Recombinant AAVs with improved tropism and specificity

Abstract

The present disclosure provides modified AAV capsid proteins comprising a targeting peptide in variable region VIII (VR.VIII) and / or a peptide segment in variable region I (VR I). The modified AAV capsid proteins can form rAAVs with preferred tropism, specificity, or biodistribution in vivo or in vitro. The rAAVs of the present disclosure can be used for gene therapy targeted to specific tissues. The present disclosure provides modified AAV capsid proteins that can form rAAVs with preferred tropism and specificity for therapeutic targets.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application Nos. 63 / 371,715, filed August 17, 2022, 63 / 371,743, filed August 17, 2022, 63 / 371,744, filed August 17, 2022, 63 / 482,754, filed February 1, 2023, 63 / 482,758, filed February 1, 2023, and 2 This application claims the benefit of US Pat. No. 63 / 482,776, filed February 1, 2023; US Pat. No. 63 / 502,525, filed May 16, 2023; US Pat. No. 63 / 502,577, filed May 16, 2023; and US Pat. No. 63 / 502,595, filed May 16, 2023, each of which is incorporated by reference herein in its entirety.

[0002] 2. Sequence Listing The present application contains a Sequence Listing that has been submitted as an XML file on triplicate CD-Rs (labeled CRF, Copy 1 and Copy 2) and mailed by USPS Priority Mail Express on August 17, 2023, which is incorporated herein by reference in its entirety. The XML file, created on August 15, 2023, is named 53019WO_CRF_sequencelisting.XML and is 137,114,243 bytes in size. [Background technology]

[0003] 3.Background Adeno-associated virus (AAV) has become the vector system of choice for in vivo gene therapy. Recombinant AAV (rAAV) engineered to deliver an increasing variety of therapeutic nucleic acids are being developed and tested in non-human primates and humans, and the FDA recently approved two rAAV gene therapy products for commercialization.

[0004] Although AAV vectors are safer and less inflammatory than other viruses, toxicity still occurs after administration of high doses of rAAV for gene therapy. Therefore, local administration of rAAV to target tissues or organs has been used to improve targeting and reduce systemic toxicity. Furthermore, various natural and synthetic AAV variants have been tested to develop AAV vectors with desired tropism and specificity.

[0005] The capsid is generally considered to be a major determinant of infectivity and host-vector related properties, such as adaptive immune response, tropism, specificity, potency, and biodistribution. Indeed, some of these properties are known to vary between native serotypes and engineered AAV variants. Over the past decade, novel synthetic AAV variants have been developed using various capsid engineering techniques, one of which involves inserting small, seven-amino acid peptides into an exposed loop of the capsid protein called variable region VIII (VRVIII). In some circumstances, inserting novel peptides into the wild-type capsid alters the tropism of the variant. For example, inserting a peptide with the sequence RGDLGLS (SEQ ID NO: 156) into the capsid of AAV9 was found to increase infection of astrocytes (see Eike Kienle's PhD thesis, Ruprecht-Karls-Universitat Heidelberg, 2014) and primary breast cancer cells (Michelfelder et al. (2009)).

[0006] However, to date, little is understood about how these capsid changes functionally alter these properties. Furthermore, AAV vectors with the desired tropism and specificity for common therapeutic targets, such as muscle, are not yet available. Summary of the Invention [Means for solving the problem]

[0007] 4. Overview The present disclosure provides modified adeno-associated virus (AAV) capsid proteins capable of forming rAAVs with preferred therapeutic target specificity and target targeting. In some embodiments, the modified adeno-associated virus (AAV) capsid proteins with preferred tropism include (i) a targeting peptide at a site within variable region VIII (VR VIII) or (ii) a peptide segment within variable region I (VR I), where the targeting peptide is X1X2X3RGDX7X8X9X 10 X1, X2, X3, X7, X8, X9 and X 10 are independently selected from any amino acid residue, and the peptide segment is P1P2P3P4P5P6P7P8NDNP 12 and P1, P2, P3, P4, P5, P6, P7, P8 and P 12 are independently selected from any amino acid residue. In some cases, the modified AAV capsid protein comprises both (i) a targeting peptide and (ii) a peptide segment. In some cases, the AAV capsid protein comprises a targeting peptide but does not comprise a peptide segment. In some cases, the AAV capsid protein comprises a peptide segment but does not comprise a targeting peptide.

[0008] Applicants have demonstrated the ability to modify AAV capsid proteins with targeting peptides selected from SEQ ID NO:1 and SEQ ID NOs:238-44858 in VR VIII. In particular, the modified AAV capsid proteins can include a targeting peptide selected from SEQ ID NOs:44859-44879, 44881-44883, 44911, 44912, 44913, and 44918-44919 in the VR VIII region. The resulting rAAV containing modified AAV capsid proteins with targeting peptides at sites within variable region VIII (VR VIII) showed better targeting with more specific expression in muscle tissue.

[0009] Furthermore, Applicant has demonstrated the ability to optimize modified AAV capsid proteins by identifying which amino acid modifications at which residues in VR I (or combinations of amino acid modifications at various residues) reduce liver tropism and increase muscle tropism (or do not achieve reduced muscle tropism compared to a control capsid, e.g., a reference capsid). Applicant has previously demonstrated that a liver-toggle mutation (i.e., G266A) is associated with liver-on or liver-off tropism. Applicant now reports modified AAV capsid proteins containing one, two, three, or more amino acid modifications (within variable region I (VR I)) that, alone or together, provide interactions for specific targeting of rAAV to target tissues. Thus, the modified AAV capsid proteins of the present disclosure can alter the tropism, specificity, and / or biodistribution of AAVs containing the modified AAV capsid proteins.

[0010] Overall, the resulting rAAVs containing modified AAV capsid proteins 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 the transgene in target tissues, e.g., muscle, and also demonstrated reduced liver tropism when administered systemically to mammalian subjects.

[0011] The use of AAV for gene therapy for muscle disorders (e.g., XLMTM) has been limited due to liver toxicity. The modified AAV capsid proteins provided herein offer improved methods for treating diseases with better safety. The modified AAV capsid proteins may be able to deliver constructs encoding therapeutic genes (e.g., MTM1) with reduced liver tropism and / or improved muscle tropism. Furthermore, the constructs may be able to drive higher and more specific MTM1 expression in targets due to appropriate expression regulatory elements (EREs) (e.g., promoter sequences) and / or codon-optimized coding sequences.

[0012] In one aspect, the disclosure includes a targeting peptide in VR VIII and a peptide segment in VR I, wherein the targeting peptide is X1X2X3RGDX7X8X9X 10 X1, X2, X3, X7, X8, X9 and X 10 are independently selected from any amino acid residue, and the peptide segment is P1P2P3P4P5P6P7P8NDNP 12 and P1, P2, P3, P4, P5, P6, P7, P8 and P 12 are independently characterized by modified adeno-associated virus (AAV) capsid proteins selected from any amino acid residue.

[0013] In another aspect, the disclosure includes one or more modifications that include (i) a targeting peptide at a site within variable region VIII (VR VIII) of a reference AAV capsid protein and (ii) a peptide segment within variable region I (VR I) of a reference AAV capsid protein, wherein the targeting peptide is X1X2X3RGDX7X8X9X 10 X1, X2, X3, X7, X8, X9 and X 10 are independently selected from any amino acid residue, and the peptide segment is P1P2P3P4P5P6P7P8NDNP 12 and P1, P2, P3, P4, P5, P6, P7, P8 and P 12 are independently characterized modified adeno-associated virus (AAV) capsid proteins comprising a sequence of a reference AAV capsid protein selected from any amino acid residue.

[0014] In some embodiments, the modified AAV capsid proteins described herein comprise (i) a targeting peptide within VR VIII and (ii) a peptide segment within VR I.

[0015] In some embodiments, the targeting peptide does not include RGDLLLS (SEQ ID NO: 1).

[0016] In some embodiments, the peptide segment does not include an alanine (A) at P6 or a threonine (T) at P8.

[0017] In some embodiments, the targeting peptide does not include RGDLLLS (SEQ ID NO: 1) and the peptide segment does not include an alanine (A) at P6 or a threonine (T) at P8.

[0018] In some embodiments, the modified AAV capsid protein has one or more modifications including an amino acid insertion, deletion, substitution, or a combination thereof compared to a reference AAV capsid protein.

[0019] In some embodiments, the modified AAV capsid protein comprises one or more modifications comprising an amino acid insertion, deletion, substitution, or a combination thereof, compared to a reference AAV capsid protein to introduce a targeting peptide within VR VIII. In some embodiments, the modified AAV capsid protein also comprises more modifications outside of VR I and VR VIII of the reference AAV capsid protein.

[0020] In some embodiments, the modified AAV capsid protein has at least 90%, 95%, 98%, 99%, or 99.5% sequence identity to the sequence of a reference AAV capsid protein.

[0021] In some embodiments, the modified AAV capsid protein is selected from VP1, VP2, and VP3.

[0022] 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, 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.

[0023] 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.

[0024] In some embodiments, the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 61, or a fragment thereof.

[0025] In some embodiments, the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 132, or a fragment thereof.

[0026] In some embodiments, the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 142, or a fragment thereof.

[0027] In some embodiments, the reference AAV capsid protein is a capsid protein selected from Anc80-55, Anc80-129, Anc80-156, Anc80-751, Anc80-1029, and Anc80-1712.

[0028] In some embodiments, the reference AAV capsid protein is a capsid protein having a sequence selected from SEQ ID NOs: 44885-44898 and 44916-44917.

[0029] In some embodiments, the AAV capsid protein has a sequence selected from SEQ ID NOs: 44900-44909.

[0030] In some embodiments, X7, X8, X9 and X 10 are independently selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V and Y.

[0031] In some embodiments, X1, X2, and X3 are independently selected from any amino acid residue.

[0032] In some embodiments, X1, X2, and X3 are amino acids identical to the amino acids at the corresponding positions in a reference AAV capsid protein.

[0033] In some embodiments, X1 is an amino acid that is identical to the amino acid at the corresponding position in a reference AAV capsid protein.

[0034] In some embodiments, X2 is an amino acid that is identical to the amino acid at the corresponding position in a reference AAV capsid protein.

[0035] In some embodiments, X3 is an amino acid that is identical to the amino acid at the corresponding position in a reference AAV capsid protein.

[0036] In some embodiments, X1 and X3 are amino acids that are identical to the amino acids at the corresponding positions in a reference AAV capsid protein.

[0037] In some embodiments, X1 and X2 are amino acids that are identical to the amino acids at the corresponding positions in a reference AAV capsid protein.

[0038] In some embodiments, X2 and X3 are amino acids that are identical to the amino acids at the corresponding positions in a reference AAV capsid protein.

[0039] In some embodiments, X1, X2, and X3 are amino acids that are identical to the amino acids at the corresponding positions in a reference AAV capsid protein.

[0040] In some embodiments, X1 is an amino acid that is not identical to the amino acid at the corresponding position in a reference AAV capsid protein.

[0041] In some embodiments, X2 is an amino acid that is not identical to the amino acid at the corresponding position in a reference AAV capsid protein.

[0042] In some embodiments, X3 is an amino acid that is not identical to the amino acid at the corresponding position in a reference AAV capsid protein.

[0043] In some embodiments, X1 and X3 are amino acids that are not identical to the amino acids at the corresponding positions in a reference AAV capsid protein.

[0044] In some embodiments, X1 and X2 are amino acids that are not identical to the amino acids at the corresponding positions in a reference AAV capsid protein.

[0045] In some embodiments, X2 and X3 are amino acids that are not identical to the amino acids at the corresponding positions in a reference AAV capsid protein.

[0046] In some embodiments, 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, hi some embodiments, the one or more modifications comprise a substitution of one or more amino acids between amino acid positions 565 and 595 of the reference AAV capsid.

[0047] In some embodiments, X 1 is selected from S, E, A, D, N, Q, or T.

[0048] 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.

[0049] 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.

[0050] In some embodiments, X2 is selected from K, E, D, A, S, F, N, V, or L. In some embodiments, X2 is selected from K, E, or D. In some embodiments, X2 is selected from N, A, or Y. In some embodiments, X2 is selected from N, Y, or S.

[0051] In some embodiments, X3 is selected from R, Q, A, D, E, F, G, H, I, K, L, M, N, P, S, T, V, W or Y.

[0052] In some embodiments, X2X3 is NR.

[0053] In some embodiments, X1X2X3 is ENR.

[0054] In some embodiments, X1X2X3 is SNR.

[0055] In some embodiments, X1X2X3 is 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, 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,<h2 style=";text-align:left;direction:ltr">EVY、EQY、QEY、QEN、SAD、AEF、EMF、SDF、ADF、ADY、AVF、TLY、SIY、SLY、AIY、DY V、AEY、ENF、EMY、QDY、ETY、EAY、TVY、SVY、QVF、QVY、TMY、DKY、ALY、AVY、TMW、E QW, 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, TT I、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、E PV、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、EV V、EVM、TPY、QEW、EIW、EPT、EIH、DFY、DQL、NDA、DIW、TDY、ELF、DVY、ESG、EPG、E IG、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、EA N、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、E GN、ENH、NGE、DYN、DNH、DNF、ESQ、ESM、ESL、SSD、ENL、EMV、QDS、TSE、SWD、ASD、<h2 style=";text-align:left;direction:ltr">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, E YM, 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, TN F、NAF、ASP、QSQ、APY、SFM、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、A FP、AEE、ADE、NIE、TYP、QIE、TFP、SYP、ELW、DVW、NEW、EPA、EVA、TLE、AWE、NME、 QME, ATE, AYA, 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, S IE、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、DQ W、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, SLWIt is selected from the group consisting of 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.,

[0056] 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、ES T、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、A NW、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、NN F, NWL, NMA, NNA, NHN, NFH, ELR, NYM, ERA, NFS, SRD, NMM, NNL, NNQ, NNS, QDR, S DK、NNI、AQH、ANA、QNH、QWG、AWA、QWA、EQK、SHS、QFA、SFA、TFA、AYG、EKQ、ERN 、EMR、EFR、AYM、EAR、EMK、EYK、EWK、QPI、QPF、NPL、SPI、SPL、NPI、SIV、SLL、S SA、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、NA M、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、N MV、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、TW M、QNW、STF、AFM、NSY、SSY、SNY、THY、TNY、QNY、THF、NSV、ATY、SKD、TNF、TWT、Q WT、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、TG H、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, Q SS、SAH、SMH、QAH、AAH、AHN、QNS、QAS、ANS、ANN、SWA、SWS、TWS、ERI、EX、QWS、 AYS、AFS、SYS、QFS、QYN、QFT、AYT、TNS、TYS、SFT、QNQ、QYS、SYT、ANQ、SWV、SN Q、ANY、TNQ、ATF、SSQ、SSN、TWA、SGQ、SSS、TWY、SMY、TGN、TGY、QWY、SQF、TGF、Q TY、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、AS F、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、N ST、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, SA T, SAI, QSL, QSI, ASI, SSL, SSV, SSI, AGI, QGM, TGM, SGM, SGL, SGV, ARL, NWE, S FL、TPY、DRP、DTK、DMK、SEY、SME、EAI、AHD、DNA、EFQ、EMH、SGE、DNQ、DNI、QLY DGT, EGN, DYN, EWV, ESM, EAL, ASD, DKA, ADF, ADY, SLY, AEY, ETY, EAY, TGA, AV M、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、A DM、SAA、SQH、NQV、ALL、ATM、QYA、STT、DGR、NHT、SHT、QHT、TTL、NHM、QHL、DNR、 QHM、QFM、QMI、AHV、THI、AHT、TNH、NHV、THV、SHI、ATV、TWI、NYV、TYI、DYR、SF I, NQF, NWM, NSL, NSP, TQY, DRS, ASP, TKD, QWI, QWN, TWN, QWV, THN, TTM, TQQ, N MQ、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、NG N、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、Q DI, DSA, DNM, DKT, DGM, SQY, TLT, QEH, DKQ, TGE, DNS, SDV, SNE, EGT, EGV, AAPSelected from the group consisting of 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, QQT, 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.,

[0057] X1X2X3, 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、EQ R、EHR、EWR、EQK、ESR、EKQ、EYR、ENR、EMK、EYK、EHK、EWK、QNI、TNI、TYI、SNY、 DRQ、AWI、QWI、DFR、EKG、QYG、QWQ、EKN、EKF、EKT、AFH、ENK、NYS、DKH、AAG、QMF 、QFH、QKD、ARE、AHQ、ADK、ADR、AHN、QNS、ANN、SWS、EKS、AFS、QFS、TNQ、TGY、N GL、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, DT K, DNA, SDS, TDH, DGL, QDV, SDM, DKT, DGM, SQY, DKS, SSD, EMV, TSE, EYV, SSE, E GM、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、N MM、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、NQ I、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、A YH、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、SM N、SAN、QAF、ASF、ASY、TAS、TAF、TFV、SMQ、SFV、TMT、QAN、SAY、EDK、AMV、QHV、T HS、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、AN H、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, S NP、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, TT V、AAL、NHL、NHM、QYL、QHL、ATL、DNR、DSR、ANL、SHL、SNL、TNL、QAL、AMI、ANI、Q NM、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、QL V、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, N ES、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, EQ H, EHH, NWV, EKY, STY, DRI, EYH, DRV, SPY, NNY, DWR, ERA, QRE, EVR, SRD, AWA, SFA, QTI, SHT, THM, AQI, AHI, SWT, ATV, TTI, SMI, QYV, NMY, SWM, Q NY, 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. ,

[0058] In some embodiments, RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 238 to 44858.

[0059] In some embodiments, RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 238 to 248.

[0060] In some embodiments, RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 238 to 338.

[0061] In some embodiments, RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 238 to 438.

[0062] In some embodiments, RGDX7X8X9X 10has an amino acid sequence selected from SEQ ID NOs: 238 to 538.

[0063] In some embodiments, RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 238 to 638.

[0064] In some embodiments, RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 238 to 738.

[0065] In some embodiments, RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 3881, 12092, 14601, 15342, 21498 and 31396.

[0066] In some embodiments, RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NO: 238.

[0067] In some embodiments, X1X2X3RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 44859-44883, 44911, 44912, 44913, 44918-44919 and 48391-157057.

[0068] In some embodiments, X1X2X3RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 44864 to 44867 and 44879 to 44883.

[0069] In some embodiments, X1X2X3RGDX7X8X9X 10 has an amino acid sequence selected from SEQ ID NOs: 44859-44878, 44911, 44912, 44913, 44918-44919 and 48391-157057.

[0070] In some embodiments, X7 is selected from R, F, H, L, Q, R, and Y.

[0071] In some embodiments, X7 is R. In some embodiments, X7 is Y or H. In some embodiments, X7 is F or Y.

[0072] In some embodiments, X8 is selected from S, G, D, I, L, N, Q, T, and V.

[0073] In some embodiments, X8 is S or G. In some embodiments, X8 is T, G or S. In some embodiments, X8 is N or Q.

[0074] In some embodiments, X9 is selected from any of the amino acids: V, S, N, G, Q, L, T, and Y.

[0075] 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, X9 is selected from S or V. In some embodiments, X9 is selected from N or S.

[0076] In some embodiments, X 10 is selected from I, V, S, L, M, R, T and Q.

[0077] In some embodiments, X 10 is I or V. In some embodiments, X 10 is V, L, or M. In some embodiments, X 10 is T or L.

[0078] In some embodiments, the targeting peptide is selected from the group consisting 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: 98 935), X1X2X3RGDFNNT (SEQ ID NO: 98943), X1X2X3RGDYSSV (SEQ ID NO: 98937), 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).

[0079] 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).

[0080] In some embodiments, the targeting peptide has the 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).

[0081] In some embodiments, the targeting peptide has the 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).

[0082] In some embodiments, X1X2X3 is selected from the group consisting of EFK, AAY, DQK, QVY, DKL, DNV, ENF, EWK, QNV, and TFM.

[0083] In some embodiments, the targeting peptide has an amino acid sequence selected from RGDRSX9I, RGDRGX9I, RGDRSX9V, or RGDRGX9V.

[0084] 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).

[0085] In some embodiments, X1 is D or E, X2 is K, E, D, A, S, F or N, and X3 is Y, V or F.

