QKY, QLT, QMM, QMQ, QMS, QMT, QNH, QNT, QNV, QPP, QQP, QQQ, QRA, QRF, QRH, QRI, QRK, QRL, QRM, QRQ, QRR, QRS, QRT, QRW, QRY, QSF, QSI, QSK, QTA, QTI, QTK, QTN, QTT, QWK, QWN, QWR, QYG, QYI, QYN, QYR, QYT, SAN, SER, SFT, SHK, SHR, SIK, SKD, SKF, SKG, SKI, SKK, SKL, SKM, SKQ, SKS, SKV, SKY, SLK, SMG, SMK, SQI, SQN, SQQ, SRA, SRF, SRG, SRH, SRI, SRM, SRN, SRQ, SRR, SRS, SRT, SRV, SRW, SRY, STK, STP, STY, SVK, SWG, TAK, TDR, TFK, THK, THR, TKF, TKG, TKI, TKL, TKM, TKN, TKR, TKV, TKW, TNT, TPS, TQN, TRA, TRE, TRG, TRI, TRK, TRM, TRN, TRR, TRS, TRT, TRY, TSA, TSS, TTY, TVK, TWH, and TYR; orAtty Docket No.: 38053-57988 / WO (112WO) b. 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, NSK, NSR, NTA, NTK, NTR, NVR, NWK, NWR, NYK, NYR, QAG, QAR, QER, QFK, QFR, QFT, QGK, QGR, QGV, QGW, QHR, QIR, QKA, QKG, QKN, QKP, QKQ, QKR, QKT, QLR, QMG, QMK, QMR, QNG, QNK, QNR, QPR, QQK, QQR, QQS, QRN, QRP, QRV, QSA, QSR, QTR, QTV, QVR, QVV, QYK, SAG, SAR, SDR, SFK, SFR, SGA, SGE, SGF, SGK, SGL, SGM, SGR, SGT, SIR, SKA, SKN, SKP, SKR, SKT, SLR, SMR, SNG, SNK, SNR, SQG, SQR, SRK, SRP, SSG, SSK, SSQ, SSR, STQ, STR, SVR, SWK, SWR, SYK, SYR, TAG, TAR, TFR, TGE, TGF, TGK, TGL, TGM, TGQ, TGR, TIK, TIR, TKE, TKK, TKP, TKQ, TKS, TKT, TKY, TLK, TLR, TMK, TMR, TNK, TNR, TPR, TQK, TQR, TRP, TRQ, TRV, TSG, TSK, TSR, TTK, TTR, TVR, TWK, TWR, and TYK.

33. The modified AAV capsid protein of claim 31, wherein a. 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, NSK, NSR, NTA, NTK, NTR, NVR, NWK, NWR, NYK, NYR, QAG, QAR, QER, QFK, QFR, QFT, QGK, QGR, QGV, QGW, QHR, QIR, QKA, QKG, QKN, QKP, QKQ, QKR, QKT, QLR, QMG, QMK, QMR, QNG, QNK, QNR, QPR, QQK, QQR, QQS, QRN, QRP, QRV, QSA, QSR, QTR, QTV, QVR, QVV, QYK, SAG, SAR, SDR, SFK, SFR, SGA, SGE, SGF, SGK, SGL, SGM, SGR, SGT, SIR, SKA, SKN, SKP, SKR, SKT, SLR,Atty Docket No.: 38053-57988 / WO (112WO) SMR, SNG, SNK, SNR, SQG, SQR, SRK, SRP, SSG, SSK, SSQ, SSR, STQ, STR, SVR, SWK, SWR, SYK, SYR, TAG, TAR, TFR, TGE, TGF, TGK, TGL, TGM, TGQ, TGR, TIK, TIR, TKE, TKK, TKP, TKQ, TKS, TKT, TKY, TLK, TLR, TMK, TMR, TNK, TNR, TPR, TQK, TQR, TRP, TRQ, TRV, TSG, TSK, TSR, TTK, TTR, TVR, TWK, TWR, and TYK, or b. 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, DLL, DLM, DLN, DLP, DLQ, DLS, DLT, DLV, DLY, DMD, DME, DMH, DMN, DMP, DMQ, DMS, DMT, DMW, DMY, DNH, DNP, DNW, DPA, DPD, DPE, DPF, DPI, DPL, DPM, DPP, DPQ, DPR, DPT, DPV, DQA, DQD, DQE, DQH, DQT, DQY, DRV, DRW, DSA, DSD, DSE, DSL, DTD, DTF, DTH, DTL, DTM, DTP, DTT, DTW, DVD, DVE, DVF, DVH, DVM, DVN, DVP, DVQ, DVT, DWI, DWL, DWM, DWS, DWY, DYA, DYE, DYF, EAD, EAE, EAH, EAI, EAL, EAS, EAV, EAW, EAY, EDA, EDD, EDE, EDF, EDG, EDK, EDL, EDM, EDN, EDP, EDQ, EDW, EDY, EEA, EED, EEE, EEF, EEL, EEM, EEQ, EEV, EEY, EFA, EFM, EFQ, EFS, EFW, EGD, EHE, EHI, EHV, EID, EIT, ELD, ELN, ELW, EMD, EMF, EMH, EML, EMW, ENI, EPA, EPL, EPM, EPQ, EPW, EQD, EQY, ESF, ESV, ESW, ETA, ETD, ETE, ETL, ETW, EVA, EVE, EVS, EVW, EWE, EWH, EWM, EWN, EWS, EWY, EYF, EYQ, EYT, EYW, NDD, NDY, NEL, NEN, NEY, NFD, NFF, NFP, NFW, NHH, NWW, NYD, QDW, QFW, QPS, QVF, QWS, SDH, SDV, SFI, SIF, SLA, SLG, SMD, SPE, SPW, SWF, SWM, SYD, TDF, TDM, TDW, TDY, TEA, THW, TLW, TWD, and TYI.