[0086] In some embodiments, X1X2X3 is 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, 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,<h2 style=";text-align:left;direction:ltr">EVY、EQY、QEY、QEN、SAD、AEF、EMF、SDF、ADF、ADY、AVF、TLY、SIY、SLY、AIY、DY V、AEY、ENF、EMY、QDY、ETY、EAY、TVY、SVY、QVF、QVY、TMY、DKY、ALY、AVY、TMW、E QW, 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, TT I、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、E PV、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、EV V、EVM、TPY、QEW、EIW、EPT、EIH、DFY、DQL、NDA、DIW、TDY、ELF、DVY、ESG、EPG、E IG、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、EA N、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、E GN、ENH、NGE、DYN、DNH、DNF、ESQ、ESM、ESL、SSD、ENL、EMV、QDS、TSE、SWD、ASD、<h2 style=";text-align:left;direction:ltr">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, E YM, 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, TN F、NAF、ASP、QSQ、APY、SFM、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、A FP、AEE、ADE、NIE、TYP、QIE、TFP、SYP、ELW、DVW、NEW、EPA、EVA、TLE、AWE、NME、 QME, ATE, AYA, 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, S IE、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、DQ W、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, SLWTPA, 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. ,

[0087] In some embodiments, X1X2X3 is 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, Q selected from the group consisting of VF, QVY, SDL, SDV, SEH, SII, SIY, SSL, SVY, SWD, SWE, TDF, TDV, TDY, TEF, TEY, TIY, TLY, and TWQ.

[0088] In some embodiments, X7 is selected from Y and H, X8 is selected from T, G and S, X9 is selected from S and V, and X 10 is selected from V, L and M.

[0089] 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).

[0090] In some embodiments, X1 is S, A, or T; X2 is N, A, or Y; and X3 is I, V, M, Q, T, Y, or K.

[0091] 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、ES T、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、A NW、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、NN F, NWL, NMA, NNA, NHN, NFH, ELR, NYM, ERA, NFS, SRD, NMM, NNL, NNQ, NNS, QDR, S DK、NNI、AQH、ANA、QNH、QWG、AWA、QWA、EQK、SHS、QFA、SFA、TFA、AYG、EKQ、ERN 、EMR、EFR、AYM、EAR、EMK、EYK、EWK、QPI、QPF、NPL、SPI、SPL、NPI、SIV、SLL、S SA、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、NA M、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、N MV、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、TW M、QNW、STF、AFM、NSY、SSY、SNY、THY、TNY、QNY、THF、NSV、ATY、SKD、TNF、TWT、Q WT、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、TG H、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, Q SS、SAH、SMH、QAH、AAH、AHN、QNS、QAS、ANS、ANN、SWA、SWS、TWS、ERI、EX、QWS、 AYS、AFS、SYS、QFS、QYN、QFT、AYT、TNS、TYS、SFT、QNQ、QYS、SYT、ANQ、SWV、SN Q、ANY、TNQ、ATF、SSQ、SSN、TWA、SGQ、SSS、TWY、SMY、TGN、TGY、QWY、SQF、TGF、Q TY、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、AS F、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、N ST、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, SA T, SAI, QSL, QSI, ASI, SSL, SSV, SSI, AGI, QGM, TGM, SGM, SGL, SGV, ARL, NWE, S FL、TPY、DRP、DTK、DMK、SEY、SME、EAI、AHD、DNA、EFQ、EMH、SGE、DNQ、DNI、QLY DGT, EGN, DYN, EWV, ESM, EAL, ASD, DKA, ADF, ADY, SLY, AEY, ETY, EAY, TGA, AV M、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、A DM、SAA、SQH、NQV、ALL、ATM、QYA、STT、DGR、NHT、SHT、QHT、TTL、NHM、QHL、DNR、 QHM、QFM、QMI、AHV、THI、AHT、TNH、NHV、THV、SHI、ATV、TWI、NYV、TYI、DYR、SF I, NQF, NWM, NSL, NSP, TQY, DRS, ASP, TKD, QWI, QWN, TWN, QWV, THN, TTM, TQQ, N MQ、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、NG N、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、Q DI, DSA, DNM, DKT, DGM, SQY, TLT, QEH, DKQ, TGE, DNS, SDV, SNE, EGT, EGV, AAPSelected from the group consisting of 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, QQT, 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.

[0092] In some embodiments, X1X2X3 is 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, QN F, QNQ, QNS, QNT, QNV, QNY, SAH, SAI, SAL, SFT, SFV, SHI, SHV, SMM, SNF, SNM, SNN, SNQ, SNV, SNY, SQI, SQV, SSL, Selected from SWQ, SWS, SYI, SYM, SYN, SYQ, TAM, TAT, TDR, TFM, THV, TNF, TNH, TNI, TNM, TNQ, TNV, TSY, TWA, and TYM.

[0093] In some embodiments, X7 is selected from F and Y, X8 is selected from N and Q, X9 is selected from N and S, and X 10 is selected from T and L.

[0094] 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).

[0095] In some embodiments, X1 is S, A, or E; X2 is N, Y, or S; and X3 is I, Q, R, V, T, M, or K.

[0096] X1X2X3, 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、EQ R、EHR、EWR、EQK、ESR、EKQ、EYR、ENR、EMK、EYK、EHK、EWK、QNI、TNI、TYI、SNY、 DRQ、AWI、QWI、DFR、EKG、QYG、QWQ、EKN、EKF、EKT、AFH、ENK、NYS、DKH、AAG、QMF 、QFH、QKD、ARE、AHQ、ADK、ADR、AHN、QNS、ANN、SWS、EKS、AFS、QFS、TNQ、TGY、N GL、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, DT K, DNA, SDS, TDH, DGL, QDV, SDM, DKT, DGM, SQY, DKS, SSD, EMV, TSE, EYV, SSE, E GM、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、N MM、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、NQ I、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、A YH、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、SM N、SAN、QAF、ASF、ASY、TAS、TAF、TFV、SMQ、SFV、TMT、QAN、SAY、EDK、AMV、QHV、T HS、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、AN H、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, S NP、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, TT V、AAL、NHL、NHM、QYL、QHL、ATL、DNR、DSR、ANL、SHL、SNL、TNL、QAL、AMI、ANI、Q NM、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、QL V、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, N ES、DWK、DLR、DIR、SDT、EAN、ETH、TYD、SYE、EAM、TND、AYD、TSP、DRT、NDH、SSF、It is selected from the group consisting of 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, 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.,

[0097] 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.

[0098] In some embodiments, RGDRX8X9X 10 is RGDRGVX 10(SEQ ID NO: 157058), RGDRGSX 10 (SEQ ID NO: 157059), RGDRGNX 10 (SEQ ID NO: 157060), RGDRGGX 10 (SEQ ID NO: 157061), RGDRGQX 10 (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).

[0099] In some embodiments, the modified sequence does not comprise an amino acid sequence selected from RGDRMVF (SEQ ID NO: 157080), RGDRTVI (SEQ ID NO: 157081), SRGDRPM (SEQ ID NO: 157082), ISLRGDR (SEQ ID NO: 157083), and RGDLLLS (SEQ ID NO: 1).

[0100] In some embodiments, the targeting peptide is 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); X1X2 X3RGDFNNL (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, X1 has a sequence selected from S, E, A, D, N, Q, or T. In some embodiments, X1 is selected from S or E. In some embodiments, X1 is S. In some embodiments, X1 is E. In some embodiments, X2 is selected from N, A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X2 is selected from N or A. In some embodiments, X2 is N. In some embodiments, X2 is A. In some embodiments, X3 is selected from R, Q, A, D, E, F, G, H, I, K, L, M, N, P, S, T, V, W, or Y. In some embodiments, 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 X3 is R. In some embodiments, X1 is E, X2 is N, and X3 is R. In some embodiments, X1 is S, X2 is N, and X3 is R.

[0101] In some embodiments, the targeting peptide is positioned between 565 and 595 within VR VIII of the modified AAV capsid protein.

[0102] In some embodiments, The reference AAV capsid protein is the capsid protein of AAV1 or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein; The reference AAV capsid protein is an AAV2 capsid protein or a modification thereof, and the targeting peptide is between Q584 and R588 or between N587 and R588 of the reference AAV capsid protein; The reference AAV capsid protein is an AAV3 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. the reference AAV capsid protein is the capsid protein of AAV4 or a modification thereof, and the targeting peptide is between G581 and N585 of the reference AAV capsid protein; The reference AAV capsid protein is an AAV5 capsid protein or a modification thereof, and the targeting peptide is between Q574 and T578 of the reference AAV capsid protein. The reference AAV capsid protein is an AAV6 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. The reference AAV capsid protein is an AAV7 capsid protein or a modification thereof, and the targeting peptide is between Q586 and T590 of the reference AAV capsid protein. The reference AAV capsid protein is an AAV8 capsid protein or a modification thereof, and the targeting peptide is between Q587 and A591 of the reference AAV capsid protein. The reference AAV capsid protein is the capsid protein of AAV9 or a modification thereof, and the targeting peptide is between H587 and A591 of the reference AAV capsid protein. The reference AAV capsid protein is the capsid protein of AAVrh10 or a modification thereof, and the targeting peptide is between Q587 and A591 of the reference AAV capsid protein; the reference AAV capsid protein is the capsid protein of AAVpo.1 or a modification thereof, and the targeting peptide is between N564 and S568 of the reference AAV capsid protein; The reference AAV capsid protein is an AAV12 capsid protein or a modification thereof, and the targeting peptide is between N589 and A593 of the reference AAV capsid protein. The reference AAV capsid protein is the capsid protein of Anc80 or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. The reference AAV capsid protein is the Anc80L65 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein; the reference AAV capsid protein is the Anc80-55 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein; the reference AAV capsid protein is the Anc80-129 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein; the reference AAV capsid protein is the Anc80-156 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein; The reference AAV capsid protein is the capsid protein of Anc80-751 or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein. the reference AAV capsid protein is the Anc80-1029 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein; or The reference AAV capsid protein is the capsid protein of Anc80-1712 or a modification thereof, and the targeting peptide is between Q585 and T589 of the reference AAV capsid protein.

[0103] In some embodiments, The reference AAV capsid protein is an AAV1 capsid protein or a modification thereof, and the targeting peptide is between D590 and P591 or between S588 and T589 of the reference AAV capsid protein; the reference AAV capsid protein is an AAV2 capsid protein or a modification thereof, and the targeting peptide is between R588 and Q588 or between N587 and R587 of the reference AAV capsid protein; the reference AAV capsid protein is an AAV3 capsid protein or a modification thereof, and the targeting peptide is between S586 and S587 or between N588 and T589 of the reference AAV capsid protein; The reference AAV capsid protein is an AAV4 capsid protein or a modification thereof, and the targeting peptide is between S584 and N585 or between S586 and N587 of the reference AAV capsid protein. the reference AAV capsid protein is an AAV5 capsid protein or a modification thereof, and the targeting peptide is between S575 and S576 or between T577 and T578 of the reference AAV capsid protein; The reference AAV capsid protein is an AAV6 capsid protein or a modification thereof, and the targeting peptide is between D590 and P591 or between S588 and T589 of the reference AAV capsid protein; The reference AAV capsid protein is an AAV7 capsid protein or a modification thereof, and the targeting peptide is between N589 and T590 of the reference AAV capsid protein. The reference AAV capsid protein is an AAV8 capsid protein or a modification thereof, and the targeting peptide is between N590 and T591 of the reference AAV capsid protein. The reference AAV capsid protein is the capsid protein of AAV9 or a modification thereof, and the targeting peptide is between Q588 and A589 of the reference AAV capsid protein. The reference AAV capsid protein is the capsid protein of AAVrh10 or a modification thereof, and the targeting peptide is between N590 and A591 of the reference AAV capsid protein. the reference AAV capsid protein is the 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 reference AAV capsid protein; The reference AAV capsid protein is an AAV12 capsid protein or a modification thereof, and the targeting peptide is between N592 and A593 or between T594 and T595 of the reference AAV capsid protein; The reference AAV capsid protein is an Anc80 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; The reference AAV capsid protein is the Anc80L65 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; the reference AAV capsid protein is the Anc80-55 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; the reference AAV capsid protein is the Anc80-129 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; the reference AAV capsid protein is the Anc80-156 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; the reference AAV capsid protein is the Anc80-751 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; the reference AAV capsid protein is the Anc80-1029 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; or The reference AAV capsid protein is the 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 reference AAV capsid protein.

[0104] In some embodiments, P1 is independently selected from asparagine (N), serine (S), or threonine (T); P2 is independently selected from serine (S) or glycine (G); P3 is independently selected from threonine (T), glutamine (Q), alanine (A), or glutamic acid (E); P4 is independently selected from serine (S), threonine (T), or alanine (A); P5 is independently selected from glycine (G) or alanine (A); P6 is independently selected from glycine (G) or alanine (A); P7 is independently selected from alanine (A) or serine (S); P8 is independently selected from serine (S) or threonine (T), and P 12 are independently selected from histidine (H), threonine (T) or alanine (A).

[0105] In some embodiments, P5, P6, or both P5 and P6 are not alanine (A).

[0106] In some embodiments, the peptide segment does not include an alanine (A) at P6 or a threonine (T) at P8.

[0107] In some embodiments, the peptide segment is P1P2P3P4GGP7P8NDNP 12 (SEQ ID NO: 44921), and 12 are independently selected from any amino acid residue.

[0108] In some embodiments, P1 is independently selected from asparagine (N) or serine (S); P2 is independently selected from serine (S) or glycine (G); P3 is independently selected from threonine (T) or glutamine (Q); P4 is independently selected from serine (S), threonine (T), or alanine (A); P7 is independently selected from alanine (A) or serine (S); P8 is independently selected from serine (S) or threonine (T), and P 12 are independently selected from histidine (H), threonine (T) or alanine (A).

[0109] In some embodiments, the peptide segment is P1P2TP4GGP7P8NDNP 12 (SEQ ID NO: 44922), and 12 are independently selected from any amino acid residue.

[0110] In some embodiments, the peptide segment is P1P2QP4GGP7P8NDNP 12 (SEQ ID NO: 44923), and 12 are independently selected from any amino acid residue.

[0111] In some embodiments, the peptide segment is P1P2TP4GGP7TNDNP 12 (SEQ ID NO: 44924), and 12 are independently selected from any amino acid residue.

[0112] In some embodiments, the peptide segment is NP2TP4GGP7P8NDNP 12 (SEQ ID NO: 44925), 12 are independently selected from any amino acid residue.

[0113] In some embodiments, the peptide segment is SP2TP4GGP7P8NDNP 12 (SEQ ID NO: 44926), and 12 are independently selected from any amino acid residue.

[0114] In some embodiments, the peptide segment is NSTSGGP7P8NDNH (SEQ ID NO: 44927), where P7 and P8 are independently selected from any amino acid residue.

[0115] In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T).

[0116] In some embodiments, the peptide segment is NSTSGGASNDNH (SEQ ID NO: 46026), NSTSGGATNDNH (SEQ ID NO: 46029), NSTSGGSSNDNH (SEQ ID NO: 46031), or NSTSGGSTNDNH (sequence number 46034) Includes.

[0117] In some embodiments, the peptide segment is NSTTGGP7P8NDNH (SEQ ID NO: 44928), where P7 and P8 are independently selected from any amino acid residue.

[0118] In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T).

[0119] In some embodiments, the peptide segment is NSTTGGASNDNH (SEQ ID NO: 46073), NSTTGGATNDNH (SEQ ID NO: 46076), NSTTGGSSNDNH (SEQ ID NO: 46079), or NSTTGGSTNDNH (SEQ ID NO: 46082) Includes.

[0120] In some embodiments, the peptide segment is SGQTGGP7P8NDNH (SEQ ID NO: 44929), where P7 and P8 are independently selected from any amino acid residue.

[0121] In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T).

[0122] In some embodiments, the peptide segment is SGQTGGASNDNH (SEQ ID NO: 46505), SGQTGGATNDNH (SEQ ID NO: 46508), SGQTGGSSNDNH (SEQ ID NO: 46511), or SGQTGGSTNDNH (sequence number 46514) Includes.

[0123] In some embodiments, the peptide segment is SGTAGGP7P8NDNT (SEQ ID NO: 44930), where P7 and P8 are independently selected from any amino acid residue.

[0124] In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T).

[0125] In some embodiments, the peptide segment is SGTAGGASNDNT (SEQ ID NO: 46554), or SGTAGGSSNDNT (sequence number 46560) Includes.

[0126] In some embodiments, the peptide segment does not include SGTAGGATNDNT (SEQ ID NO: 46557) or SGTAGGSTNDNT (SEQ ID NO: 46563).

[0127] In some embodiments, the peptide segment is SGTSGGP7P8NDNA (SEQ ID NO: 44931), where P7 and P8 are independently selected from any amino acid residue.

[0128] In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T).

[0129] In some embodiments, the peptide segment is SGTSGGASNDNA (SEQ ID NO: 46600), SGTSGGATNDNA (SEQ ID NO: 46603), SGTSGGSSNDNA (SEQ ID NO: 46606), or SGTSGGSTNDNA (SEQ ID NO: 46609) Includes.

[0130] In some embodiments, the peptide segment is SGTTGGP7P8NDNT (SEQ ID NO: 44932), where P7 and P8 are independently selected from any amino acid residue.

[0131] In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T).

[0132] In some embodiments, the peptide segment is SGTTGGASNDNT (SEQ ID NO: 46650), SGTTGGATNDNT (sequence number 46653), SGTTGGSSNDNT (SEQ ID NO: 46656), or SGTTGGSTNDNT (sequence number 46659) Includes.

[0133] In some embodiments, the peptide segment is SSTAGGP7P8NDNA (SEQ ID NO: 44933), where P7 and P8 are independently selected from any amino acid residue.

[0134] In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T).

[0135] In some embodiments, the peptide segment is SSTAGGASNDNA (SEQ ID NO: 47128), SSTAGGATNDNA (SEQ ID NO: 47131), SSTAGGSSNDNA (SEQ ID NO: 47134), or SSTAGGSTNDNA (SEQ ID NO: 47137) Includes.

[0136] In some embodiments, the peptide segment is SSTAGGASNDNA (SEQ ID NO: 47128).

[0137] In some embodiments, the peptide segment is SSTAGGATNDNA (SEQ ID NO: 47131).

[0138] In some embodiments, the peptide segment is NSTSGASTNDNA (SEQ ID NO: 48390).

[0139] In some embodiments, the peptide segment is selected from the peptide segments shown in Tables 20 or 28.

[0140] In some embodiments, variable region I (VR I) corresponds to amino acid residues between about 259 and about 275 of the modified capsid protein.

[0141] In some embodiments, the peptide segment is located between S261 and Y274 of the AAV9 capsid protein (SEQ ID NO: 61).

[0142] In some embodiments, the peptide segment is located between S260 and Y273 of the Anc80 capsid protein (SEQ ID NO: 132).

[0143] In some embodiments, the peptide segment is located between S260 and Y273 of the Anc80L65 capsid protein (SEQ ID NO: 142).

[0144] In some embodiments, the peptide segment is located between S260 and Y273 of the AAV2 capsid protein (SEQ ID NO: 55).

[0145] In some embodiments, the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919, and the peptide segment has an amino acid sequence selected from SEQ ID NOs: 46026, 46029, 46031, 46035, 46073, 46076, 46079, 46082, 46505, 46508, 46511, 46514, 46554, 46560, 46609, 46600, 46603, 46606, 46650, 46653, 46656, 46659, 47128, 47131, 47134, and 47137.

[0146] In some embodiments, the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919, and the peptide segment has the amino acid sequence SGTAGGASNDNT (SEQ ID NO: 46554).

[0147] In some embodiments, the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919, and the peptide segment has the amino acid sequence SGTSGSTNDNA (SEQ ID NO: 46609).

[0148] In some embodiments, the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919, and the peptide segment has the amino acid sequence SGTTGGSTNDNT (SEQ ID NO: 46659).

[0149] In some embodiments, the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919, and the peptide segment has the amino acid sequence SGTTGGSTNDNT (SEQ ID NO: 47128).

[0150] In some embodiments, the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919, and the peptide segment has the amino acid sequence NSTSGGSSNDNA (SEQ ID NO: 48388).

[0151] In some embodiments, the targeting peptide has an amino acid sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919, and the peptide segment has the amino acid sequence NSTSGASTNDNA (SEQ ID NO: 48390).

[0152] In some embodiments, the targeting peptide has the amino acid sequence ENRRGDFNNL (SEQ ID NO: 44864) and the peptide segment has the amino acid sequence SGTTGGSTNDNT (SEQ ID NO: 46659).

[0153] In some embodiments, the targeting peptide has the amino acid sequence ENRRGDFNNL (SEQ ID NO: 44864) and the peptide segment has the amino acid sequence SGTTGGSSNDNT (SEQ ID NO: 46656).

[0154] In some embodiments, the targeting peptide has the amino acid sequence ENRRGDFNNL (SEQ ID NO: 44864) and the peptide segment has the amino acid sequence SSTAGGASNDNA (SEQ ID NO: 47128).

[0155] In some embodiments, the targeting peptide has the amino acid sequence SAQRGDLLLS (SEQ ID NO: 44882) and the peptide segment has the amino acid sequence SSTAGGATNDNA (SEQ ID NO: 47131).

[0156] In some embodiments, the targeting peptide has the amino acid sequence SAQRGDLLLS (SEQ ID NO: 44882) and the peptide segment has the amino acid sequence SGTTGGSTNDNT (SEQ ID NO: 46659).

[0157] In some embodiments, the targeting peptide has the amino acid sequence SAQRGDLLLS (SEQ ID NO: 44882) and the peptide segment has the amino acid sequence SGTTGGSSNDNT (SEQ ID NO: 46656).

[0158] In some embodiments, the targeting peptide has the amino acid sequence SAQRGDLLLS (SEQ ID NO: 44882) and the peptide segment has the amino acid sequence SSTAGGASNDNA (SEQ ID NO: 47128).

[0159] In some embodiments, the targeting peptide has the amino acid sequence ENRRGDFQNT (SEQ ID NO: 44866) and the peptide segment has the amino acid sequence SSTAGGATNDNA (SEQ ID NO: 47131).

[0160] In some embodiments, the targeting peptide has the amino acid sequence ENRRGDFQNT (SEQ ID NO: 44866) and the peptide segment has the amino acid sequence SGTTGGSTNDNT (SEQ ID NO: 46659).

[0161] In some embodiments, the targeting peptide has the amino acid sequence ENRRGDFQNT (SEQ ID NO: 44866) and the peptide segment has the amino acid sequence SGTTGGSSNDNT (SEQ ID NO: 46656).

[0162] In some embodiments, the targeting peptide has the amino acid sequence SNRRGDFNNT (SEQ ID NO: 44883) and the peptide segment has the amino acid sequence SSTAGGASNDNA (SEQ ID NO: 47128).

[0163] In another aspect, the disclosure includes a targeting peptide at a site within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 44864-44867, 44879-44883, 44911, 44912, 44913, and 44918-44919, and 1, wherein the peptide segment has a sequence selected from SEQ ID NOs: 46026, 46029, 46031, 46035, 46073, 46076, 46079, 46082, 46505, 46508, 46511, 46514, 46554, 46560, 46609, 46600, 46603, 46606, 46650, 46653, 46656, 46659, 47128, 47131, 47134, and 47137.

[0164] In another aspect, the disclosure features a polynucleotide encoding any of the modified AAV capsid proteins described herein.

[0165] In another aspect, the disclosure features a vector including any of the polynucleotides described herein.

[0166] In some embodiments, the vector also comprises a promoter operably linked to the polynucleotide.

[0167] In another aspect, the disclosure features a host cell containing any of the modified AAV capsid proteins described herein, any of the polynucleotides described herein, or any of the vectors described herein.

[0168] In another aspect, the disclosure features a recombinant AAV virion (rAAV), any of the modified AAV capsid proteins described herein.

[0169] In some embodiments, the AAV virion also comprises an exogenous polynucleotide.

[0170] In some embodiments, the exogenous polynucleotide comprises a homology-directed repair template.

[0171] In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA editing guide RNA.

[0172] In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

[0173] In some embodiments, the therapeutic protein is MTM1 or a fragment thereof.

[0174] In some embodiments, the expressible polypeptide comprises the sequence of SEQ ID NO: 165 or a fragment thereof.

[0175] In some embodiments, the expressible polypeptide comprises a sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 166-170.

[0176] In some embodiments, the exogenous polynucleotide further comprises a regulatory sequence. In some embodiments, the regulatory sequence comprises an expression regulatory element (ERE). In some embodiments, the ERE comprises a CAG promoter. In some embodiments, the ERE comprises a sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 171-173.

[0177] In another aspect, the disclosure features a pharmaceutical composition including any of the modified AAV capsid proteins described herein or any of the rAAV virions described herein.

[0178] In another aspect, the disclosure features a method for treating, ameliorating, or preventing a disease or condition in a subject, the method including administering a therapeutically effective amount of any of the AAV virions described herein or any of the pharmaceutical compositions described herein.

[0179] In some embodiments of any of the methods of treating, ameliorating, or preventing a disease described herein, the disease is a muscle disease and / or the condition is muscle degeneration.

[0180] In some embodiments of any of the methods of treating, ameliorating, or preventing a disease described herein, the muscle is a striated muscle, preferably a cardiac or skeletal muscle or a diaphragm.

[0181] In some embodiments of any of the methods of treating, ameliorating, or preventing a disease described herein, the muscle disease is muscular dystrophy, cardiomyopathy, myotonia, muscle atrophy, myoclonus-dystonia, mitochondrial myopathy, rhabdomyolysis, fibromyalgia, and / or myofascial pain syndrome.

[0182] In some embodiments, any of the modified adeno-associated virus (AAV) capsid proteins described herein for use in the treatment and / or prevention of muscle diseases and / or muscle degeneration.

[0183] In another aspect, the disclosure features an AAV virion comprising any of the modified AAV capsid proteins described herein or any of the rAAV virions described herein for use in the treatment and / or prevention of muscle diseases and / or in muscle regeneration.

[0184] In another aspect, the disclosure features a pharmaceutical composition including any of the modified AAV capsid proteins described herein and / or any of the rAAV virions described herein for use in the treatment and / or prevention of muscle diseases and / or in muscle regeneration.

[0185] In another aspect, the disclosure features a method of transferring an exogenous polynucleotide into a muscle cell, the method including administering to a subject any of the rAAV virions described herein.

[0186] In some embodiments, administration results in the transfer of the exogenous polynucleotide into muscle cells at a muscle:liver infection ratio of greater than 1, as measured by genome copies of AAV virions.

[0187] In some embodiments, the muscle:liver infection ratio ranges from 1-100.

[0188] In some embodiments, the muscle:liver infection ratio ranges from 1-10.

[0189] In some embodiments, the muscle:liver infection ratio ranges from 2-8.

[0190] In some embodiments, the administration results in expression of the exogenous polynucleotide in muscle cells at a muscle:liver expression ratio of greater than 10.

[0191] In some embodiments, the muscle:liver expression ratio is in the range of 10-100.

[0192] In some embodiments, the muscle:liver expression ratio is in the range of 20-80.

[0193] In some embodiments, the muscle:liver expression ratio, as measured by mRNA transcript expression, ranges from 50 to 80.

[0194] In some embodiments, the muscle:liver expression ratio, as measured by protein expression, ranges from 10 to 50.

[0195] In some embodiments, the muscle cells are selected from triceps surae, biceps, cardiac, and quadriceps.

[0196] In another aspect, the disclosure features the use of any of the modified AAV capsid proteins described herein and / or any of the AAV virions described herein to transfer an exogenous polynucleotide into a muscle cell.

[0197] In some embodiments, the use is a non-therapeutic use, preferably the use is an in vitro use, hi some embodiments, the muscle cells are selected from triceps surae, biceps, cardiac and quadriceps.

[0198] Accordingly, one aspect of the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising: (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.

[0199] In some embodiments, the AAV capsid proteins are selected from one or more of 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 AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; 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; Anc80-55, Anc80-129, Anc80-156, Anc80-751, Anc80-1029, Anc80-1712, 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, or a fragment thereof.

[0200] In some embodiments, the 7-mer peptide is inserted into the reference AAV capsid protein between amino acid positions 565 and 595. In some embodiments, (i) the reference AAV capsid protein is an AAV1 capsid protein and the 7-mer peptide is inserted between D590 and P591 or between S588 and T589 of the capsid protein, (ii) the reference AAV capsid protein is an AAV2 capsid protein and the 7-mer peptide is inserted between R588 and Q589 or between N587 and R588 of the capsid protein, or (iii) the reference AAV capsid protein is an AAV3b capsid protein. (iv) the reference AAV capsid protein is an AAV4 capsid protein, and a 7-mer peptide is inserted between S584 and N585 or between S586 and N587 of the capsid protein; (v) the reference AAV capsid protein is an AAV5 capsid protein, and a 7-mer peptide is inserted between S575 and S576 or between N588 and T589 of the capsid protein. (vi) the reference AAV capsid protein is an AAV6 capsid protein, and a 7-mer peptide is inserted between D590 and P591 or between S588 and T589 of the capsid protein; (vii) the reference AAV capsid protein is an AAV7 capsid protein, and a 7-mer peptide is inserted between N589 and T590 of the capsid protein; (viii) the reference AAV capsid protein is an AAV8 capsid protein. (ix) the reference AAV capsid protein is the capsid protein of AAV9, and a 7-mer peptide is inserted between Q588 and A589 of the capsid protein; (x) the reference AAV capsid protein is the capsid protein of AAVrh10, and a 7-mer peptide is inserted between N590 and A591 of the capsid protein; (xi) the reference AAV capsid protein is AAVpo.(xii) the reference AAV capsid protein is an AAV12 capsid protein, wherein a 7-mer peptide is inserted between N567 and S568 or between N569 and T570 of the capsid protein; or (xiii) the reference AAV capsid protein is an AAV12 capsid protein, wherein a 7-mer peptide is inserted between N592 and A593 or between T594 and T595 of the capsid protein.

[0201] In some embodiments, the modified AAV capsid protein has the sequence of SEQ ID NO: 158.

[0202] In some embodiments, the reference AAV capsid protein is a liver-toggle mutant of the capsid protein 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; 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; 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 liver-toggle mutant of a capsid protein having a sequence selected from SEQ ID NOs: 54-152, or a fragment thereof.

[0203] In some embodiments, the modified AAV capsid protein comprises an alanine (A) amino acid residue at the amino acid position corresponding to position 266 in Anc80, or a lysine (K) amino acid residue at the amino acid position corresponding to position 168 in Anc80.

[0204] In some embodiments, the modified AAV capsid protein comprises a glycine (G) amino acid residue at the amino acid position corresponding to position 266 in Anc80, or an arginine (R) amino acid residue at the amino acid position corresponding to position 168 in Anc80.

[0205] In some embodiments, the reference AAV capsid protein is a liver toggle mutant of the capsid protein of AAV9 that comprises an alanine (A) amino acid residue at amino acid position 267 and a threonine (T) amino acid residue at amino acid position 269. In some embodiments, the modified AAV capsid protein comprises the sequence of SEQ ID NO: 159.

[0206] In some embodiments, the modified AAV capsid protein comprises a glycine (G) amino acid residue at the amino acid position corresponding to position 266 in Anc80, or a lysine (K) amino acid residue at the amino acid position corresponding to position 168 in Anc80.

[0207] In another aspect, the disclosure provides (i) a liver-toggle mutant of a reference AAV capsid protein comprising a) an alanine (A) or glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80, or b) a lysine (K) or arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80, and (ii) a modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide inserted at a site within VR VIII of the liver-toggle mutant.

[0208] In some embodiments, the liver-toggle mutant comprises a) an alanine (A) amino acid residue at the amino acid position corresponding to position 266 in Anc80, or b) a lysine (K) amino acid residue at the amino acid position corresponding to position 168 in Anc80. In some embodiments, the liver-toggle mutant comprises a) an alanine (A) amino acid residue at the amino acid position corresponding to position 266 in Anc80, and b) a lysine (K) amino acid residue at the amino acid position corresponding to position 168 in Anc80.

[0209] In some embodiments, the liver-toggle mutant comprises a) a glycine (G) amino acid residue at the amino acid position corresponding to position 266 in Anc80, or b) an arginine (R) amino acid residue at the amino acid position corresponding to position 168 in Anc80. In some embodiments, the liver-toggle mutant comprises a) a glycine (G) amino acid residue at the amino acid position corresponding to position 266 in Anc80, and b) an arginine (R) amino acid residue at the amino acid position corresponding to position 168 in Anc80.

[0210] In some embodiments, the targeting peptide is a 7-mer peptide having the sequence RGDX1X2X3X4, where X1-X4 are independently selected amino acid residues. In some embodiments, X1, X2, and X3 are independently selected from L, G, V, and A, and X4 is selected from S, V, A, G, and L. In some embodiments, X1, X2, and X3 are independently selected from L, V, and A, and at least two of X1, X2, and X3 are independently L. In some embodiments, X2 is L. In some embodiments, the 7-mer peptide has the sequence RGDLLLS (SEQ ID NO: 1).

[0211] In some embodiments, the targeting peptide is the 7-mer peptide TLAVPFK (SEQ ID NO: 53). In some embodiments, the targeting peptide has a sequence selected from SEQ ID NOs: 2-51 and 53.

[0212] In some embodiments, the reference AAV capsid protein is a capsid protein 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; 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; 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 having a sequence selected from SEQ ID NOs: 54-152, or a fragment thereof.

[0213] In some embodiments, the reference AAV capsid polypeptide is an AAV9 capsid protein.

[0214] In some embodiments, the liver-toggle mutant comprises an alanine (A) amino acid residue at position 267. In some embodiments, the liver-toggle mutant comprises a threonine (T) amino acid residue at position 269. In some embodiments, the liver-toggle mutant comprises an alanine (A) amino acid residue at position 267 and a threonine (T) amino acid residue at position 269.

[0215] In some embodiments, the targeting peptide is inserted between amino acid positions 565 and 595 of the liver-toggle mutant. In some embodiments, (i) the reference AAV capsid protein is an AAV1 capsid protein, and the targeting peptide is inserted between D590 and P591 or between S588 and T589 of the liver-toggle mutant, (ii) the reference AAV capsid protein is an AAV2 capsid protein, and the targeting peptide is inserted between R588 and Q589 or between N587 and R588 of the liver-toggle mutant, or (iii) the reference AAV capsid protein is an AAV3b capsid protein. (iv) the reference AAV capsid protein is an AAV4 capsid protein, and the targeting peptide is inserted between S584 and N585 or between S586 and N587 of the liver-toggle mutant; (v) the reference AAV capsid protein is an AAV5 capsid protein, and the targeting peptide is inserted between S575 and S576 or between N588 and T589 of the liver-toggle mutant. (vi) the reference AAV capsid protein is an AAV6 capsid protein, and the targeting peptide is inserted between D590 and P591 or between S588 and T589 of the liver-toggle mutant; (vii) the reference AAV capsid protein is an AAV7 capsid protein, and the targeting peptide is inserted between N589 and T590 of the liver-toggle mutant; (viii) the reference AAV capsid protein is an AAV8 capsid protein. (ix) the reference AAV capsid protein is the capsid protein of AAV9, and the targeting peptide is inserted between Q588 and A589 of the liver-toggle mutant; (x) the reference AAV capsid protein is the capsid protein of AAVrh10, and the targeting peptide is inserted between N590 and A591 of the liver-toggle mutant; (xi) the reference AAV capsid protein is AAVpo.(xii) the reference AAV capsid protein is an AAV12 capsid protein and the targeting peptide is inserted between N592 and A593 or between T594 and T595 of the liver-toggle mutant.

[0216] In some embodiments, the liver-toggle mutant comprises a sequence selected from NSTSGASS (SEQ ID NO: 160), NSTSGGST (SEQ ID NO: 161) and NSTSGAST (SEQ ID NO: 162).

[0217] In some embodiments, the liver-toggle mutant of the reference AAV capsid protein comprises a) an alanine (A) amino acid residue at the amino acid position corresponding to position 266 in Anc80 and b) a lysine (K) amino acid residue at the amino acid position corresponding to position 168 in Anc80.

[0218] In some embodiments, the liver-toggle mutant of the reference AAV capsid protein comprises: a) an alanine (A) amino acid residue at the amino acid position corresponding to position 267 in AAV9; and b) a threonine (T) amino acid residue at the amino acid position corresponding to position 269 in AAV9.

[0219] In some embodiments, the liver-toggle mutant further comprises: a) an alanine (A) amino acid residue at the amino acid position corresponding to position 504 in AAV9; and b) an alanine (A) amino acid residue at the amino acid position corresponding to position 505 in AAV9.

[0220] In some embodiments, the modified AAV capsid protein comprises the sequence of SEQ ID NO: 159.

[0221] In yet another aspect, the present disclosure provides a polynucleotide encoding the modified AAV capsid protein disclosed herein. In one aspect, the present disclosure relates to a vector comprising the polynucleotide. In some embodiments, the vector further comprises a promoter operably linked to the polynucleotide. Also disclosed herein is a host cell comprising the modified AAV capsid protein, polynucleotide, or vector.

[0222] One aspect of the present disclosure provides a recombinant AAV virion (rAAV) comprising the modified AAV capsid protein disclosed herein. In some embodiments, the rAAV virion further comprises an exogenous polynucleotide. In some embodiments, the exogenous polynucleotide comprises a template for homologous recombination 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.

[0223] Another aspect of the present disclosure provides a pharmaceutical composition comprising a modified AAV capsid protein or an AAV virion.

[0224] Further disclosed are methods for treating, alleviating, or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of an AAV virion or pharmaceutical composition of the present disclosure. In some embodiments, the disease is a muscle disease and / or the condition is muscle degeneration. In some embodiments, the muscle is a striated muscle, preferably a cardiac or skeletal muscle or diaphragm. In some embodiments, the muscle disease is a muscular dystrophy, cardiomyopathy, myotonia, muscle atrophy, myoclonus-dystonia, mitochondrial myopathy, rhabdomyolysis, fibromyalgia, and / or myofascial pain syndrome.

[0225] In one aspect, the present disclosure provides a modified adeno-associated virus (AAV) capsid protein for use in the treatment and / or prevention of muscle diseases and / or muscle degeneration. Further disclosed are AAV virions comprising the modified AAV capsid protein for use in the treatment and / or prevention of muscle diseases and / or muscle regeneration. Also disclosed are pharmaceutical compositions comprising the modified AAV capsid protein and / or AAV virions for use in the treatment and / or prevention of muscle diseases and / or muscle regeneration. Additionally, provided herein is the use of an AAV capsid polypeptide and / or an AAV virion for transferring an active compound into muscle cells. In some embodiments, the use is non-therapeutic, and preferably, the use is in vitro.

[0226] In one aspect, the present disclosure provides a method of transferring an exogenous polynucleotide into a muscle cell, the method comprising administering an AAV virion of the present disclosure to a subject. In some embodiments, the administration results in transfer of the exogenous polynucleotide into the muscle cell at a muscle:liver infection ratio of greater than 1, as measured by genome copies of the AAV virion. In some embodiments, the muscle:liver infection ratio is in the range of 1 to 100. In some embodiments, the muscle:liver infection ratio is in the range of 1 to 10. In some embodiments, the muscle:liver infection ratio is in the range of 2 to 8.

[0227] In some embodiments, administration results in expression of the exogenous polynucleotide in muscle cells at a muscle:liver expression ratio of greater than 10. In some embodiments, the muscle:liver expression ratio is in the range of 10 to 100. In some embodiments, the muscle:liver expression ratio is in the range of 20 to 80. In some embodiments, the muscle:liver expression ratio is in the range of 50 to 80, as measured by mRNA transcript expression. In some embodiments, the muscle:liver expression ratio is in the range of 10 to 50, as measured by protein expression.

[0228] In some embodiments, the muscle cells are selected from triceps surae, biceps, cardiac, and quadriceps.

[0229] In another aspect, the disclosure provides an rAAV whose genome comprises 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, and optionally, 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: 167-170.

[0230] In some embodiments, the MTM1 sequence encodes a protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the MTM1 protein comprises an amino acid sequence having at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the MTM1 protein comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 164.

[0231] In some embodiments, the MTM1 sequence encodes a protein comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 165. In some embodiments, the MTM1 protein comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 165. In some embodiments, the MTM1 protein comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 165. In some embodiments, the MTM1 protein comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 165.

[0232] In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 166. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 166. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 166. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 166. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 166.

[0233] In some embodiments, the MTM1 coding sequence is codon-optimized for expression in human cells. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 167-170. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 167-170. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having at least 98% sequence identity to any one of SEQ ID NOs: 167-170. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having at least 99% sequence identity to any one of SEQ ID NOs: 167-170. In some embodiments, the MTM1 coding sequence comprises a nucleotide sequence having 100% sequence identity to any one of SEQ ID NOs: 167-170. In some embodiments, the sequence identity is to SEQ ID NO: 167. In some embodiments, the sequence identity is to SEQ ID NO: 168. In some embodiments, the sequence identity is to SEQ ID NO: 169. In some embodiments, the sequence identity is to SEQ ID NO:169.

[0234] In some embodiments, the rAAV comprises a hybrid expression control element (ERE) comprising a CMV enhancer and a chicken beta-actin promoter operably linked to an MTM1 coding sequence.

[0235] In some embodiments, the ERE comprises (a) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 171 and a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 172, or (b) a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 173. In some embodiments, the ERE comprises (a) a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 171 and a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 172, or (b) a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 173. In some embodiments, the ERE comprises (a) a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 171 and a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 172, or (b) a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 173. In some embodiments, the ERE comprises (a) a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 171 and a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 172, or (b) a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 173. In some embodiments, the ERE comprises (a) a nucleotide sequence having 100% sequence identity to SEQ ID NO: 171 and a nucleotide sequence having 100% sequence identity to SEQ ID NO: 172, or (b) a nucleotide sequence having 100% sequence identity to SEQ ID NO: 173.

[0236] In some embodiments, the rAAV further comprises a chimeric intron formed from an intron sequence derived from chicken beta-actin and / or human beta-herpesvirus and / or human beta-globin, and / or operably linked to the MTM1 coding sequence.