34. The modified AAV capsid protein of claim 31, wherein X1X2X3is 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,Atty Docket No.: 38053-57988 / WO (112WO) 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, DLP, DLQ, DLS, DLT, DLV, DLY, DMD, DME, DMH, DMN, DMP, DMQ, DMS, DMT, DMW, DMY, DNH, DNP, DNW, DPA, DPD, DPE, DPF, DPI, DPL, DPM, DPP, DPQ, DPR, DPT, DPV, DQA, DQD, DQE, DQH, DQT, DQY, DRV, DRW, DSA, DSD, DSE, DSL, DTD, DTF, DTH, DTL, DTM, DTP, DTT, DTW, DVD, DVE, DVF, DVH, DVM, DVN, DVP, DVQ, DVT, DWI, DWL, DWM, DWS, DWY, DYA, DYE, DYF, EAD, EAE, EAH, EAI, EAL, EAS, EAV, EAW, EAY, EDA, EDD, EDE, EDF, EDG, EDK, EDL, EDM, EDN, EDP, EDQ, EDW, EDY, EEA, EED, EEE, EEF, EEL, EEM, EEQ, EEV, EEY, EFA, EFM, EFQ, EFS, EFW, EGD, EHE, EHI, EHV, EID, EIT, ELD, ELN, ELW, EMD, EMF, EMH, EML, EMW, ENI, EPA, EPL, EPM, EPQ, EPW, EQD, EQY, ESF, ESV, ESW, ETA, ETD, ETE, ETL, ETW, EVA, EVE, EVS, EVW, EWE, EWH, EWM, EWN, EWS, EWY, EYF, EYQ, EYT, EYW, NDD, NDY, NEL, NEN, NEY, NFD, NFF, NFP, NFW, NHH, NWW, NYD, QDW, QFW, QPS, QVF, QWS, SDH, SDV, SFI, SIF, SLA, SLG, SMD, SPE, SPW, SWF, SWM, SYD, TDF, TDM, TDW, TDY, TEA, THW, TLW, TWD, and TYI.

35. The modified AAV capsid protein of claim 1, a. wherein X7X8X9X10is FNNT and P1P2P3P4P5P6P7P8NDNP12is SSTAGGATNDNA (SEQ ID NO: 47131), or b. wherein X7X8X9X10is FNNL and P1P2P3P4P5P6P7P8NDNP12is SSTAGGASNDNA (SEQ ID NO: 47128).

36. The modified AAV capsid protein of claim 35, wherein 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,Atty Docket No.: 38053-57988 / WO (112WO) DKD, DKE, DKH, DKQ, DKW, DLA, DLF, DLG, DLH, DLI, DLL, DLM, DLN, DLP, DLQ, DLS, DLT, DLV, DLY, DMD, DME, DMH, DMN, DMP, DMQ, DMS, DMT, DMW, DMY, DNH, DNP, DNW, DPA, DPD, DPE, DPF, DPI, DPL, DPM, DPP, DPQ, DPR, DPT, DPV, DQA, DQD, DQE, DQH, DQT, DQY, DRV, DRW, DSA, DSD, DSE, DSL, DTD, DTF, DTH, DTL, DTM, DTP, DTT, DTW, DVD, DVE, DVF, DVH, DVM, DVN, DVP, DVQ, DVT, DWI, DWL, DWM, DWS, DWY, DYA, DYE, DYF, EAD, EAE, EAH, EAI, EAL, EAS, EAV, EAW, EAY, EDA, EDD, EDE, EDF, EDG, EDK, EDL, EDM, EDN, EDP, EDQ, EDW, EDY, EEA, EED, EEE, EEF, EEL, EEM, EEQ, EEV, EEY, EFA, EFM, EFQ, EFS, EFW, EGD, EHE, EHI, EHV, EID, EIT, ELD, ELN, ELW, EMD, EMF, EMH, EML, EMW, ENI, EPA, EPL, EPM, EPQ, EPW, EQD, EQY, ESF, ESV, ESW, ETA, ETD, ETE, ETL, ETW, EVA, EVE, EVS, EVW, EWE, EWH, EWM, EWN, EWS, EWY, EYF, EYQ, EYT, EYW, NDD, NDY, NEL, NEN, NEY, NFD, NFF, NFP, NFW, NHH, NWW, NYD, QDW, QFW, QPS, QVF, QWS, SDH, SDV, SFI, SIF, SLA, SLG, SMD, SPE, SPW, SWF, SWM, SYD, TDF, TDM, TDW, TDY, TEA, THW, TLW, TWD, and TYI.

37. The modified AAV capsid protein of claim 35, wherein X1X2X3 is not selected from a. 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, NFN, NFQ, NFT, NHK, NHR, NHT, NKA, NKK, NKM, NKN, NKQ, NKR, NKS, NKW, NLQ, NLT, NNF, NPG, NPK, NPN, NQN, NQS, NRA, NRF, NRG, NRH, NRI, NRL, NRM, NRR, NRS, NRV, NRW, NRY, NSH, NSN, NSP, NSQ, NSS, NTS, NTY, NVK, NYM, NYN, QAK, QEK, QFG, QGD, QHK, QIK, QIQ, QKF, QKI, QKK, QKL, QKM, QKV, QKW, QKY, QLT, QMM, QMQ, QMS, QMT, QNH, QNT, QNV, QPP, QQP, QQQ, QRA, QRF, QRH, QRI, QRK, QRL, QRM, QRQ, QRR, QRS, QRT, QRW, QRY, QSF, QSI, QSK, QTA, QTI, QTK, QTN, QTT, QWK, QWN, QWR, QYG, QYI, QYN, QYR,Atty Docket No.: 38053-57988 / WO (112WO) QYT, SAN, SER, SFT, SHK, SHR, SIK, SKD, SKF, SKG, SKI, SKK, SKL, SKM, SKQ, SKS, SKV, SKY, SLK, SMG, SMK, SQI, SQN, SQQ, SRA, SRF, SRG, SRH, SRI, SRM, SRN, SRQ, SRR, SRS, SRT, SRV, SRW, SRY, STK, STP, STY, SVK, SWG, TAK, TDR, TFK, THK, THR, TKF, TKG, TKI, TKL, TKM, TKN, TKR, TKV, TKW, TNT, TPS, TQN, TRA, TRE, TRG, TRI, TRK, TRM, TRN, TRR, TRS, TRT, TRY, TSA, TSS, TTY, TVK, TWH, and TYR; or b. 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, NSK, NSR, NTA, NTK, NTR, NVR, NWK, NWR, NYK, NYR, QAG, QAR, QER, QFK, QFR, QFT, QGK, QGR, QGV, QGW, QHR, QIR, QKA, QKG, QKN, QKP, QKQ, QKR, QKT, QLR, QMG, QMK, QMR, QNG, QNK, QNR, QPR, QQK, QQR, QQS, QRN, QRP, QRV, QSA, QSR, QTR, QTV, QVR, QVV, QYK, SAG, SAR, SDR, SFK, SFR, SGA, SGE, SGF, SGK, SGL, SGM, SGR, SGT, SIR, SKA, SKN, SKP, SKR, SKT, SLR, SMR, SNG, SNK, SNR, SQG, SQR, SRK, SRP, SSG, SSK, SSQ, SSR, STQ, STR, SVR, SWK, SWR, SYK, SYR, TAG, TAR, TFR, TGE, TGF, TGK, TGL, TGM, TGQ, TGR, TIK, TIR, TKE, TKK, TKP, TKQ, TKS, TKT, TKY, TLK, TLR, TMK, TMR, TNK, TNR, TPR, TQK, TQR, TRP, TRQ, TRV, TSG, TSK, TSR, TTK, TTR, TVR, TWK, TWR, and TYK.