[0237] In some embodiments, the chimeric intron comprises a nucleotide sequence derived from human beta globin, optionally comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 174. In some embodiments, the chimeric intron comprises a nucleotide sequence derived from human beta globin comprising SEQ ID NO: 174.

[0238] In some embodiments, the chimeric intron comprises a nucleotide sequence derived from a human beta-herpesvirus, optionally comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 175. In some embodiments, the nucleotide sequence is derived from a human beta-herpesvirus comprising SEQ ID NO: 175.

[0239] In some embodiments, the chimeric intron is formed from introns derived from a human beta-herpesvirus and a rabbit beta-globin. In some embodiments, the chimeric intron comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 176. In some embodiments, the chimeric intron comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 176. In some embodiments, the chimeric intron comprises a nucleotide sequence having at least 98% sequence identity to SEQ ID NO: 176. In some embodiments, the chimeric intron comprises a nucleotide sequence having at least 99% sequence identity to SEQ ID NO: 176. In some embodiments, the chimeric intron comprises a nucleotide sequence having 100% sequence identity to SEQ ID NO: 176. In some embodiments, the chimeric intron comprises the nucleotide sequence of SEQ ID NO: 176.

[0240] In some embodiments, the rAAV comprises an unmodified or modified AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; 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; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; Anc110 or an Anc80DI capsid protein.

[0241] In some embodiments, the rAAV comprises an unmodified or modified rAAV9 capsid protein.In some embodiments, the rAAV comprises VP1, VP2, and / or VP3 capsid proteins comprising an amino acid sequence having at least 90% sequence identity to the corresponding proteins in AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; 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; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; Anc110; or Anc80DI.

[0242] In some embodiments, the rAAV comprises VP1, VP2, and / or VP3 capsid proteins comprising an amino acid sequence having at least 95% sequence identity to the corresponding proteins in AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; 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; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; Anc110; or Anc80DI.

[0243] In some embodiments, the rAAV comprises VP1, VP2, and / or VP3 capsid proteins comprising an amino acid sequence having at least 98% sequence identity to the corresponding proteins in AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; 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; Anc8o; Anc81; Anc82; Anc83; Anc84; Anc94; AncI13; Anc126; Anc127; Anc80L27; Anc80L59; Anc80L60; AncI80L62; Anc80L65; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; Anc110; or Anc80DI.

[0244] In some embodiments, the rAAV comprises VP1, VP2, and / or VP3 capsid proteins comprising an amino acid sequence having at least 99% sequence identity to the corresponding protein in AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; 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; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; Anc110; or Anc80DI.

[0245] In some embodiments, the rAAV comprises VP1, VP2, and / or VP3 capsid proteins comprising an amino acid sequence having 100% sequence identity to the corresponding protein in AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; 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; Anc80-55; Anc80-129; Anc80-156; Anc80-751; Anc80-1029; Anc80-1712; Anc110; or Anc80DI.

[0246] In some embodiments, the rAAV comprises a modified AAV capsid protein that comprises at least one liver-toggle mutation compared to a reference capsid protein.

[0247] In some embodiments, the reference capsid protein is a VP1, VP2, and / or VP3 protein. In some embodiments, the reference AAV capsid protein is a capsid protein having any one of SEQ ID NOs: 54-152, or a fragment thereof.

[0248] In some embodiments, at least one liver-toggle mutation comprises an alanine (A) or glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80 and / or a lysine (K) or arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80.

[0249] In some embodiments, at least one liver-toggle mutation comprises an alanine (A) amino acid residue at an amino acid position corresponding to position 266 in Anc80 and / or a lysine (K) amino acid residue at an amino acid position corresponding to position 168 in Anc80.

[0250] In some embodiments, at least one liver-toggle mutation comprises an alanine (A) amino acid residue at an amino acid position corresponding to position 266 in Anc80 and / or an arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80.

[0251] In some embodiments, at least one liver-toggle mutation comprises a glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80 and / or a lysine (K) amino acid residue at an amino acid position corresponding to position 168 in Anc80.

[0252] In some embodiments, at least one liver-toggle mutation comprises a glycine (G) amino acid residue at an amino acid position corresponding to position 266 in Anc80 and / or an arginine (R) amino acid residue at an amino acid position corresponding to position 168 in Anc80.

[0253] In some embodiments, at least one liver-toggle mutation comprises an alanine (A) at the amino acid position corresponding to position 267 in AAV9. In some embodiments, at least one liver-toggle mutation comprises a threonine (T) at the amino acid position corresponding to position 269 in AAV9.

[0254] In some embodiments, the capsid protein is a modified AAV9 capsid protein, and optionally, the capsid protein is a modified AAV9 VP1 capsid protein.

[0255] In some embodiments, the liver-toggle mutation comprises an alanine (A) amino acid residue at the amino acid position corresponding to position 267 in AAV9 and a threonine (T) amino acid residue at the amino acid position corresponding to position 269 in AAV9.

[0256] In some embodiments, the liver-toggle mutation further comprises an alanine (A) amino acid residue at the amino acid position corresponding to position 504 in AAV9 and / or an alanine (A) amino acid residue at the amino acid position corresponding to position 505 in AAV9.

[0257] In some embodiments, the liver-toggle mutant comprises the sequence NSTSGASS (SEQ ID NO: 160), NSTSGGST (SEQ ID NO: 161), or NSTSGAST (SEQ ID NO: 162). In some embodiments, the rAAV capsid protein has the sequence of SEQ ID NO: 159. In some embodiments, the rAAV capsid protein has the sequence of SEQ ID NO: 163.

[0258] In some embodiments, the one or more liver toggle mutations comprise one or more amino acid substitutions at one or more of Q263, S264, G265, A266, S267, N268, H271, N382, G383, S384, Q385, S446, R471, W502, T503, D528, D529, Q589, K706 and V708 compared to the AAV2 reference capsid protein (SEQ ID NO: 1 of WO2021 / 050614, incorporated herein by reference).

[0259] In some embodiments, the one or more liver toggle mutations comprise the amino acid substitution S446R compared to the reference capsid protein. In some embodiments, the one or more liver toggle mutations comprise the amino acid substitution R471A compared to the reference capsid protein. In some embodiments, the one or more liver toggle mutations comprise the amino acid substitution V708T or V708A compared to the reference capsid protein.

[0260] In some embodiments, the rAAV comprises a modified AAV capsid protein that includes at least one muscle-targeting element compared to a reference capsid protein, in some embodiments, the reference capsid protein is a VP1, VP2, and / or VP3 protein.

[0261] In some embodiments, the muscle-targeting element is a 7-mer peptide having the sequence RGDX1X2X3X4 (SEQ ID NO: 52), where X1-X4 are independently selected amino acid residues. In some embodiments, X1, X2, and X3 are independently selected from L, G, V, and A, and X4 is selected from S, V, A, G, and L. In some embodiments, X1, X2, and X3 are independently selected from L, V, and A, and at least two of X1, X2, and X3 are independently L. In some embodiments, X2 is L.

[0262] In some embodiments, the muscle-targeting element is a 7-mer peptide having the sequence RGDX1X2X3X4 (SEQ ID NO: 52), where X1, X2, X3, and X4 are independently selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-44858. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-338. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-438. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-538. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-638. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-738. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-838. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-938. In some embodiments, the 7-mer has a sequence selected from SEQ ID NOs: 238-1038.

[0263] In some embodiments, the 7-mer peptide has the sequence RGDLLLS (SEQ ID NO: 1). In some embodiments, the targeting peptide is the 7-mer peptide TLAVPFK (SEQ ID NO: 53). In some embodiments, the targeting peptide is a peptide having any one of SEQ ID NOs: 2-51 and 53.

[0264] In some embodiments, the muscle-targeting element consists of a 7-mer peptide having a sequence selected from SEQ ID NOs: 238-44858 inserted into a site within VR VIII of an AAV capsid protein. In some embodiments, the muscle-targeting element consists of a 7-mer peptide having the sequence RGDLLLS (SEQ ID NO: 1) inserted into a site within VR VIII of an AAV capsid protein. In some embodiments, the 7-mer peptide is inserted into an amino acid position between 565 and 595 of a reference AAV capsid protein.

[0265] In some embodiments, the reference AAV capsid protein is an AAV1 capsid protein, and the 7-mer muscle-targeting peptide is inserted between D590 and P591 or between S588 and T589 of the capsid protein; the reference AAV capsid protein is an AAV2 capsid protein, and the 7-mer muscle-targeting peptide is inserted between R588 and Q589 or between N587 and R588 of the capsid protein; and the reference AAV capsid protein is an AAV3b capsid protein. The reference AAV capsid protein is an AAV4 capsid protein, and the 7-mer muscle-targeting peptide is inserted between S586 and S587 or between N588 and T589 of the capsid protein. The reference AAV capsid protein is an AAV4 capsid protein, and the 7-mer muscle-targeting peptide is inserted between S584 and N585 or between S586 and N587 of the capsid protein. The reference AAV capsid protein is an AAV5 capsid protein, and the 7-mer muscle-targeting peptide is inserted between S575 and S576 of the capsid protein. or between T577 and T578 of the capsid protein is an AAV6 capsid protein; a 7-mer muscle-targeting peptide is inserted between D590 and P591 or between S588 and T589 of the capsid protein is an AAV7 capsid protein; and a 7-mer muscle-targeting peptide is inserted between N589 and T590 of the capsid protein is an AAV8 capsid protein. The reference AAV capsid protein is the capsid protein of AAV9, where the 7-mer muscle-targeting peptide is inserted between N590 and T591 of the capsid protein. The reference AAV capsid protein is the capsid protein of AAVrh10, where the 7-mer muscle-targeting peptide is inserted between Q588 and A589 of the capsid protein. The reference AAV capsid protein is the capsid protein of AAVpo.The reference AAV capsid protein is the capsid protein of AAV11, and a 7-mer muscle-targeting peptide is inserted between N567 and S568 or between N569 and T570 of the capsid protein, or the reference AAV capsid protein is the capsid protein of AAV12, and a 7-mer muscle-targeting peptide is inserted between N592 and A593 or between T594 and T595 of the capsid protein.

[0266] In some embodiments, the muscle-targeting peptide is inserted into a site within VR VIII of the liver-toggle mutant capsid, optionally wherein the liver-toggle mutant capsid is as described in any one of embodiments 49 to 62. In some embodiments, the muscle-targeting peptide is inserted into an amino acid position between 565 and 595 of the liver-toggle mutant.

[0267] In some embodiments, the reference AAV capsid protein is an AAV1 capsid protein, and the targeting peptide is inserted between D590 and P591 or between S588 and T589 of the liver-toggle mutant; the reference AAV capsid protein is an AAV2 capsid protein, and the targeting peptide is inserted between R588 and Q589 or between N587 and R588 of the liver-toggle mutant; the reference AAV capsid protein is an AAV3b capsid protein. The reference AAV capsid protein is an AAV4 capsid protein, and the targeting peptide is inserted between S584 and N585 or between S586 and N587 of the liver-toggle mutant. The reference AAV capsid protein is an AAV5 capsid protein, and the targeting peptide is inserted between S575 and S576 of the liver-toggle mutant. or between T577 and T578 of the liver-toggle mutant is an AAV6 capsid protein, and the targeting peptide is inserted between D590 and P591 or between S588 and T589 of the liver-toggle mutant is an AAV7 capsid protein, and the targeting peptide is inserted between N589 and T590 of the liver-toggle mutant is an AAV8 capsid protein. The reference AAV capsid protein is the capsid protein of AAV9, and the targeting peptide is inserted between N590 and T591 of the liver-toggle mutant. The reference AAV capsid protein is the capsid protein of AAVrh10, and the targeting peptide is inserted between N590 and A591 of the liver-toggle mutant. The reference AAV capsid protein is the capsid protein of AAVpo.The reference AAV capsid protein is the capsid protein of AAV11, and the targeting peptide is inserted between N567 and S568 or between N569 and T570 of the liver-toggle mutant, or the reference AAV capsid protein is the capsid protein of AAV12, and the targeting peptide is inserted between N592 and A593 or between T594 and T595 of the liver-toggle mutant.

[0268] In some embodiments, the capsid protein has the sequence of SEQ ID NO: 158. In some embodiments, the rAAV capsid protein has the sequence of SEQ ID NO: 159.

[0269] In some embodiments, the ERE comprises a constitutive promoter, which is a Rous sarcoma virus (RSV) LTR promoter (optionally including an RSV enhancer), a cytomegalovirus (CMV) promoter (optionally including a CMV enhancer), an SV40 promoter, a dihydrofolate reductase (DHFR) promoter, a beta-actin promoter, a phosphoglycerol kinase 1 (PGK1) promoter (optionally a minimal PGK1 promoter), or an EF1 alpha promoter (optionally including an intron).

[0270] In some embodiments, the ERE comprises an inducible promoter. In some embodiments, the inducible promoter is a tetracycline or rapamycin inducible promoter. In some embodiments, the ERE comprises a muscle-specific promoter. In some embodiments, the muscle-specific promoter is a desmin promoter (optionally a CpG-depleted desmin promoter), a CKM promoter derivative, or an MTM1 promoter. In some embodiments, the promoter is a human promoter.

[0271] In some embodiments, the rAAV comprises a rabbit globin polyA sequence 3' to the MTM1 coding sequence, and optionally the rabbit globin polyA sequence has at least 90% sequence identity to SEQ ID NO: 177. In some embodiments, the rabbit globin polyA sequence has at least 95% sequence identity to SEQ ID NO: 177. In some embodiments, the rabbit globin polyA sequence has at least 98% sequence identity to SEQ ID NO: 177. In some embodiments, the rabbit globin polyA sequence has at least 99% sequence identity to SEQ ID NO: 177. In some embodiments, the rabbit globin polyA sequence has 100% sequence identity to SEQ ID NO: 177.

[0272] In some embodiments, the genome of the rAAV comprises AAV-derived inverted terminal repeat sequences (ITRs). In some embodiments, the ITRs are derived from AAV serotype 2. In some embodiments, the rAAV comprises a first ITR having at least 90% sequence identity to SEQ ID NO: 178 and a second ITR having at least 90% sequence identity to SEQ ID NO: 179. In some embodiments, the first ITR has at least 95% sequence identity to SEQ ID NO: 178 and the second ITR has at least 95% sequence identity to SEQ ID NO: 179. In some embodiments, the first ITR has at least 98% sequence identity to SEQ ID NO: 178 and the second ITR has at least 98% sequence identity to SEQ ID NO: 179. In some embodiments, the first ITR has at least 99% sequence identity to SEQ ID NO: 178 and the second ITR has at least 99% sequence identity to SEQ ID NO: 179. In some embodiments, the first ITR has 100% sequence identity to SEQ ID NO:178 and the second ITR has 100% sequence identity to SEQ ID NO:179.

[0273] In some embodiments, the rAAV comprises a heterologous splice acceptor sequence 5' to the MTM1 coding sequence. In some embodiments, the heterologous splice acceptor sequence is derived from human beta globin exon 3. In some embodiments, the heterologous splice acceptor sequence comprises the nucleotide sequence of SEQ ID NO: 180.

[0274] In one aspect, the disclosure provides an rAAV comprising a modified AAV capsid protein comprising at least one liver-toggle mutation and / or one muscle-targeting element, optionally wherein the modified capsid protein comprises the amino acid sequence of SEQ ID NO: 158, SEQ ID NO: 159, or SEQ ID NO: 163, and wherein the genome comprises a first ITR sequence, a hybrid expression regulatory element (ERE) comprising a CMV enhancer and a chicken beta-actin promoter, optionally wherein the ERE comprises the nucleotide sequence of SEQ ID NO: 173, an MTM1 coding sequence operably linked to the ERE, and a second ITR sequence.

[0275] In some embodiments, the rAAV further comprises a chimeric intron between the ERE and the MTM1 coding sequence, where optionally, the chimeric intron comprises the nucleotide sequence of SEQ ID NO: 176. In some embodiments, the rAAV further comprises a splice acceptor site 5' to the MTM1 coding sequence, where optionally, the splice acceptor site comprises the nucleotide sequence of SEQ ID NO: 180. In some embodiments, the rAAV further comprises a polyadenylation sequence 3' to the MTM1 coding sequence, where optionally, the polyadenylation sequence comprises the nucleotide sequence of SEQ ID NO: 177.

[0276] In some embodiments, the MTM1 coding sequence is codon-optimized for expression in human cells, and optionally the MTM1 coding sequence comprises the nucleotide sequence of SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169 or SEQ ID NO: 170.

[0277] In some embodiments, the rAAV has a genome that is self-complementary, and optionally, the genome is completely self-complementary.

[0278] The present disclosure further provides a pharmaceutical composition comprising the rAAV described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is in unit dose form.

[0279] In some embodiments, the pharmaceutical composition comprises 1×10 10 ~1×10 16 and / or rAAV concentrations of 1 x 10 10 vg / ml ~ 1 × 10 16 vg / ml.

[0280] In some embodiments, the pharmaceutical composition is formulated for parenteral administration, eg, systemic (eg, intravenous), intramuscular, or subcutaneous administration.

[0281] The present disclosure further discloses host cells engineered to produce the rAAVs described herein. In some embodiments, the host cells contain a recombinant nucleic acid vector comprising a polynucleotide expressing one or more capsid proteins of the rAAV, a functional rep gene, and an MTM coding sequence operably linked to an AAV ITR and an expression regulatory element (ERE), optionally a hybrid ERE comprising a CMV enhancer and a chicken beta-actin promoter.

[0282] In another aspect, the present disclosure provides a method for treating, ameliorating, or preventing X-linked myotubular myopathy in a subject, comprising administering a therapeutically effective amount of an rAAV or pharmaceutical composition described herein. In some embodiments, the effective dose is 1×10 10 ~1×10 16 In some embodiments, the effective dose comprises a rAAV with a genome copy number (GC) of 1 x 10 15 GC or less. In some embodiments, the effective dose is 5×10 14 GC or less. In some embodiments, the effective dose is 1 x 10 14GC or less. In some embodiments, the effective dose is 5×10 13 GC or less. In some embodiments, the effective dose is 1 x 10 13 GC or less.

[0283] In some embodiments, administration is parenteral. In some embodiments, administration is systemic (e.g., intravenous). In some embodiments, administration is intramuscular. In some embodiments, administration is subcutaneous.

[0284] In yet another aspect, the present disclosure provides an rAAV or pharmaceutical composition described herein for use in treating and / or preventing X-linked myotubular myopathy. In some embodiments, the rAAV or pharmaceutical composition is for use in expressing myotubularin in muscle cells.

[0285] Without being bound by theory, it is believed that the rAAVs of the present disclosure have an improved therapeutic index due to higher MTM1 expression levels per administered viral genome and / or reduced off-target (e.g., liver) targeting or expression per administered viral genome compared to control rAAVs whose genomes include an MTM1 coding sequence under the control of the desmin promoter and / or include an unmodified capsid protein. 5. A brief description of some figures in the drawing

[0286] These and other features, aspects and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Brief explanation of the drawings]

[0287] [Figure 1] Figure 1 illustrates the structure of the AAV VP1 protein, highlighting certain variable regions (VR I, VR III, VR IV). The locations of the liver toggle (mut1) in VR I and the peptide insert (deco1) in VR VIII are indicated.

[0288] [Figure 2-1] Figures 2A-2C provide sequence alignments of the VP1 sequences of certain AAV variants using AAV2 VP1 as a reference. The locations of residue 168, the liver toggle site, mut1 (Figure 2A), and the targeting peptide insertion site (Figure 2B) are indicated. Figures 2A-2B disclose, in order of appearance, SEQ ID NOs: 55, 54, 58, 56, 64, 59, 60, 89, 111, 61, 63, 62, and 57, respectively. [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0289] [Figure 3-1] Figures 3A-3D provide sequence alignments of ancestral AAV VP1 sequences using AAV2 as a reference. The locations of the liver toggle site, residue 168 (Figure 3A) and residue 266 (Figure 3B), and the targeting peptide insertion site (Figure 3C) are indicated. Sequences of one or more representative members of each of the Anc80, Anc81, Anc82, Anc83, Anc84, Anc94, Ac110, Anc113, Anc126, and Anc127 libraries were used for alignment. Figures 3A-3D disclose SEQ ID NOs: 217-237, respectively, in order of appearance. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above.

[0290] [Figure 4] Figures 4A-4J show immunohistochemistry data obtained from the experiment described in Example 2 below in the Examples section. Anti-GFP immunohistochemistry was performed on liver and skeletal muscle (quadriceps) tissue cross sections from mice injected with vehicle (Figure 4A), AAV9 (Figure 4B), AAVmut1 (Figure 4C), AAVdeco1 (Figure 4D), or AAVmut1-deco1 (Figure 4E) and vehicle (Figure 4F), AAV9 (Figure 4G), AAVmut1 (Figure 4H), AAVdeco1 (Figure 4I), or AAVmut1-deco1 (Figure 4J).

[0291] [Figure 5] Figures 5A-5B show mRNA expression in various tissues of C57BL / 6 mice treated with different AAV vectors, as measured by RT-ddPCR. The y-axis represents the copy ratio of eGFP mRNA transcripts to RPP30 mRNA, and the x-axis represents the AAV vector and dose injected into the experimental animals. Each graph shows eGFP expression in the liver (Figure 5A) and quadriceps muscle (Figure 5B).

[0292] [Figure 6-1] Figures 6A-6E show eGFP mRNA expression in various tissues of C57BL / 6 mice treated with different AAV vectors, as measured by RT-ddPCR. The y-axis represents the ratio of eGFP to RPP30 mRNA copies, and the x-axis represents the AAV vector and dose injected into the experimental animals. Each graph shows eGFP expression in the liver (Figure 6A), heart (Figure 6B), triceps surae (Figure 6C), quadriceps (Figure 6D), or diaphragm (Figure 6E). [Figure 6-2] Same as above. [Figure 6-3] Same as above.

[0293] [Figure 7] Figures 7A-7D show eGFP vector genome (DNA) and eGFP expression (mRNA) in liver and quadriceps tissues of C57BL / 6 mice treated with vehicle, AAVMut1, and AAVMut1-deco1 AAV vectors. DNA data is shown in Figures 7A and 7B, with eGFP genome copies measured by RT-ddPCR plotted at days 14 and 28, respectively. The y-axis represents vector genome (copies per DPG), and the x-axis represents vehicle and AAV vector. mRNA data is shown in Figures 7C and 7D, with eGFP expression measured by RT-ddPCR plotted at days 14 and 28, respectively. The y-axis represents the ratio of eGFP to RPP30 mRNA copies, and the x-axis represents AAV vector.

[0294] [Figure 8] Figure 8 shows eGFP mRNA expression in various tissues of BalbC mice treated with vehicle, AAVmut1, and AAVmut1-deco1 AAV vectors, as measured by RT-ddPCR. The y-axis represents the copy ratio of eGFP to RPP30 mRNA, and the x-axis represents the AAV vector and dose injected into the experimental animals. The graphs show eGFP expression in the liver (left) and quadriceps muscle (right).

[0295] [Figure 9] 9A and 9B show exemplary IHC tissue analyses obtained from Run 1 samples from NHPs. Liver tissue is shown in FIG. 9A, with the left side showing tissue obtained from an AAV9 vector-treated NHP and the right side showing tissue obtained from an AAVmut1_deco1 vector-treated NHP. Exemplary IHC quadriceps tissue is shown in FIG. 9B, with the left side obtained from an AAV9 vector-treated NHP and the right side obtained from an AAVmut1_deco1 vector-treated NHP.

[0296] [Figure 10] Figure 10 shows the % GFP-positive cells in 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_deco1 vectors.

[0297] [Figure 11] FIG. 11 shows the % GFP-positive cells (average from runs 1 and 2) in various skeletal muscle and liver tissues in slides obtained from NHPs administered vehicle, AAV9, or AAVmut1_deco1 vectors.

[0298] [Figure 12]FIG. 12 shows the % GFP-positive cells per animal (average from runs 1 and 2) in various skeletal muscle and liver tissues in slides obtained from NHPs administered vehicle, AAV9, or AAVmut1_deco1 vectors.

[0299] [Figure 13] FIG. 13 shows the mean combined quantification (average from runs 1 and 2) of % GFP-positive cells per animal in various skeletal muscle and liver tissues obtained from NHPs administered vehicle, AAV9, or AAVmut1_deco1 vectors.

[0300] [Figure 14] FIG. 14 shows the % GFP-positive cells (average from runs 1 and 2) in various cardiac tissues obtained from NHPs administered vehicle, AAV9, or AAVmut1_deco1 vectors.

[0301] [Figure 15] FIG. 15 shows the % GFP-positive cells per animal (average from runs 1 and 2) in various myocardium obtained from NHPs administered vehicle, AAV9, or AAVmut1_deco1 vectors.

[0302] [Figure 16] FIG. 16 shows the mean % GFP-positive cells per animal (average from runs 1 and 2) in ventricular wall, atrium, and interventricular septum slides obtained from NHPs administered vehicle, AAV9, or AAVmut1_deco1 vectors.

[0303] [Figure 17]Figures 17A-17C show the mean % GFP-positive cells per NHP animal (average from Runs 1 and 2) in various tissues administered with vehicle and AAV9 and AAVmut1_deco1 vectors. Figure 17A shows the mean % GFP-positive cells per animal in liver tissue. Figure 17B shows the mean % GFP-positive cells per animal in various skeletal muscle tissues. Figure 17C shows the mean % GFP-positive cells per animal in various heart tissues.

[0304] [Figure 18-1] Figures 18A-18D show the results of DNA samples analyzed for vector genome biodistribution in liver and quadriceps tissues using a duplex ddPCR method targeting the transgene (eGFP) and reference gene (RPP30). Results are shown in Figure 18A (liver), Figure 18B (quadriceps), Figure 18C (biceps), and Figure 18D (heart). The x-axis represents the AAV vector (wild-type AAV9 on the left and AAVmut1deco1 on the right of each plot) and indicates whether the sample was taken from the left or right side of the organ / animal. [Figure 18-2] Same as above.

[0305] [Figure 19-1] Figures 19A-19D show the results of mRNA transcript analysis measured by eGFP copies relative to RPP30 mRNA. Figure 19A (liver), Figure 19B (quadriceps), Figure 19C (biceps), and Figure 19D (heart) are illustrated. The x-axis represents the AAV vector (wild-type AAV9 on the left and AAVmut1deco1 on the right) and indicates whether the sample was taken from the left or right side of the organ / animal. [Figure 19-2] Same as above.

[0306] [Figure 20]Figure 20 shows human MTM1 protein expression in RD cells. Human MTM protein expression levels were determined using an automated JESS-ProteinSimple instrument. Each bar represents the JESS peak area value before (blue) or after (orange) normalization to total protein load. Data were obtained from a single run using a 1:4 dilution as described in the Western protocol.

[0307] [Figure 21] FIG. 21 provides the study design to evaluate the distribution patterns of Anc80 variants in non-human primates as described in Example 8.

[0308] [Figure 22] FIG. 22 provides a scatter plot of Anc80 variant counts from the Anc80 library showing the enrichment of each variant in muscle (y-axis) and liver (x-axis).

[0309] [Figure 23] Figure 23 shows the negative log fold change in tissues for liver-off variants (variants with "A" at P3) of the Anc80 library (left) and liver-on variants (variants with "G" at P3) of the Anc80 library (right) compared to test article input (Anc80 variants of the Anc80 library) at day 28. Test article, as used herein, refers to the administered vector pool.

[0310] [Figure 24] Figure 24 shows the effect of P3 on the log fold change in tissue enrichment in Group 2 primates G62N in the quadriceps muscle at day 28. A significant difference in the log fold change in tissue enrichment in the quadriceps muscle was found between the "liver off" "0" and "liver on" "1" Anc80 variants. On the x-axis, "0" represents the amino acid residue "G" and "1.0" represents the amino acid "A."

[0311] [Figure 25]Figure 25 shows the effect of P3 on the log fold change of tissue enrichment in quadriceps in Group 2 primates G66E at day 28 in the quadriceps. A significant difference in the log fold change of tissue enrichment in quadriceps was found between the "liver off" "0" and "liver on" "1" Anc80 variants. On the x-axis, "0" represents the amino acid residue "G" and "1.0" represents the amino acid "A."

[0312] [Figure 26] Figure 26 shows the effect of P3 on the mean log fold change in tissue enrichment in quadriceps muscle in both primates in Group 2 at day 28 in the quadriceps muscle. A significant difference in the log fold change in tissue enrichment in quadriceps muscle was found between the "liver off" "0" and "liver on" "1" Anc80 variants. On the x-axis, "0" represents the amino acid residue "G" and "1.0" represents the amino acid "A."

[0313] [Figure 27] Figure 27 shows a four-quadrant liver plot from a G62N primate on day 28. The upper left (UL) quadrant had a higher biodistribution of 74 Anc80 variants in quadriceps muscle with liver toggle on "liver on." The upper right (UR) quadrant had a higher biodistribution of 3 Anc80 variants in quadriceps muscle with liver toggle on and off. The lower left (LR) quadrant had a lower biodistribution of 56 Anc80 variants in quadriceps muscle with liver toggle on. The lower right (LL) quadrant had a higher distribution of 891 Anc80 variants in quadriceps muscle with liver toggle on "liver on" and off.

[0314] [Figure 28]Figure 28 shows a four-quadrant liver plot from a G66E primate on day 28. The upper left (UL) quadrant had a higher biodistribution of the 66 Anc80 variant in quadriceps muscle with liver toggle on "liver on." The upper right (UR) quadrant had a higher biodistribution of the 125 Anc80 variant in liver toggle on and off. The lower left (LL) quadrant had a lower biodistribution of the 271 Anc80 variant in quadriceps muscle with liver toggle on and off. The lower right (LR) quadrant had a lower distribution of the 566 Anc80 variant in quadriceps muscle with liver toggle on "liver on."

[0315] [Figure 29] Figure 29 provides a flowchart depicting comparative data among animals in each of Groups 1 and 2. When tissue enrichment data from two primates in each group were averaged, 13 variant pairs were found to have effects on liver toggling. Variability is common in AAV transduction, especially at low doses in primates. To use limited data from two primates to evaluate each variant at low doses in the ANc80 library, the average fold enrichment of each variant was used rather than simply relying on enrichment from each animal.

[0316] [Figure 30] Figure 30 shows the average liver toggle data between both non-primate animals G62N and G66E in Group 2. The upper left (UL) quadrant had a higher biodistribution of 69 Anc80 variants in quadriceps muscle with liver toggle on "liver on." The upper right (UR) quadrant had a higher biodistribution of 13 Anc80 variants in quadriceps muscle with liver toggle on and off. The lower left (LL) quadrant had a lower biodistribution of 687 Anc80 variants in quadriceps muscle with liver toggle on and off. The lower right (LR) quadrant had a lower distribution of 257 Anc80 variants in quadriceps muscle with liver toggle on "liver on."

[0317] [Figure 31]Figure 31 shows a fingerprint plot of the top 100 Anc80 variants, positions P1 (amino acid 168 of the Anc80 capsid), P2 (amino acid 205), P3 (amino acid 266), P4, … P10 (amino acid 587), and P11 (amino acid 609) on the Anc80 scaffold. Anc80 variants are color-coded by the toggle amino acid at that particular position (P1-P11). "0" represents green, and "1" represents red. Variants with different amino acids at the variable positions are color-coded. Variants were sorted by liver-onset fold change of tissue enrichment in quadriceps muscle and ranked in descending order. As shown, position P3 contains the amino acid G in red for all 100 Anc80 variants.

[0318] [Figure 32] Figure 32 shows linear modeling of positional pattern assessment on the mean log fold change of P1-P11 variant pairs. Positional pattern assessment provides tissue enrichment scores for the mean log fold change at every amino acid position (P1-P11). The analysis shows that variation at P3, P5, P6, and P10 has some pattern of influence on the mean log fold change (log(FC)) of quadriceps muscle.

[0319] [Figure 33-1] Figure 33 provides the binary codes described in Figure 31 for liver toggle variant pairs in the Anc80 library of Example 8. P3 represents the "liver off" and "liver on" liver toggle variant pairs. As shown in the bottom left scatter plot, some Anc80 variants were more abundant in muscle (LR) only in "liver off," which was dependent on liver toggling, while other variants performed well in both "liver on" and "liver off" conditions (UR). [Figure 33-2] Same as above.

[0320] [Figure 34]Figure 34 provides the amino acid positions of 50,625 RGD-targeting peptides in the RGD-targeting peptide library of "Y1, Y2, Y3, Y4, Y5, Y6, and Y7" listed in Table 27. In the X1X2X3RGDX7X8X9X10 described in the present disclosure, Y1 represents the amino acid residue "R," Y2 represents the amino acid residue "G," and Y3 represents the amino acid residue "D." Y4 is represented as "X7" in X1X2X3RGDX7X8X9X10, Y5 is represented as "X8" in X1X2X3RGDX7X8X9X10, Y6 is represented as "X9" in X1X2X3RGDX7X8X9X10, and Y7 is represented as "X10" in X1X2X3RGDX7X8X9X10. Exemplary amino acid modifications of the Y4, Y5, Y6 and Y7 regions of the targeting peptide include those described herein.

[0321] [Figure 35] Figure 35 shows a density plot of the density of vector DNA at a particular enrichment score ((Log2(fold)) of AAV9 capsids with specific amino acids at 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. Different colors represent the amino acid at a particular position.

[0322] [Figure 36-1]Figure 36A shows muscle-targeting sequence motifs identified from the experiments described in Example 11. Figure 36B is a scatter plot showing muscle enrichment by the distribution of scaled enrichment scores (inverse coefficient of variation (ICV)) of the variants tested in Example 11. Red dots on the scatter plot represent variants with muscle-targeting sequence motifs. The mean-ICV plot shows the relationship between the mean enrichment score of each variant in the library and the inverse CV (ICV) of the enrichment score. The ICV plot is useful for selecting variants with high mean tissue enrichment scores and low variability (high ICV). Figure 36C compares the scaled enrichment score distributions of variants with and without muscle-targeting motifs. The cumulative density of variants at a particular tissue enrichment score (Log2(fold)) is shown. Lines are colored by the probability of the amino acid at a particular position. [Figure 36-2] Same as above.

[0323] [Figure 37-1] Figure 37A shows sequence motifs identified from the top 10 muscle-enhancing variants listed in Table 27 that were shown to have significant sequence similarity. Figure 37B is a scatter plot showing the muscle enrichment scores (inverse coefficient of variation (ICV)) of the variants, with the top muscle-targeting peptides in red. The mean-ICV plot shows the relationship between the mean enrichment score of each variant in the library and the inverse CV (ICV) of the enrichment score. The ICV plot is useful for selecting variants with high mean tissue enrichment scores and low variability (high ICV). Figure 37C is a network plot in which lines connect dots representing variants with similar sequences. Eight of the top 10 variants were connected in the sequence similarity network. Figure 37C shows the sequence similarity network of SEQ ID NO: 238 ("ATLVT013XX38181"), a top targeting peptide for muscle targeting. [Figure 37-2] Same as above.

[0324] [Figure 37-3] Figure 37D shows sequence motifs that are enriched in muscle-tropic capsids.

[0325] FIG. 37E shows a network comparison of the targeting peptides described herein to literature myopeptides.

[0326] [Figure 38-1] Figure 38A provides a network plot, and Figure 38B provides a scatter plot showing the tissue enrichment scores for each tissue region evaluated. The network analysis and scatter plot show that SEQ ID NO:238 ("ATLVT013XX38181") is superior to wild-type AAV9, AAV9deco1 (AAV9 capsid with deco1 peptide), and AAV9myo3E (AAV9 capsid with myo3E peptide) capsids in muscle tropism, and reduces liver toxicity due to the liver-detargeting phenotype Mut1 compared to wild-type AAV9. The targeting peptide SEQ ID NO:238 ("ATLVT013XX38181") consistently ranked high in target enrichment in all tissues except the triceps. [Figure 38-2] Same as above.

[0327] [Figure 38-3] Figure 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 containing the M3 (MYODV6 (SEQ ID NO: 44864)) targeting peptide located in VR VIII between amino acids 588 and 589 or the M2 (RGDRSVV (SEQ ID NO: 239)) targeting peptide located in VR VIII. Muscle tissues analyzed included the ventricular wall of the heart, biceps femoris, diaphragm, tibialis anterior, and triceps brachii. Higher tissue enrichment score values ​​represent higher tropism.

[0328] [Figure 39] Figure 39 shows the probabilities of the top 100 targeting peptides at positions Y4, Y5, Y6 and Y7, where position 4 (Y4) is rich in the amino acid "R" and position 7 (Y7) is rich in the amino acids "V, I and L".

[0329] [Figure 40] Figure 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. Six of the seven Anc80 variants with deco1 inserted into VR VIII enhanced muscle delivery of the Anc80 variant.

[0330] [Figure 41] In Figure 41, the inventors created additional targeting peptide variants for insertion, substitution, or modification within the VR8 region of an AAV capsid by modifying the three amino acids (referred to as a "triplet") before the amino acid "RGD" in a targeting peptide having the amino acid sequence of SEQ ID NO: 44910 and the four amino acids (referred to as a "quad" or "quadruplet") after the "RGD" in a targeting peptide having the amino acid sequence of SEQ ID NO: 44910. Additionally, the inventors created additional targeting peptides for insertion, substitution, or modification within the VR8 region of an AAV capsid by modifying the three amino acids before the amino acid "RGD" in a targeting peptide having the amino acid sequence of SEQ ID NO: 44880 and the four amino acids after the amino acid "RGD" in a targeting peptide having the amino acid sequence of SEQ ID NO: 44880.

[0331] Figure 41 shows a list of 20 targeting peptide variants (myoDV1-myoDV10 and myoCD5-myoCD14). Figure 41 shows the tissue enrichment scores of each of the 20 targeting peptide variants in muscle tissues (diaphragm, flexor digitorum profundus, cardiac left ventricular wall, tibialis anterior, and triceps brachii). A higher tissue enrichment score indicates a higher muscle tropism, with MYODV6 and MYODV8 having the highest average tissue enrichment scores across all muscle tissue regions.

[0332] [Figure 42]Figure 42 shows a network plot illustrating the log2 fold change (tissue enrichment score) of the targeting peptides 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) in each of the analyzed muscle tissues. 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 the amino acid residue "SNR" (triplet) before "RGD" in the targeting peptide). Furthermore, MYODV6 and MYODV8 each have a higher overall log2 fold change of tissue enrichment in each muscle tissue compared to the targeting peptide having the amino acid sequence of SEQ ID NO: 44880.

[0333] [Figure 43] Figure 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 containing either the H1 (RGDLIGR (SEQ ID NO: 1422)) targeting peptide located in VR VIII between amino acids 588 and 589 or the H2 (RGDQSTL (SEQ ID NO: 3052)) targeting peptide located in VR VIII between amino acids 588 and 589. Muscle tissues analyzed included the biceps femoris, diaphragm, tibialis anterior, triceps brachii, and ventricular wall of the heart. Higher tissue enrichment score values ​​represent greater tropism.

[0334] [Figure 44]Figure 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 containing the S1 (RGDISRT (SEQ ID NO: 263)) targeting peptide located in VR VIII between amino acids 588 and 589 or the S2 (RGDRSQT (SEQ ID NO: 251)) targeting peptide located in VR VIII between amino acids 588 and 589. Muscle tissues analyzed included the ventricular wall of the heart, biceps femoris, diaphragm, tibialis anterior, and triceps brachii. Higher tissue enrichment score values ​​represent greater tropism.

[0335] [Figure 45] Figure 45 shows a plot of the median GFP+ cell percentage in liver and the indicated muscle tissues. Capsids tested included AAV9 and a targeting peptide containing the M3 ("DV6" ENRRGDFNNL (SEQ ID NO: 44864)) located in VR VIII (the M3 targeting peptide was inserted between 585 and 589, resulting in the replacement of amino acids at positions 586, 587, and 588 with three amino acids from the targeting peptide), a targeting peptide containing the M1 ("38181" SAQRGDRGQI (SEQ ID NO: 44911)) located in VR VIII (the M1 targeting peptide was inserted between 585 and 589, resulting in the replacement of amino acids at positions 586, 587, and 588 with three amino acids from the targeting peptide), and a mut1 VR I substitution, or a wild-type VR I with a VR I substitution. The AAV9 contained the M1 ("38181" SAQRGDRGQI (SEQ ID NO: 44911)) targeting peptide located in VIII (the M1 targeting peptide was inserted between 585 and 589, resulting in the replacement of amino acids at positions 586, 587, and 588 with three amino acids from the targeting peptide). A higher percentage of GFP+ cells indicates greater targeting.

[0336] [Figure 46]Figure 46 shows representative IHC images of anti-GFP staining of the indicated tissues (quadriceps, heart, and liver) for various AAV capsid proteins (AAV9, AAV9 with SEQ ID NO: 44880, AAV9 with the M3 targeting peptide (SEQ ID NO: 48391), and AAV9 with the M1 targeting peptide (SEQ ID NO: 44864). Images were taken at 10x magnification. The IHC images in Figure 46 served, in part, as the basis for the quantification in Figure 45.