38. The modified AAV capsid protein of claim 1, wherein X1X2X3RGDX7X8X9X10 has a sequence selected from SEQ ID NOs: 98944 to 157056.

39. The modified AAV capsid protein of claim 1 or claim 38, wherein P1P2P3P4P5P6P7P8NDNP12 is selected from: NSTSGASTNDNA (SEQ ID NO: 48390); SGTTGGSSNDNT (SEQ ID NO: 46656);Atty Docket No.: 38053-57988 / WO (112WO) SSTAGGASNDNA (SEQ ID NO: 47128); and SSTAGGATNDNA (SEQ ID NO: 47131).

40. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10is NYQRGDFQNT (SEQ ID NO: 145855) and P1P2P3P4P5P6P7P8NDNP12is SGTTGGSSNDNT (SEQ ID NO: 46656).

41. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10 is SYQRGDFQNT (SEQ ID NO: 146577) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656).

42. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10 is ADRRGDFNNT (SEQ ID NO: 146987) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656).

43. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10 is NNQRGDFNNT (SEQ ID NO: 148096) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656).

44. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10 is ASSRGDFNNL (SEQ ID NO: 149579) and P1P2P3P4P5P6P7P8NDNP12 is SGTTGGSSNDNT (SEQ ID NO: 46656).

45. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10 is SGQRGDRGQI (SEQ ID NO: 124229) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390).

46. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10 is TGYRGDRGQI (SEQ ID NO: 124587) and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390).

47. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10 is QGQRGDRGQI (SEQ ID NO: 126394) and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390).Atty Docket No.: 38053-57988 / WO (112WO) 48. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10is SGVRGDRGVV (SEQ ID NO: 126741) and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390).

49. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10is TGFRGDARTL (SEQ ID NO: 99313) and P1P2P3P4P5P6P7P8NDNP12is NSTSGASTNDNA (SEQ ID NO: 48390).

50. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10is QGIRGDRSQT (SEQ ID NO: 136490) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390).

51. The modified AAV capsid protein of claim 38 or 39, wherein X1X2X3RGDX7X8X9X10 isSAQRGDRGVV (SEQ ID NO: 48394) and P1P2P3P4P5P6P7P8NDNP12 is NSTSGASTNDNA (SEQ ID NO: 48390).

52. The modified AAV capsid protein of any one of the preceding claims, having at least 90%, 95%, 98%, 99% or 99.5% sequence identity to the sequence of the reference AAV capsid protein.

53. The modified AAV capsid protein of any one of the preceding claims, wherein the AAV capsid protein is selected from VP1, VP2 and VP3.

54. The modified AAV capsid protein of any one of the preceding claims, 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; 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; rh.13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu.9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.1-C; hu.18-C; hu.3-C; hu.25-C; hu.15-C; hu.16-C; hu.11-C; hu.10-C; hu.4-C; rh.54-D; rh.48- D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E;Atty Docket No.: 38053-57988 / WO (112WO) rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu.17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; Anc113; Anc126; Anc127; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc110; and Anc80DI.

55. The modified AAV capsid protein of any one of the preceding claims, 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.

56. The modified AAV capsid protein of claim 55, wherein the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 61 or a fragment thereof.

57. The modified AAV capsid protein of claim 55, wherein the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 132 or a fragment thereof.

58. The modified AAV capsid protein of claim 55, the reference AAV capsid protein is a capsid protein having a sequence of SEQ ID NO: 142 or a fragment thereof.

59. The modified AAV capsid protein of any one of the preceding claims, wherein the targeting peptide is positioned between 565 and 595 within VR VIII of the modified AAV capsid protein.

60. The modified AAV capsid protein of claim 59, wherein: (i) 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; (ii) 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; (iii) 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 AAVAtty Docket No.: 38053-57988 / WO (112WO) capsid protein, thereby replacing residues S586, S587, and N588 of the reference capsid protein; (iv) 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, thereby replacing residues D582, Q583, and S584 of the reference capsid protein; (v) the reference AAV capsid protein is a capsid protein of AAV5 or a modification thereof and the targeting peptide is between Q574 and T578 of the reference AAV capsid protein, thereby replacing residues S575, S576, and S577 of the reference capsid protein; (vi) 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, thereby replacing residues S586, S587, and S588 of the reference capsid protein; (vii) 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, thereby replacing residues A587, A588, and N589 of the reference capsid protein; (viii) the reference AAV capsid protein is a capsid protein of AAV8 or a modification thereof and the targeting peptide is between Q587 and A591 of the modified AAV capsid protein thereby replacing residues Q588, Q589, and N590 of the reference capsid protein; (ix) 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 thereby replacing residues S586, A587, and Q588 of the reference capsid protein; (x) 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 thereby replacing residues Q588, Q589, and N590 of the reference capsid protein;Atty Docket No.: 38053-57988 / WO (112WO) (xi) 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 thereby replacing residues N565, Q566, and N567 of the reference capsid protein; (xii) 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 thereby replacing residues N590, Q591, and N592 of the reference capsid protein; (xiii) 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 thereby replacing residues S586, S587, and N588 of the reference capsid protein; (xiv) 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 thereby replacing residues S586, A587, and N588; (xv) 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 thereby replacing residues S586, S587, and N588 of the reference capsid protein; (xvi) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein; (xvii) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein; (xviii) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein;Atty Docket No.: 38053-57988 / WO (112WO) (xix) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein; or (xx) 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 thereby replacing residues S586, A587, and N588 of the reference capsid protein.

61. The modified AAV capsid protein of any one of the preceding claims, wherein variable region I (VR I) corresponds to amino acid residues between about position 259 to about position 275 of the modified capsid protein.

62. The modified AAV capsid protein of any one of claims 1 to 61, wherein the peptide segment is at a position between S261 and Y274 of an AAV9 capsid protein (SEQ ID NO: 61).

63. The modified AAV capsid protein of any one of claims 1 to 61, wherein the peptide segment is at a position between S260 and Y273 of an Anc80 capsid protein (SEQ ID NO: 132).

64. The modified AAV capsid protein of any one of claims 1 to 61, wherein the peptide segment is at a position between S260 and Y273 of an Anc80L65 capsid protein (SEQ ID NO: 142).

65. A polynucleotide encoding the modified AAV capsid protein of any one of claims 1 to 64.

66. A vector comprising the polynucleotide of claim 65.

67. The vector of claim 66, further comprising a promoter operably linked to the polynucleotide.

68. A host cell comprising the modified AAV capsid protein of any one of claims 1 to 64, the polynucleotide of claim 65, or the vector of claim 66 or 67.Atty Docket No.: 38053-57988 / WO (112WO) 69. A recombinant AAV virion (rAAV) comprising the modified AAV capsid protein of any one of claims 1 to 64.

70. The rAAV virion of claim 69, further comprising an exogenous polynucleotide.

71. The rAAV virion of claim 70, wherein the exogenous polynucleotide comprises a template for homology directed repair.

72. The rAAV virion of claim 70, wherein the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA-editing guide RNA.

73. The rAAV virion of claim 70, wherein the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

74. The rAAV virion of claim 73, wherein the therapeutic protein is MTM1 or a fragment thereof.

75. The rAAV virion of claim 74, wherein the expressible polypeptide comprises the sequence of SEQ ID NO: 165 or a fragment thereof.

76. The rAAV virion of claim 74, wherein 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.

77. The rAAV virion of any one of claims 70-76, wherein the exogenous polynucleotide further comprises a regulatory sequence.