[0337] [Figure 47] Figure 47 shows plots of ddPCR data for cells from liver and the indicated muscle tissues. Capsids tested included AAV9 and a targeting peptide containing the M3 ("DV6" ENRRGDFNNL (SEQ ID NO: 44864)) located in VR VIII (the M3 targeting peptide was inserted between 585 and 589, replacing amino acids 586, 587, and 588 with three amino acids from the targeting peptide), a targeting peptide containing the M1 ("38181" SAQRGDRGQI (SEQ ID NO: 44911)) located in VR VIII (the M1 targeting peptide was inserted between 585 and 589, replacing amino acids 586, 587, and 588 with three amino acids from the targeting peptide), and a mut1 VR I substitution, or a wild-type VR I with a VR I substitution. The AAV9 contained an M1 ("38181" SAQRGDRGQI (SEQ ID NO: 44911)) targeting peptide located in VIII (the M1 targeting peptide was inserted between 585 and 589, resulting in the replacement of amino acids at positions 586, 587, and 588 with three amino acids from the targeting peptide).

[0338] [Figure 48] Figure 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 genome copies (DNA) per DPG in various tissues of C57BL / 6 mice treated with different AAV vectors, as measured by RT-ddPCR.

[0339] [Figure 49-1] Figure 49A provides a peptide segment within variable region I (VRI) of the AAV9 capsid protein, modifications made at various amino acid positions within the peptide segment (P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12) and at various positions of the capsids present in the AAV-Lib1 capsid library.

[0340] [Figure 49-2] Figure 49B provides a sequence alignment of variable region I in certain AAV variants, using AAV2 variable region I as a numbering reference.

[0341] [Figure 50] Figure 50 shows vector genomes (DNA) in the livers of C57BL / 6 mice treated with the AAV-Lib1 library. Log2-fold DNA levels are shown for each of the 3,456 capsid variants (variant ID on the x-axis) in addition to AAV9 and AAVmut1. The Log2-fold DNA levels of AAV9 and AAVmut1 are specifically identified in Figure 50.

[0342] [Figure 51] Figure 51A is a schematic diagram showing the amino acid residues in VRI, with the box highlighting position P6 and including two amino acid residues at P6 of the AAV-Lib1 library; alanine (A) and glycine (G).

[0343] Figure 51B shows an AAV containing alanine (A) at P6 (amino acid position 266). -Lib1 Density plot of the amount of vector DNA (Log2(fold)) in the liver for the sum of all capsids in and AAV containing glycine (G) at P6 (amino acid position 266) -Lib1 Figure 1 shows a density plot of the amount of vector DNA (Log2(fold)) in the liver for the sum of all capsids in the liver.

[0344] Figure 51C shows an AAV containing alanine (A) at P6 (amino acid position 266). -Lib1 The amount of vector DNA in the liver (Log2(fold)) for the sum of all capsids in AAV containing glycine (G) at P6 (amino acid position 266) -Lib1 Figure 11 is an empirical cumulative distribution function (eCDF) plot showing the amount of vector DNA (Log2(fold)) in the liver relative to the sum of all capsids in the liver.

[0345] [Figure 52-1] Figure 52A is a schematic diagram showing the amino acid residues in the VRI, with boxes highlighting positions of interest: P1, P3, P5, P6, P8, and P12. Additionally, Figure 52A provides an exemplary formula for calculating the enrichment score of modified capsid proteins in the AAV-Lib1 library.

[0346] [Figure 52-2] Figure 52B is an empirical cumulative distribution function (eCDF) plot showing the amount of vector DNA (Log2(fold)) in the liver for the sum of all capsids in the AAV-Lib1 library containing either alanine (A), glutamic acid (E), glutamine (Q), or threonine (T) at P3 (amino acid position 263).

[0347] [Figure 52-3] Figure 52C is an empirical cumulative distribution function (eCDF) plot showing the amount of vector DNA (Log2(fold)) in the liver for the sum of all capsids in the AAV-Lib1 library containing either alanine (A) or glycine (G) at P5 (amino acid position 265).

[0348] [Figure 52-4] Figure 52D is an empirical cumulative distribution function (eCDF) plot showing the amount of vector DNA (Log2(fold)) in the liver for the sum of all capsids in the AAV-Lib1 library containing either alanine (A) or glycine (G) at P6 (amino acid position 266).

[0349] [Figure 53] Figure 53A is an enrichment plot showing the amount of vector DNA in the liver for the sum of all capsids in the AAV-Lib1 library containing either alanine (A) or glycine (G) at P6 (amino acid position 266).

[0350] Figure 53B shows AAVs with P5P6 combinations of AA, AG, GA, or GG. -Lib1 FIG. 10 is an enrichment plot showing the amount of vector DNA in the liver relative to the sum of all capsids in the library.

[0351] Figure 53C shows AAVs with P3, P5, and P6 combinations 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). -Lib1 10 is an enrichment plot showing the amount of vector DNA in the liver relative to the sum of all capsids in the library.

[0352] [Figure 54] Figure 54 shows the output of a network analysis of data from the AAV-Lib1 library. Each circle represents a capsid variant, and the lines connecting the capsids indicate sequence similarity.

[0353] [Figure 55] Figure 55 is an illustration of the structure of the AAV VP1 protein with positions P3, P5, P6 and P8 in the VRI highlighted.

[0354] [Figure 56] Figure 56A is an empirical cumulative distribution function (eCDF) plot showing RNA expression (Log2(fold)) in liver for the sum of all capsids in the AAV-Lib1 library containing alanine (A), glutamic acid (E), glutamine (Q), or threonine (T) at P3 (amino acid position 263).

[0355] Figure 56B shows AAVs containing alanine (A), glutamic acid (E), glutamine (Q), or threonine (T) at P3 (amino acid position 263). -Lib1 10 is an empirical cumulative distribution function (eCDF) plot showing RNA expression (Log2(fold)) in heart for all capsids in the library combined.

[0356] Figure 56C shows AAVs containing either alanine (A) or glycine (G) at P5 (amino acid position 265). -Lib1 10 is an empirical cumulative distribution function (eCDF) plot showing RNA expression (Log2(fold)) in liver for all capsids in the library combined.

[0357] Figure 56D shows AAVs containing either alanine (A) or glycine (G) at P5 (amino acid position 265). -Lib1 10 is an empirical cumulative distribution function (eCDF) plot showing RNA expression (Log2(fold)) in heart for all capsids in the library combined.

[0358] Figure 56E shows AAVs containing either alanine (A) or glycine (G) at P6 (amino acid position 266). -Lib1 10 is an empirical cumulative distribution function (eCDF) plot showing RNA expression (Log2(fold)) in liver for all capsids in the library combined.

[0359] Figure 56F shows AAVs containing either alanine (A) or glycine (G) at P6 (amino acid position 266). -Lib1 10 is an empirical cumulative distribution function (eCDF) plot showing RNA expression (Log2(fold)) in heart for all capsids in the library combined.

[0360] [Figure 57-1]Figure 57A shows the enrichment in liver DNA (x-axis) plotted against the enrichment in liver RNA (y-axis) of capsids containing various combinations of amino acid modifications at P3, P5, and P6. The positions of the AAVmut1 and AAV9 controls are indicated by arrows and corresponding text.

[0361] [Figure 57-2] Figure 57B shows the enrichment in liver DNA (x-axis) plotted against the enrichment in heart RNA (y-axis) of capsids containing various combinations of amino acid modifications at P3, P5, and P6. The positions of AAVmut1 and AAV9 controls are indicated by arrows and corresponding text.

[0362] [Figure 58-1] Figure 58A shows the enrichment in liver DNA (x-axis) plotted against the enrichment in liver RNA (y-axis) of capsids with the combination of threonine (T), glycine (G), and glycine (G) amino acid modifications at P3, P5, and P6, respectively. The positions of the AAVmut1 and AAV9 controls are indicated by arrows and corresponding text.

[0363] Figure 58B shows the enrichment in liver DNA (x-axis) plotted against the enrichment in heart RNA (y-axis) of capsids with the combined amino acid modifications of threonine (T), glycine (G), and glycine (G) at P3, P5, and P6, respectively. mut1 and the positions of the AAV9 control are indicated by arrows and corresponding text.

[0364] Figure 58C shows the enrichment in liver DNA (x-axis) plotted against the enrichment in heart RNA (y-axis) of capsids with threonine (T), glycine (G), and a combination of glycine (G) amino acid modifications at P3, P5, and P6, respectively. The subpopulations of capsids with threonine (T) (yellow dots) or asparagine (N) / serine (S) (blue dots) at position P1 are color-coded. AAV mut1and the positions of the AAV9 control are indicated by arrows and corresponding text.

[0365] [Figure 58-2] Figure 58D shows the enrichment in liver RNA (x-axis) plotted against the enrichment in heart RNA (y-axis) for capsids with threonine (T), glycine (G), and a combination of glycine (G) amino acid residues at P3, P5, and P6, respectively. Each point represents a capsid with TGG at P3, P5, and P6. Capsids with specific amino acid residues (or combinations of amino acid residues) (e.g., asparagine (N), glycine (G), threonine (T), serine (S), and / or alanine (A)) at positions P1, P2, P4, and P12 are identified and shaded accordingly. Specific subpopulations with NGTT, NSTT, SGAA, and SGAT at positions P1, P2, P4, and P12 are identified. The positions of AAVmut1 and AAV9 controls are indicated by arrows and corresponding text.

[0366] Figure 58E shows the enrichment in liver RNA (x-axis) plotted against the enrichment in heart RNA (y-axis) of capsids with the amino acid residue combination glutamine (Q), glycine (G), and glycine (G) at positions P3, P5, and P6, respectively. Each point represents a capsid with TGG at positions P3, P5, and P6. 12 Subpopulations have been identified that contain serine (S), glycine (G), threonine, and histidine (H) in AAV. mut1 and the positions of the AAV9 control are indicated by arrows and corresponding text.

[0367] [Figure 58-3] Figure 58F shows the average enrichment in liver RNA (x-axis) plotted against the enrichment in heart RNA (y-axis) for all AAV-Lib1 capsids that have the amino acid residue combinations threonine (T), glycine (G), and glycine (G) at P3, P5, and P6, respectively. The positions of the AAVmut1 and AAV9 controls are indicated by the corresponding text.

[0368] Figure 58G shows the enrichment in liver RNA (x-axis) plotted against the enrichment in heart RNA (y-axis) of 24 selected capsids that have a combination of threonine (T), glycine (G), and glycine (G) amino acid residues at P3, P5, and P6, respectively. The 24 selected capsids correspond to Group 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). mut1 and the positions of the AAV9 controls are indicated by the corresponding letters.

[0369] [Figure 58-4] Figures 58H-58I show the enrichment of liver RNA (x-axis) plotted against the enrichment of heart RNA (y-axis) for six groups, each group having a combination of threonine (T), glycine (G), and glycine (G) amino acid residues at P3, P5, and P6, respectively. Figure 58H includes a filled circle identifying the VR1 mini-Lib peptide segment in the key. Figure 58I includes a filled circle identifying the VR1 mini-Lib peptide segment in the key.

[0370] [Figure 59] Figure 59 shows vector genome (DNA) levels in the livers of NHPs treated with the AAV-Lib1 library. Log2-fold DNA levels of each of the 3,456 capsid variants (variant ID on the x-axis) are shown, in addition to AAV9 and AAVmut1. The Log2-fold DNA levels of AAV9 and AAVmut1 are specifically identified in Figure 59.

[0371] [Figure 60]Figure 60 is a box plot showing the distribution of liver RNA log2FC enrichment scores from capsids in all sequence groups defined by positions P3, P5, P6, P8 (see also positions P3, P5, P6, P8 in VR I).

[0372] [Figure 61] Figure 61 is a plot showing the liver enrichment of rAAV from AAV-Lib1 in mice (y-axis) versus the liver enrichment of rAAV from AAV-Lib1 in NHPs (x-axis). The correlation is demonstrated by a 0.72 R^2 value. The capsids plotted in Figure 61 include capsids with a peptide segment containing the P3TP5GP6G motif.

[0373] [Figure 62-1] Figures 62A-62E show non-limiting examples of targeting peptide combinations containing a variable triplet, a constant RGD, a variable quad, and optionally, a peptide segment in the AAV-Lib3 library in Example 27. Figure 62A shows the possible amino acid residues in the variable triplet corresponding to X1, X2, and X3, with color indicating the type of amino acid. Figure 62B shows the constant RGD quad present in each targeting peptide. Figure 62C shows the amino acid residue category (genera) and the specific quad. Figure 62D shows non-limiting examples of peptide segments present in VR I. Figure 62E shows the amino acids at positions X7, X8, X9, and X10, with color indicating the type of amino acid residue. [Figure 62-2] Same as above.

[0374] [Figure 63-1]Figures 63A-63B show enrichment plots (Avg_logFC vs. inverse CV (coefficient of variation)) of capsids in the AAV-Lib2 library. Figure 63A is an enrichment plot showing capsid performance in muscle (i.e., all muscle tissue) for all 50,500 capsids in the AAV-Lib2 library. Figure 63B shows an enrichment plot of capsids from the AAV-Lib2 library with quads selected from FNNL, FNNT, and RGQI. [Figure 63-2] Same as above.

[0375] [Figure 64] Figure 64 shows an enrichment plot of the amount of vector RNA in muscle tissue for the sum of all capsids in the AAV-Lib2 library with the indicated quad sequences.

[0376] [Figure 65] Figure 65 shows a heat map of the tissue enrichment scores in muscle tissue for each capsid in the AAV-Lib2 library. The AAV-Lib2 library contains approximately 50,500 capsids with one of 20 quad and one of XX triplet sequences. Data for each capsid appears as a cell in the heat map, with rows representing quads and columns representing triplets. Data was grouped using hierarchical clustering. Clustering identified Group I, Group II, and Group III.

[0377] [Figure 66-1] Figure 66A shows the enrichment scores averaged across all muscle tissues for each capsid in the AAV-Lib2 library (Group 1) with quads selected from RGQI, RSVV, and RGVV. The top 100 capsids are identified by the larger of the two points on the plot.

[0378] [Figure 66-2]Figure 66B shows the motif plot of the top 100 capsid targeting peptides in the AAV-Lib2 library (Group 1) with quads selected from RGQI, RSVV, and RGVV. The data used to generate the motif plot was averaged across all muscle tissues. [Figure 66-3] Same as above.

[0379] [Figure 66-4] Figure 66C shows a plot of the unique variant counts of the indicated triplets of the top 100 capsids in the AAV-Lib2 library (group 1) with quads selected from RGQI, RSVV, and RGVV.

[0380] [Figure 67-1] Figure 67A shows the enrichment scores averaged across all muscle tissues for each capsid in the AAV-Lib2 library (group 2) with quads selected from HGVL, YSSV, YSTM, and YTSV. The top 100 capsids are identified by the larger of the two points on the plot.

[0381] [Figure 67-2] Figure 67B shows a motif plot of the top 100 capsid targeting peptides in the AAV-Lib2 library (Group 2) with quads selected from HGVL, YSSV, YSTM, and YTSV. The data used to generate the motif plot was averaged across all muscle tissues.

[0382] [Figure 67-3] Figure 67C shows a plot of unique variant counts of the indicated triplets of the top 100 capsids in the AAV-Lib2 library (group 2) with quads selected from HGVL, YSSV, YSTM, and YTSV. [Figure 67-4] Same as above.

[0383] [Figure 68-1]Figure 68A shows the enrichment scores averaged across all muscle tissues for each capsid in the AAV-Lib2 library with quads selected from FNNT, FNNL, FQNT, and YNSL (group 3). The top 100 capsids are identified by the larger of the two points on the plot.

[0384] [Figure 68-2] Figure 68B shows a motif plot of the top 100 capsid targeting peptides in the AAV-Lib2 library (group 3) with quads selected from FNNT, FNNL, FQNT, and YNSL. The data used to generate the motif plot was averaged across all muscle tissues.

[0385] [Figure 68-3] Figure 68C shows a plot of unique variant counts of the indicated triplets of the top 100 capsids in the AAV-Lib2 library with quads selected from FNNT, FNNL, FQNT, and YNSL (group 3). [Figure 68-4] Same as above.

[0386] [Figure 69] Figure 69 shows an enrichment plot of the amount of vector RNA in liver tissue for the sum of all capsids in the AAV-LIB2 library with the indicated quad sequences.

[0387] [Figure 70-1] Figure 70A shows the liver tissue enrichment scores for each capsid in the AAV-Lib2 library with quads selected from RGQI, RSVV, RGVV, RQGI, and RSQT. The top 100 capsids are identified by the larger of the two points on the plot.

[0388] [Figure 70-2]Figure 70B shows a motif plot of the top 100 capsid targeting peptides in the AAV-Lib2 library (from Figure 70A) with quads selected from RGQI, RSVV, RGVV, RQGI, and RSQT. The data used to generate the motif plot was averaged across liver tissues.

[0389] [Figure 71-1] Figure 71A shows enrichment score plots (inverse VC relative to mean MN_FC) for biceps femoris, quadriceps, diaphragm, heart-atrium, heart-ventricle, and liver for each capsid in the AAV-Lib2 library. The top 10 performing capsids are identified by darker shaded circles. [Figure 71-2] Same as above. [Figure 71-3] Same as above.

[0390] [Figure 71-4] Figure 71B shows a table of the enrichment scores of the top 10 capsids containing the indicated targeting peptides.

[0391] [Figure 71-5] Figure 71C shows a table of amino acid residues (at each position of the targeting peptide described in Figure 71B and also shown in Figure 71A).

[0392] [Figure 72-1] Figure 72A shows the enrichment score plot (LogFC mean vs. inverse CV (variant counts)) of capsids with the indicated quads. Targeting peptides with specific triplets have been identified.

[0393] [Figure 72-2] Figure 72B shows a table of the amino acid residues at each position of the targeting peptides described in Figure 72A.

[0394] [Figure 73-1]Figure 73A shows the enrichment score plot (LogFC mean vs. inverse CV (variant counts)) of capsids with the indicated quads. Targeting peptides with specific triplets have been identified.

[0395] [Figure 73-2] Figure 73B shows a table of the amino acid residues (and the properties (eg, polarity) of each amino acid residue) of the targeting peptide described in Figure 73A.

[0396] [Figure 74-1] Figure 74A shows the enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV-Lib2 library. Ten capsids are highlighted.

[0397] [Figure 74-2] Figure 74B shows the enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV-Lib2 library. Ten capsids are highlighted.

[0398] [Figure 75-1] Figure 75A shows the enrichment scores in skeletal muscle versus heart for each of the capsids in the AAV-Lib2 library.

[0399] [Figure 75-2] Figure 75B shows the enrichment scores in skeletal muscle versus the enrichment scores in heart for each capsid in the AAV-Lib2 library with the quad "LIGR."

[0400] [Figure 75-3] Figure 75C shows the enrichment scores in skeletal muscle versus the enrichment scores in heart for each capsid in the AAV-Lib2 library with quad "QSTL".

[0401] [Figure 75-4]Figure 75D shows the enrichment scores in skeletal muscle relative to the enrichment scores in heart for each capsid in the AAV-Lib2 library with the quad "RGVV."

[0402] [Figure 75-5] Figure 75E shows the enrichment scores in heart relative to the enrichment scores in skeletal muscle for each of the capsids in the AAV-Lib2 library with the indicated quads. DETAILED DESCRIPTION OF THE INVENTION

[0403] 6. Detailed Description 6.1.Definition "AAV" is an adeno-associated virus and may be used to refer to the virus itself or its derivatives. The term encompasses all subtypes, serotypes, and pseudotypes, as well as both naturally occurring and recombinant forms, unless otherwise required.

[0404] The term "AAV capsid protein" or simply "capsid protein" refers to the VP1, VP2, or VP3 capsid proteins. In some embodiments, the AAV capsid protein is the 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.

[0405] The term "modified AAV capsid protein" or simply "modified capsid protein" refers to a capsid protein that is modified relative to a naturally occurring or synthetic / artificial capsid protein referred to as a "reference AAV capsid protein" or "reference capsid protein." A reference AAV capsid protein, as used herein, can be the VP1, VP2, or VP3 capsid protein of a naturally occurring AAV variant or a non-naturally occurring VP1, VP2, or VP3 capsid protein known in the art.

[0406] The term "targeting peptide," as used herein, refers to a sequence of 10 amino acids (X1X2X3RGDX7X8X9X) within variable region VIII (VRVIII) of a modified AAV capsid protein that is introduced by one or more modifications described herein. 10 ) AAVs containing modified capsid proteins with targeting peptides may have different localization and distribution in target cells, tissues, or organs than AAVs with capsid proteins without targeting peptides.

[0407] The term "peptide segment," as used herein, refers to a portion of variable region I (VR I) of an AAV capsid protein comprising 10, 11, or 12 amino acids. In a preferred embodiment, the peptide segment is located between amino acids 250 and 280 of the AAV capsid protein. The modified AAV capsid proteins provided herein include peptide segments having a sequence that differs from the corresponding sequence of a reference AAV capsid protein by having one or more modifications. The peptide segment is a sequence of P1P2P3P4P5P6P7P8P9P within VR I. 10 P 11 P 12 where "Pn" refers to the position in the peptide segment.

[0408] As used herein, the term "amino acid position" in an AAV capsid protein refers to the position of an amino acid residue in the AAV VP1 protein sequence, counting from the first amino acid at the N-terminus.As used herein, the term "amino acid" includes 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 a preferred embodiment, the amino acid is a naturally occurring L-α-amino acid.

[0409] For the avoidance of doubt, as used herein, an insertion site indicated as being at amino acid position X means that the targeting peptide is inserted between amino acids X and X+1, i.e., the targeting peptide is inserted after the indicated amino acid.

[0410] The term "liver-off" is used herein to describe an AAV that has lower liver tropism or reduced liver biodistribution when administered to a mammalian subject compared to other AAV variants. The term "liver-off" is also used to describe modifications in AAV capsid proteins that reduce liver tropism or liver biodistribution when administered to a mammalian subject. The term "liver-off" is also used to describe AAV capsid species with variable region 1 (VRI) that reduce liver tropism or liver biodistribution when administered to a mammalian subject.

[0411] The term "liver-on" is used herein to describe an AAV that has greater liver tropism or greater liver biodistribution when administered to a mammalian subject compared to other AAV variants. The term "liver-on" is also used to describe modifications in an AAV capsid protein that increase liver tropism or liver biodistribution when administered to a mammalian subject.

[0412] The term "CAG," when used in reference to a promoter or ERE, refers to a promoter or ERE having a chicken beta-actin promoter and a CMV enhancer sequence.

[0413] The term "constitutive" promoter or ERE, as used herein, refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0414] The term "expression regulatory element" or "ERE," as used herein in the context of the rAAV of the present disclosure, refers to a nucleic acid sequence that is required for expression of an MTM1 coding sequence operably linked to the ERE. In some instances, the ERE sequence may be a core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements that are required for expression of a gene product, e.g., an exon sequence.

[0415] The term "functional fragment," in the context of myotubularin or MTM1, refers to a biologically functional fragment of myotubularin or MTM1. As understood in the art, a biologically functional fragment is a portion or portions of a full-length sequence that retains the biological function of the full-length sequence. An exemplary functional fragment corresponds to amino acids 29-486 of SEQ ID NO: 165 (also disclosed herein as SEQ ID NO: 164). Biological functions of MTM1 include the ability to cleave or hydrolyze endogenous phosphoinositide substrates known in the art or artificial phosphoinositide substrates in in vitro assays (i.e., phosphoinositide phosphatase activity), recruit and / or associate with other proteins, such as the GTPase Rab5, the PI3-kinase Vps34, or Vps15 (i.e., with appropriate localization), or treat a myotubular muscle disease.

[0416] The term "functional variant," in the context of myotubularin or MTM1, refers to different splice isoforms, variants, fusion proteins, and modified forms of a wild-type MTM1 polypeptide or functional fragments thereof. Such isoforms, biologically active fragments or variants, fusion proteins, and modified forms of an MTM1 polypeptide retain at least one biological function of a full-length MTM1 protein (e.g., the protein of SEQ ID NO: 165).

[0417] The term "inducible" promoter or ERE, as used herein, refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell only when an inducer substantially corresponding to the promoter is present in the cell.

[0418] As used herein, the term "internalizing moiety" refers to a moiety that can interact with a target tissue or cell type to deliver an attached molecule to the cell (i.e., penetrate the desired cell and transport it across the cell membrane). In certain embodiments, the MTM1 polypeptide encoded by the rAAV of the present disclosure can be a fusion protein comprising an internalizing moiety. In some embodiments, the internalizing moiety selectively, but not necessarily exclusively, targets and penetrates muscle cells. In certain embodiments, the internalizing moiety has limited cross-reactivity and thus preferentially targets specific cell or tissue types. 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 translocation across the cell membrane via the ENT2 transporter. Exemplary internalizing moieties are disclosed in U.S. Patent No. 9,447,394 B2, the contents of which are incorporated herein by reference.

[0419] The term "inverted terminal repeat" (or "ITR") refers to polynucleotide sequences found at the ends of the AAV genome that form hairpins, which contribute to the genome's ability to self-prime (allowing primase-independent synthesis of a complementary second DNA strand) and provide for encapsidation of the genome into an AAV particle. The ITRs can be wild-type ITRs or variants thereof.

[0420] As used herein, the terms "liver-toggle mutant," "liver-toggle mutant of a reference AAV capsid protein," and the like refer to a capsid protein that contains a sequence that differs from a reference AAV capsid protein by having one or more mutations (e.g., amino acid substitutions) that alter its tropism, specificity, or distribution in the liver compared to the reference AAV capsid protein when administered to a mammalian subject (such sequence differences are referred to herein as "liver toggle mutations"). The mammalian subject may be a human, non-human primate (NHP), mouse, rat, bird, rabbit, guinea pig, hamster, livestock (including pigs and sheep), dog, or cat. Exemplary liver toggle mutations are disclosed in WO2019 / 217911 and WO2021 / 050614, the entire contents of which are incorporated herein by reference. In some embodiments, the liver-toggle mutation comprises (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, the liver-toggle mutant of a reference AAV capsid protein is a capsid protein that comprises a sequence that differs from the reference AAV capsid protein by having an alanine (A) amino acid residue at an amino acid position corresponding to position 267 in the AAV9 VP1 protein and a threonine (T) amino acid residue at an amino acid position corresponding to position 269 in AAV9 VP1. In still further embodiments, the liver toggle mutation comprises a sequence that differs from a reference AAV capsid protein by having, in each case, any combination of: (i) arginine (R) instead of serine (S) at position 446, (ii) alanine (A) instead of arginine (R) at position 471, and (iii) threonine (T) or alanine (A) instead of valine (V) at position 708, numbered according to the AAV2 reference capsid protein (SEQ ID NO: 1 of WO2021 / 050614, incorporated herein by reference).

[0421] The term "modification" when combined with amino acid residue(s) or modified sequence refers to insertion(s), deletion(s) and / or substitution(s).

[0422] 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.

[0423] 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.

[0424] The terms "operably linked" and "operatively linked" refer to the functional association of a nucleic acid sequence with nucleotide regulatory sequences, such as promoters, enhancers, transcription and translation stop sites, and other signal sequences, indicating that two or more DNA segments are joined together so that they function in concert for their intended purpose. For example, operably linking a nucleic acid sequence, usually DNA, to a regulatory sequence or promoter region refers to a physical and functional association between the DNA and the regulatory sequence or promoter such that transcription of such DNA is initiated from the regulatory sequence or promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0425] The term "parenteral" administration of a composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0426] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0427] The term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, excipient, stabilizer, and adjuvant. For examples of carriers, excipients, stabilizers, and adjuvants, see Remington: The Science and Practice of Pharmacy, 22nd Revised Ed., Pharmaceutical Press, 2012.

[0428] The abbreviation "rAAV" refers to the recombinant adeno-associated virus particle that is composed of at least one AAV capsid protein and encapsidated polynucleotide, and is sometimes referred to herein as "genome".rAAV can comprise a genome that includes heterologous polynucleotide (i.e., polynucleotide other than wild-type AAV genome), for example, the heterologous polynucleotide that encodes the gene that is delivered to mammalian cell, for example, the MTM1 gene.Heterologous nucleotide can also be referred to as transgene.

[0429] The term "self-complementary" rAAV vector or genome, as used herein, refers to 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 rAAV ITRs. The absence of a TR prevents replication initiation at the vector end where the TR is not present. Generally, a fully self-complementary rAAV vector generates a single-stranded inverted repeat genome using wild-type (wt) AAV TRs at each end and a mutant TR (mTR) in the middle. Thus, a fully self-complementary rAAV genome is typically a single-stranded polynucleotide having, from 5' to 3', a first ITR sequence, a heterologous sequence (e.g., an MTM1 coding sequence and / or ERE), a second ITR sequence, a second heterologous sequence complementary to the first heterologous sequence, and a third ITR sequence. A "partially self-complementary" rAAV genome refers to a single-stranded polynucleotide having a self-complementary region that is complementary, in the 5' to 3' or 3' to 5' direction, to a first ITR sequence, a heterologous sequence (e.g., an MTM1 coding sequence and / or an ERE), a second ITR sequence, and a portion of the heterologous sequence, and has a length that is less than the full-length heterologous sequence.

[0430] The term "tissue-specific" promoter or ERE, as used herein, refers to a nucleotide sequence that, when operably linked to a polynucleotide, encodes or is specified by a gene, such that the gene product is produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0431] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease, condition, or its symptoms are completely or partially prevented, and / or may be therapeutic, in that a disease or condition and / or adverse effects that may result from the disease or condition are partially or completely cured. "Treatment," as used herein, encompasses any treatment of a mammalian, particularly a human, disease or condition, including (a) preventing the disease or condition from occurring in a subject who may be predisposed to, but has not yet been diagnosed as having, the disease or condition; (b) inhibiting the disease or condition (e.g., halting its development); or (c) alleviating the disease or condition (e.g., causing regression of the disease or condition, providing improvement in one or more symptoms).

[0432] The terms "vector," "AAV vector," and "rAAV vector" refer to a rAAV that contains a heterologous polynucleotide, e.g., a transgene.

[0433] The term "variable region" or "VR," as used herein, refers to one or more of the nine sequence-variable regions (e.g., VRI-VRIX) in the AAV capsid protein previously defined by comparison and alignment of various AAV capsid proteins. See, for example, 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). VRs are known to contain amino acids that contribute to subtle differences in surface topology and distinct functional phenotypes in receptor binding, transduction efficiency, antigen reactivation, and the like. The relative locations of VR I, VR IV, and VR VIIII are illustrated in Figure 1, although the specific locations of the variable regions within the capsid protein may vary depending on the capsid protein and / or sequence alignment method.

[0434] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this method and composition belongs.Methods and materials similar or equivalent to those described herein can be used in the implementation or testing of method and composition, and suitable methods and materials are described below.In addition, materials, methods and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents and other references described herein are incorporated by reference in their entirety. 6.2. Modified AAV Capsid Proteins

[0435] 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 is X1X2X3RGDX7X8X9X 10 X1, X2, X3, X7, X8, X9 and X 10 are independently selected from any amino acid residue, and the peptide segment is P1P2P3P4P5P6P7P8NDNP 12 and P1, P2, P3, P4, P5, P6, P7, P8 and P 12 are independently selected from any amino acid residue. In some embodiments, the modified AAV capsid protein comprises both (i) a targeting peptide and (ii) a peptide segment. In some embodiments, the AAV capsid protein comprises a targeting peptide but does not comprise a peptide segment. In some embodiments, the AAV capsid protein comprises a peptide segment but does not comprise a targeting peptide.

[0436] 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) of the reference AAV capsid protein and (ii) a sequence of a reference AAV capsid protein comprising one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the targeting peptide is X1X2X3RGDX7X8X9X 10 X1, X2, X3, X7, X8, X9 and X 10 are independently selected from any amino acid residue, and the peptide segment is P1P2P3P4P5P6P7P8NDNP 12 and P1, P2, P3, P4, P5, P6, P7, P8 and P 12 are independently selected from any amino acid residue.

[0437] In some embodiments, the modified AAV capsid protein does not comprise RGDLLLS (SEQ ID NO: 1).

[0438] In some embodiments, the modified AAV capsid protein has a peptide segment that does not contain an alanine (A) at P6 or a threonine (T) at P8.

[0439] In some embodiments, the modified AAV capsid does not include RGDLLLS (SEQ ID NO: 1) and the peptide segment does not include an alanine (A) at P6 and a threonine (T) at P8.

[0440] In some embodiments, the modified AAV capsid protein has one or more amino acid insertions, deletions, substitutions, or a combination thereof compared to a reference AAV capsid.

[0441] 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 a targeting peptide within VR VIII of the reference AAV capsid protein.

[0442] In some embodiments, the capsid protein further comprises one or more modifications outside of VR I and VR VIII of the reference AAV capsid protein.

[0443] In some embodiments, the reference AAV capsid protein comprises one or more modifications including an amino acid insertion, deletion, substitution, or a combination thereof to introduce a targeting peptide within VR VIII (e.g., VR VIII corresponds to amino acids between positions 565 and 595 of the reference AAV capsid protein).

[0444] In some embodiments, the reference AAV capsid protein comprises one or more modifications outside of VR I of the reference capsid protein (e.g., VR I corresponds to amino acids between 259 and 275 of the reference AAV capsid protein). In some embodiments, the reference AAV capsid protein comprises one or more modifications outside of VR VIII of the reference AAV capsid protein (e.g., VR VIII corresponds to amino acids between 565 and 595 of the reference AAV capsid protein). In some embodiments, the reference AAV capsid protein comprises one or more modifications outside of VR I (e.g., VR I corresponds to amino acids between 259 and 275 of the reference AAV capsid protein) and VR VIII (e.g., VR VIII corresponds to amino acids between 565 and 595 of the reference AAV capsid protein) of the reference AAV capsid protein.

[0445] In some embodiments, the one or more modifications are amino acid insertions, deletions, substitutions, or combinations thereof to introduce a targeting peptide into the VR VIII of the reference AAV capsid protein. In some embodiments, the one or more modifications are amino acid insertions, substitutions, or combinations in a region outside the VR VIII region of the reference AAV capsid protein. In some embodiments, the one or more modifications outside the VR VIII are modifications in the VR I region of the reference AAV capsid protein. In some embodiments, the one or more modifications result in a liver-off phenotype. In some embodiments, the one or more modifications outside the VR VIII are liver toggle mutations. 6.2.1. Targeting Peptides

[0446] In some embodiments, the targeting peptide has the sequence X1X2X3RGDX7X8X9X 10 X1, X2, X3, X7, X8, X9 and X 10 are independently selected from any amino acid residue. 10are independently selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, and Y. In some embodiments, X, X, X, and X 10 are independently selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T and V.

[0447] 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.

[0448] In some embodiments, X1, X2, and X3 are independently selected from any amino acid residue.

[0449] In yet other embodiments, X1, X2, and X3 are unaltered from the amino acids at the corresponding positions in the reference AAV capsid protein, in which case X1, X2, and X3 are identical to the amino acids at the corresponding positions in the reference AAV capsid protein.

[0450] In still other embodiments, X1 and X2 are unaltered from the amino acid at the corresponding position in the reference AAV capsid protein. In still other embodiments, X1 is unaltered from the amino acid at the corresponding position in the reference AAV capsid protein. In still other embodiments, X2 is unaltered from the amino acid at the corresponding position in the reference AAV capsid protein. In some embodiments, X1, X2, and / or X3 are natural amino acid residues in the reference AAV capsid protein.

[0451] X1, X2, and X3 may be introduced by amino acid substitution, insertion, mutation, and / or deletion of 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 X3 in the reference AAV capsid may be deleted or substituted. In some embodiments, the 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 X3 in the reference AAV capsid is deleted or substituted. In some embodiments, the corresponding sites X1 and X2 in the reference AAV capsid are deleted or substituted. In some embodiments, the corresponding sites X2 and X3 in the reference AAV capsid are deleted or substituted. In some embodiments, the corresponding sites X1 and X3 in the reference AAV capsid are deleted or substituted. In some embodiments, the corresponding site X in the reference AAV capsid is deleted or substituted. 1、 X2 and X3 are deleted or substituted.

[0452] In some embodiments, X1 is 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, X1 is E.

[0453] 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, X2 is selected from K, E, D, A, S, F, N, V ​​or L. In some embodiments, X2 is selected from K, E or D. In some embodiments, X2 is selected from N, A or Y. In some embodiments, X2 is selected from N, Y or S.

[0454] In some embodiments, X3 is selected from R, Q, A, D, E, F, G, H, I, K, L, M, N, P, S, T, V, W, or Y. In some embodiments, X2 is N. In some embodiments, X2 is A. 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 X3 is R. In some embodiments, X1 is E, X2 is N, and X3 is R. In some embodiments, X1 is S, X2 is N, and X3 is 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.

[0455] In some embodiments, X2X3 is NR. In some embodiments, X1X2X3 is ENR. In some embodiments, X1X2X3 is SNR.

[0456] In some embodiments, X1X2X3 is 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, 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,<h2 style=";text-align:left;direction:ltr">EVY、EQY、QEY、QEN、SAD、AEF、EMF、SDF、ADF、ADY、AVF、TLY、SIY、SLY、AIY、DY V、AEY、ENF、EMY、QDY、ETY、EAY、TVY、SVY、QVF、QVY、TMY、DKY、ALY、AVY、TMW、E QW, 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, TT I、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、E PV、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、EV V、EVM、TPY、QEW、EIW、EPT、EIH、DFY、DQL、NDA、DIW、TDY、ELF、DVY、ESG、EPG、E IG、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、EA N、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、E GN、ENH、NGE、DYN、DNH、DNF、ESQ、ESM、ESL、SSD、ENL、EMV、QDS、TSE、SWD、ASD、<h2 style=";text-align:left;direction:ltr">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, E YM, 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, TN F、NAF、ASP、QSQ、APY、SFM、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、A FP、AEE、ADE、NIE、TYP、QIE、TFP、SYP、ELW、DVW、NEW、EPA、EVA、TLE、AWE、NME、 QME, ATE, AYA, 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, S IE、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、DQ W、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, SLWIt is selected from the group consisting of 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.,

[0457] 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, EYI, 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.,

[0458] 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、ES T、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、A NW、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、NN F, NWL, NMA, NNA, NHN, NFH, ELR, NYM, ERA, NFS, SRD, NMM, NNL, NNQ, NNS, QDR, S DK、NNI、AQH、ANA、QNH、QWG、AWA、QWA、EQK、SHS、QFA、SFA、TFA、AYG、EKQ、ERN 、EMR、EFR、AYM、EAR、EMK、EYK、EWK、QPI、QPF、NPL、SPI、SPL、NPI、SIV、SLL、S SA、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、NA M、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、N MV、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、TW M、QNW、STF、AFM、NSY、SSY、SNY、THY、TNY、QNY、THF、NSV、ATY、SKD、TNF、TWT、Q WT、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、TG H、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, Q SS、SAH、SMH、QAH、AAH、AHN、QNS、QAS、ANS、ANN、SWA、SWS、TWS、ERI、EX、QWS、 AYS、AFS、SYS、QFS、QYN、QFT、AYT、TNS、TYS、SFT、QNQ、QYS、SYT、ANQ、SWV、SN Q、ANY、TNQ、ATF、SSQ、SSN、TWA、SGQ、SSS、TWY、SMY、TGN、TGY、QWY、SQF、TGF、Q TY、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、AS F、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、N ST、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, SA T, SAI, QSL, QSI, ASI, SSL, SSV, SSI, AGI, QGM, TGM, SGM, SGL, SGV, ARL, NWE, S FL、TPY、DRP、DTK、DMK、SEY、SME、EAI、AHD、DNA、EFQ、EMH、SGE、DNQ、DNI、QLY DGT, EGN, DYN, EWV, ESM, EAL, ASD, DKA, ADF, ADY, SLY, AEY, ETY, EAY, TGA, AV M、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、A DM、SAA、SQH、NQV、ALL、ATM、QYA、STT、DGR、NHT、SHT、QHT、TTL、NHM、QHL、DNR、 QHM、QFM、QMI、AHV、THI、AHT、TNH、NHV、THV、SHI、ATV、TWI、NYV、TYI、DYR、SF I, NQF, NWM, NSL, NSP, TQY, DRS, ASP, TKD, QWI, QWN, TWN, QWV, THN, TTM, TQQ, N MQ、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、NG N、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、Q DI, DSA, DNM, DKT, DGM, SQY, TLT, QEH, DKQ, TGE, DNS, SDV, SNE, EGT, EGV, AAPSelected from the group consisting of 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, QQT, 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.,

[0459] In some embodiments, X1X2X3 is selected from the group consisting of ANY, SNI, AAI, AAM, ANT, AST, AYQ, EHK, ENK, ENR, SFQ, SSI, TAY, TDK, TNT, AAF, AAL, AAY, ADK, AFA, ANF, ANI, ANQ, ANS, AQM, ARE, ASV, AYH, AYT, EMK, EWK, NNM, QAF, QAI, QAM, QAT, QAY, QFT, QGM, QHL, QNF, QNQ, QNS, QNT, QNV, QNY, SAH, SAI, SAL, SFT, SFV, SHI, SHV, SMM, SNF, SNM, SNN, SNQ, SNV, SNY, SQI, SQV, SSL, SWQ, SWS, SYI, SYM, SYN, SYQ, TAM, TAT, TDR, TFM, THV, TNF, TNH, TNI, TNM, TNQ, TNV, TSY, TWA, and TYM.