78. The rAAV virion of claim 77, wherein the regulatory sequence comprises expression regulatory elements (EREs).

79. The rAAV virion of claim 78, wherein the EREs comprise a CAG promoter.

80. The rAAV virion of claim 78, 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.Atty Docket No.: 38053-57988 / WO (112WO) 81. A pharmaceutical composition comprising the modified AAV capsid protein of any one of claims 1 to 64 or the AAV virion of any one of claims 69-80.

82. 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 claims 69-80 or the pharmaceutical composition of claim 81.

83. The method of treating or ameliorating or preventing a disease of claim 82, wherein the disease is a muscular disease and / or the condition is muscle degeneration.

84. The method of treating or ameliorating or preventing a disease of claim 83, wherein the muscle is a striated muscle, preferably heart or a skeletal muscle or diaphragm.

85. The method of treating or ameliorating or preventing a disease of claim 84, wherein 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.

86. The modified adeno-associated virus (AAV) capsid protein of any one of claims 1 to 64, for use in treating and / or preventing a muscular disease and / or muscle degeneration.

87. A recombinant AAV (rAAV) virion comprising the modified AAV capsid protein of any one of claims 1 to 64 or the rAAV virion of any one of claims 69-80 for use in treating and / or preventing a muscular disease and / or in muscle regeneration.

88. A pharmaceutical composition comprising the modified AAV capsid protein of any one of claims 1 to 64, and / or the rAAV virion of any one of claims 69-80 for use in treating and / or preventing a muscular disease and / or in muscle regeneration.

89. A method of transferring an exogenous polynucleotide into a muscle cell, comprising the step of administering the rAAV virion of any one of claims 69-80 to a subject.

90. The method of claim 89, 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 rAAV virion.Atty Docket No.: 38053-57988 / WO (112WO) 91. The method of claim 90, wherein the muscle:liver infection ratio ranges from 1 to 100.

92. The method of claim 91, wherein the muscle:liver infection ration ranges from 1 to 10.

93. The method of claim 92, wherein the muscle:liver infection ratio ranges from 2 to 8.

94. The method of any one of claims 89-93, wherein the administration results in expression of the exogenous polynucleotide in the muscle cell, at a muscle:liver expression ratio of greater than 10.

95. The method of claim 94, wherein the muscle:liver expression ratio ranges from 10 to 100.

96. The method of claim 95, wherein the muscle:liver expression ratio ranges from 20 to 80.

97. The method of any one of claims 89-96, wherein the muscle:liver expression ratio ranges from 50 to 80 when measured by mRNA transcript expression.

98. The method of any one of claims 89-97, wherein the muscle:liver expression ratio ranges from 10 to 50 when measured by protein expression.

99. The method of any one of claims 89-98, wherein the muscle cell is selected from triceps surae, biceps, heart and quadricep.

100. Use of the modified AAV capsid protein of any one of claims 1 to 64, and / or the rAAV virion of any one of claims 69-80 for transferring an exogenous polynucleotide into a muscle cell.

101. The use of claim 100, wherein the use is a non-therapeutic use, preferably wherein the use is an in vitro use.

102. The use of claim 100, wherein the muscle cell is selected from triceps surae, biceps, heart and quadricep.

103. 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 of any one of claims 1 to 64 or the rAAV virion of any one of claims 69-80 to a subject.Atty Docket No.: 38053-57988 / WO (112WO) 104. The method of claim 103, wherein a. the target cell is heart and X7X8X9X10is FVQR, IHNL, KERF, LDEL, LIGR, QSTL, RTNL, or VRTL; b. the target cell is muscle and X7X8X9X10is ARTL, FNNL, FNNT, FQNT, HVNL, RGQI, RGVV, RNVV, RQGI, or RSVV; or c. the target cell is skeletal muscle and X7X8X9X10 is ISRT or RSQT.

105. The method or use of any one of claims 82 to 104, comprising administering 1e12 vg / kg to 1e14 vg / kg dose of rAAV to the subject.

106. The method or use of claim 105, comprising administering 1.5e12 vg / kg to 1.5e13 vg / kg dose of rAAV to the subject.

107. The method or use of claim 106, comprising administering 1e13 vg / kg dose of rAAV to the subject.

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