[0460] X1X2X3, 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、EQ R、EHR、EWR、EQK、ESR、EKQ、EYR、ENR、EMK、EYK、EHK、EWK、QNI、TNI、TYI、SNY、 DRQ、AWI、QWI、DFR、EKG、QYG、QWQ、EKN、EKF、EKT、AFH、ENK、NYS、DKH、AAG、QMF 、QFH、QKD、ARE、AHQ、ADK、ADR、AHN、QNS、ANN、SWS、EKS、AFS、QFS、TNQ、TGY、N GL、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, DT K, DNA, SDS, TDH, DGL, QDV, SDM, DKT, DGM, SQY, DKS, SSD, EMV, TSE, EYV, SSE, E GM、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、N MM、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、NQ I、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、A YH、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、SM N、SAN、QAF、ASF、ASY、TAS、TAF、TFV、SMQ、SFV、TMT、QAN、SAY、EDK、AMV、QHV、T HS、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、AN H、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, S NP、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, TT V、AAL、NHL、NHM、QYL、QHL、ATL、DNR、DSR、ANL、SHL、SNL、TNL、QAL、AMI、ANI、Q NM、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、QL V、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, N ES、DWK、DLR、DIR、SDT、EAN、ETH、TYD、SYE、EAM、TND、AYD、TSP、DRT、NDH、SSF、It is selected from the group consisting of 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, 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.

[0461] 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.

[0462] In some embodiments, the targeting peptide is a 7-mer peptide having an amino acid sequence selected from SEQ ID NOs: 238-44858 (RGDX7X8X9X 10 ) in which case X1X2X3 can be an amino acid at the corresponding position in a reference AAV capsid. For example, X1X2X3 can be a naturally occurring amino acid at the corresponding position in a reference AAV capsid. In some embodiments, the targeting peptide is a 7-mer peptide (RGDX7X8X9X) having an amino acid sequence with at least 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 238-44858. 10 ) is included.

[0463] In some embodiments, the modified AAV capsid protein is a 7-mer peptide having an amino acid sequence selected from SEQ ID NOs: 238-247 (RGDX7X8X9X 10 ) in which X1X2X3 can be the amino acid at the corresponding position in a reference AAV capsid. In some embodiments, X1X2X3 has a sequence that differs from the sequence at the corresponding position in a reference AAV capsid. In some embodiments, any one of the corresponding positions X1, X2, and X3 in a 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 a 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.

[0464] In some embodiments, the targeting peptide is a 7-mer peptide having an amino acid sequence selected from SEQ ID NOs: 3881, 12092, 14601, 15342, 21498, and 31396 (RGDX7X8X9X 10 ) is included.

[0465] In some embodiments, the targeting peptide is a 7-mer peptide having the amino acid sequence of SEQ ID NO: 238 (RGDX7X8X9X 10 ) is included.

[0466] 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 the amino acid sequence of 44859. In some embodiments, the targeting peptide comprises the amino acid sequence of 44860. In some embodiments, the targeting peptide comprises the amino acid sequence of 44861. In some embodiments, the targeting peptide comprises the amino acid sequence of 44862. In some embodiments, the targeting peptide comprises the amino acid sequence of 44863. In some embodiments, the targeting peptide comprises the amino acid sequence of 44864. In some embodiments, the targeting peptide comprises the amino acid sequence of 44865. In some embodiments, the targeting peptide comprises the amino acid sequence of 44866. In some embodiments, the targeting peptide comprises the amino acid sequence of 44867. In some embodiments, the targeting peptide comprises the amino acid sequence of 44868. In some embodiments, the targeting peptide comprises the amino acid sequence of 44869. In some embodiments, the targeting peptide comprises the amino acid sequence of 44870. In some embodiments, the targeting peptide comprises the amino acid sequence of 44871. In some embodiments, the targeting peptide comprises the amino acid sequence of 44872. In some embodiments, the targeting peptide comprises the amino acid sequence of 44873. In some embodiments, the targeting peptide comprises the amino acid sequence of 44874. In some embodiments, the targeting peptide comprises the amino acid sequence of 44875. In some embodiments, the targeting peptide comprises the amino acid sequence of 44876. In some embodiments, the targeting peptide comprises the amino acid sequence of 44877. In some embodiments, the targeting peptide comprises the amino acid sequence of 44878. In some embodiments, the targeting peptide comprises the amino acid sequence of 44879. In some embodiments, the targeting peptide comprises the amino acid sequence of 44880. In some embodiments, the targeting peptide comprises the amino acid sequence of 44881. In some embodiments, the targeting peptide comprises the amino acid sequence of 44882.In some embodiments, the targeting peptide comprises the amino acid sequence of 44883. In some embodiments, the targeting peptide comprises the amino acid sequence of 44911. In some embodiments, the targeting peptide comprises the amino acid sequence of 44912. In some embodiments, the targeting peptide comprises the amino acid sequence of 44913. In some embodiments, the targeting peptide comprises the amino acid sequence of 44918. In some embodiments, the targeting peptide comprises the 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 an amino acid sequence selected from SEQ ID NOs: 44859-44883, 44911, 44912, 44913, and 44918-44919.

[0467] 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%, or 99% sequence identity to an 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 the amino acid sequence of SEQ ID NO: 44864. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44865. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44866. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44867. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44879. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44880. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44881. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44882. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44883. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44911. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44912. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44913. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44918. In some embodiments, the targeting peptide comprises the amino acid sequence of SEQ ID NO: 44919.

[0468] In some embodiments, the targeting peptide comprises a sequence X7 selected from R, F, H, L, Q, R and Y. In some embodiments, X7 is R. In some embodiments, X7 is Y or H. In some embodiments, X7 is F or Y.

[0469] In some embodiments, X8 is selected from S, G, D, I, L, N, Q, T and V. In some embodiments, X8 is S or G. In some embodiments, X8 is S. In some embodiments, X8 is G. In some embodiments, X8 is T, G or S. In some embodiments, X8 is N or Q. In some embodiments, X9 is selected from S or V.

[0470] 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, X9 is selected from N or S.

[0471] In some embodiments, X 10 is selected from I, V, S, L, M, R, T and Q. In some embodiments, X 10 is I or V. In some embodiments, X 10 is I. X 10 is V. In some embodiments, X 10 is V, L, or M. In some embodiments, X 10 is T or L.

[0472] In some embodiments, the targeting peptide is RGDRX8X9X 10 In some embodiments, X8 is S or G. In some embodiments, X8 is S. In some embodiments, X8 is G. In some embodiments, X9 is selected from any amino acid residue. In some embodiments, X9 is selected from V, S, N, G and Q. In some embodiments, X9 is V. In some embodiments, X9 is S. In some embodiments, X9 is N. In some embodiments, X9 is G. In some embodiments, X9 is Q. In some embodiments, X 10 is selected from I, V, S, L and Q. In some embodiments, X 10 is I. In some embodiments, X 10 is V. In some embodiments, X10 is S. In some embodiments, X 10 is L. In some embodiments, X 10 is Q. In some embodiments, X 10 is I or V.

[0473] In some embodiments, the targeting peptide is selected from the group consisting 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: 98 935), X1X2X3RGDFNNT (SEQ ID NO: 98943), X1X2X3RGDYSSV (SEQ ID NO: 98937), 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).

[0474] 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).

[0475] In some embodiments, the targeting peptide has the 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).

[0476] In some embodiments, the targeting peptide has the 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).

[0477] In some embodiments, X1X2X3 is selected from the group consisting of EFK, AAY, DQK, QVY, DKL, DNV, ENF, EWK, QNV, and TFM.

[0478] In certain embodiments, the modified sequence comprises a 7-mer peptide selected from RGDRSX9I, RGDRGX9I, RGDRSX9V, or RGDRGX9V.

[0479] 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).

[0480] In some embodiments, X1X2X3 is 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, Q selected from the group consisting of VF, QVY, SDL, SDV, SEH, SII, SIY, SSL, SVY, SWD, SWE, TDF, TDV, TDY, TEF, TEY, TIY, TLY, and TWQ.

[0481] In some embodiments, X1X2X3 is selected from the group consisting of ETI, DQN, DLL, EKW, DNN, EYS, or TVF.

[0482] In some embodiments, X7 is selected from Y and H, X8 is selected from T, G and S, X9 is selected from S and V, and X 10 is selected from V, L and M.

[0483] 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).

[0484] In some embodiments, X1X2X3 is ANY, SNI, AAI, AAM, ANT, AST, AYQ, EHK, ENK, ENR, SFQ, SSI, TAY, TDK, TNT, AAF, AAL, AAY, A DK, 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.

[0485] In some embodiments, X1X2X3 is selected from TVF, APM, AQI, TTS, NQF, NFL, or SMN.

[0486] In some embodiments, X7 is selected from F and Y, X8 is selected from N and Q, X9 is selected from N and S, and X 10 is selected from T and L.

[0487] 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).

[0488] In some embodiments, X1X2X3 is 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, NY Q, 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, selected from the group consisting of SNA, SNE, SNF, SNI, SNQ, SNT, SRE, SST, SWQ, SWT, SYG, SYY, TFQ, THL, THQ, THV, TMI, TNL, TNS, TSH, TSQ, and TWQ.

[0489] In some embodiments, X1X2X3 is selected from the group consisting of TVF, APM, AQI, TTS, NQF, NFL, or SMN.

[0490] In certain embodiments, the modified sequence is RGDRGVX 10 (SEQ ID NO: 157058), RGDRGSX 10 (SEQ ID NO: 157059), RGDRGNX 10 (SEQ ID NO: 157060), RGDRGGX 10 (SEQ ID NO: 157061), RGDRGQX 10(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).

[0491] In certain embodiments, the targeting peptide comprises the sequence of H1 (RGDLIGR (SEQ ID NO: 1422)) or the sequence of H2 (RGDQSTL (SEQ ID NO: 3052)). In some embodiments, the targeting peptide comprises the sequence of the H1 (RGDLIGR (SEQ ID NO: 1422)) targeting peptide located in VR VIII between amino acids 588 and 589 or the sequence of the H2 (RGDQSTL (SEQ ID NO: 3052)) targeting peptide located in VR VIII between amino acids 588 and 589. In such embodiments, the modified AAV capsid protein comprising the H1 or H2 targeting peptide has increased tissue enrichment (tropism) in cardiac muscle tissue compared to skeletal muscle tissue.

[0492] In certain embodiments, the targeting peptide comprises the sequence of the S1 (RGDISRT (SEQ ID NO: 263)) targeting peptide located in VR VIII or the sequence of S2 (RGDRSQT (SEQ ID NO: 251)). In some embodiments, the targeting peptide comprises the sequence of the S1 (RGDISRT (SEQ ID NO: 263)) targeting peptide located in VR VIII between amino acids 588 and 589 or the sequence of the S2 (RGDRSQT (SEQ ID NO: 251)) targeting peptide located in VR VIII between amino acids 588 and 589. In such embodiments, the modified AAV capsid protein comprising the S1 or S2 targeting peptide has increased tissue enrichment (tropism) in skeletal tissue compared to myocardial tissue.

[0493] In certain embodiments, the modified sequence does not include a 7-mer peptide selected from RGDRMVF, RGDRTVI, SRGDRPM, and ISLRGDR. In some embodiments, the modified sequence does not include a 7-mer peptide having the amino acid sequence of SEQ ID NO: 1 (RGDLLLS). In certain embodiments, the modified sequence does not include a 7-mer peptide of RGDRMVF. In certain embodiments, the modified sequence does not include a 7-mer peptide of RGDRTVI. In certain embodiments, the modified sequence does not include a 7-mer peptide selected from SRGDRPM or ISLRGDR. In some embodiments, the modified sequence does not include a 7-mer peptide of RGDRMVF, RGDRTVI, SRGDRPM, and ISLRGDR. In some embodiments, the modified sequence does not include a 7-mer peptide of X1SLRGDR, where X1 is any amino acid residue. In some embodiments, the modified sequence does not include a 7-mer peptide of X1X2LRGDR, where X1 and X2 are independently any amino acid residue.

[0494] In some embodiments, the targeting peptide is 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, X1 is selected from S, E, A, D, N, Q, or T. In some embodiments, X1 is selected from S or E. In some embodiments, X1 is S. In some embodiments, X1 is E. In some embodiments, X2 is selected from N, A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. In some embodiments, X2 is selected from N or A. In some embodiments, X2 is N. In some embodiments, X2 is A. In some embodiments, X3 is selected from R, Q, A, D, E, F, G, H, I, K, L, M, N, P, S, T, V, W, or Y. In some embodiments, X3 is selected from R or Q. In some embodiments, X3 is Q. In some embodiments, X3 is R. In some embodiments, X2 is N and X3 is R. In some embodiments, X1 is E, X2 is N, and X3 is R. In some embodiments, X1 is S, X2 is N, and X3 is R.

[0495] In some embodiments, the modified AAV capsid protein further comprises one or more mutations outside of VRVIII. The mutations outside of VRVIII may include (1) an alanine (A) or glycine (G) amino acid residue at the amino acid position corresponding to position 266 in Anc80 VP1 and / or (2) a lysine (K) or arginine (R) amino acid residue at the amino acid position corresponding to position 168 in Anc80 VP1. The modified AAV capsid protein may include an alanine (A) amino acid residue at amino acid position 267 and a threonine (T) amino acid residue at amino acid position 269 in AAV9 VP1.

[0496] In some embodiments, the modified AAV capsid protein further comprises one or more deletions or substitutions within VRVIII. In some embodiments, the modified AAV capsid protein further comprises 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 comprises one or more deletions or substitutions within VRVIII followed by the insertion of a targeting peptide. In some embodiments, the modified AAV capsid protein further comprises one or more substitutions within VRVIII followed by the insertion of a targeting peptide. In some embodiments, the modified AAV capsid protein further comprises one or more substitutions within VRVIII. In some embodiments, the modified AAV capsid protein further comprises one or more substitutions between positions 565 and 595 of the reference capsid protein. In some embodiments, the modified AAV capsid protein further comprises one or more substitutions within VRVIII followed by the insertion of a targeting peptide.

[0497] In some embodiments, the targeting peptide can enhance targeting of AAV to the brain, muscle, spinal cord, eye, liver, heart, muscle, or other organs, hi some embodiments, the targeting peptide can decrease targeting of AAV to the brain, muscle, spinal cord, eye, liver, heart, muscle, or other organs.

[0498] Exemplary targeting peptide sequences that can be used in 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.

[0499] 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% sequence 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 of 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 an amino acid sequence selected from SEQ ID NOs: 44855-44858.

[0500] 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, X7 is 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, X 10 is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V and Y.

[0501] In some embodiments, X7 is 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, X8 is G. In some embodiments, X9 is selected from V, M, P, S, and D. In some embodiments, X9 is V. In some embodiments, X 10 is selected from S, N, L, H and M. In some embodiments, X 10 is S.

[0502] In some embodiments, X7 is L. In further embodiments, X8 is G. In further embodiments, X9 is L. In further embodiments, X 10 is S.

[0503] In some embodiments, X7 is A. In further embodiments, X8 is V. In further embodiments, X9 is G. In further embodiments, X 10 is V.

[0504] In some embodiments, X7 is L. In further embodiments, X8 is L. In further embodiments, X9 is L. In further embodiments, X 10 is S.

[0505] In some embodiments, X7 is L, X8 is selected from G, L and V, X9 is selected from L and G, and / or X 10 is selected from S, V and L. In a further embodiment, X7 is L, X8 is G or L, and / or X 10 is S. In certain embodiments, X8 and X9 are G or L.

[0506] In certain embodiments, X7, X8, and X9 are independently selected from L, V, and A, and at least two of X7, X8, and X9 are independently L. In some embodiments, X7, X8, and X9 are L. In certain embodiments, X8 is L.

[0507] In some embodiments, the targeting peptide is 10 and has the sequence X7X8X9X 10 is a quad selected for enhanced muscle targeting. In some embodiments, the targeting peptide is 10 and has the sequence X7X8X9X 10 is a quad selected for enhanced targeting to specific muscles (e.g., biceps, quadriceps, diaphragm, heart).

[0508] In some embodiments, the targeting peptide is 10 and has the sequence X7X8X9X 10 is a quad selected for enhanced targeting to skeletal muscle. In some embodiments, the quad is FNNL, YNSL, RQGI, FQNT, YVGL, YSSV, YTSM, RSVV, YTSV, RDYL, or FNNT.

[0509] In some embodiments, the targeting peptide is 10 and has the sequence X7X8X9X 10 is a quad selected for enhanced targeting to the myocardium. In some embodiments, the quad is LIGR, QSTL, or RGVV.

[0510] In some embodiments, the targeting peptide is 10 and has the sequence X7X8X9X 10 is a quad selected for enhanced liver targeting. In some embodiments, the quad is RGVV, RSVV, RGQI, RSQT, RQGI, FQNT, HGVL, YTSV, YTSM, LTVT, FNNT, YSSV, or HVNL.

[0511] In certain embodiments, the targeting peptide comprises 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) and / or a sequence containing at most two, preferably at most one, amino acid substitutions compared to one of the above 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).

[0512] In some embodiments, the targeting peptide comprises the sequence RGDLLLS (SEQ ID NO: 1).

[0513] 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.

[0514] 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 selected from SEQ ID NOs: 238-937. In some embodiments, the targeting peptide comprises a sequence selected from SEQ ID NOs: 238-1037.

[0515] In some embodiments, the targeting peptide is a targeting peptide disclosed in US2017 / 0166926, which is incorporated by reference in its entirety.

[0516] The targeting peptide can have any of the sequences provided herein selected from SEQ ID NOs: 2-51 and 53. In some embodiments, the targeting peptide is the 7-mer peptide TLAVPFK (SEQ ID NO: 53).

[0517] 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, the entireties of which are incorporated by reference herein. 6.2.2. Targeting Peptide Sites

[0518] The modified AAV capsid proteins of the present disclosure contain a targeting peptide within VR VIII of the reference AAV capsid protein (Figure 1).

[0519] Preferably, the targeting peptide is at a site exposed on the exterior of the capsid, preferably based on structural predictions and / or experimental data, and more preferably, the targeting peptide is at a site exposed on the exterior of the AAV capsid in a manner that does not interfere with the activity of said protein in capsid assembly.

[0520] The position of a targeting peptide in an AAV capsid protein that "corresponds" to a position in the AAV9 capsid protein can be established by those skilled in the art by known methods, preferably by aligning the amino acids of the capsid protein. In some embodiments, the position of the targeting peptide corresponds to amino acid 588 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a position corresponding to amino acid 580 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a position corresponding to amino acid 581 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a position corresponding to amino acid 582 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a position corresponding to amino acid 583 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a position corresponding to amino acid 584 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 588 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 585 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 586 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 587 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 588 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 587 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 586 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 585 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 589 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is located at a site corresponding to amino acid position 590 of the AAV9 VP1 capsid protein.In other words, X1 of the targeting peptide is at a site corresponding to amino acid 591 of the AAV9 VP1 capsid protein. In other words, X1 of the targeting peptide is at a site corresponding to amino acid 592 of the AAV9 VP1 capsid protein.

[0521] The location of the targeting peptide can be any one of those described in WO2019 / 207132, which is incorporated by reference in its entirety. Some of the sites are provided below in Table 1 and highlighted in Figures 2A-2C and 3A-3D. In Table 1, preferred sites are indicated by "-" relative to the wild-type VP1 capsid polypeptide. [Table 1-1] [Table 1-2]

[0522] Table 1 shows three exemplary insertion sites. For insertion sites 1 and 2, the targeting peptide is inserted between two amino acid positions, and the insertion site is indicated 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 mutation or substitution of one or more amino acids, followed by insertion of the targeting peptide. For example, for insertion site 3 in Table 1, the three amino acids located between the insertion sites are substituted or mutated before insertion of the targeting peptide. For example, for AAV1 capsid protein insertion site 3, the targeting peptide is inserted between positions Q585 and T589 after the amino acids "SSS" (S586, S587, and S588) located between Q585 and T589 are deleted, and is indicated as "Q-SSS-T".

[0523] In some embodiments, the targeting peptide is between 560 and 600 in VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is between 565 and 595 in VR VIII of the modified AAV capsid protein.

[0524] In some embodiments, the targeting peptide is between 570 and 610 in VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is between 580 and 610 in VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is between 580 and 595 in VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is between 582 and 600 in VR VIII of the modified AAV capsid protein.

[0525] In some embodiments, the reference AAV capsid protein is an AAV1 capsid protein 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 an AAV1 capsid protein 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 an AAV1 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0526] In some embodiments, the reference AAV capsid protein is an AAV2 capsid protein 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 an AAV2 capsid protein 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 an AAV2 capsid protein or a modification thereof, and the targeting peptide is between Q584 and R588.

[0527] In some embodiments, the reference AAV capsid protein is an AAV3 capsid protein 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 an AAV3 capsid protein 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 an AAV3 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0528] In some embodiments, the reference AAV capsid protein is an AAV4 capsid protein, 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 an AAV4 capsid protein or a modification thereof, and the targeting peptide is between 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 an AAV4 capsid protein or a modification thereof, and the targeting peptide is between G581 and N585 of the modified AAV capsid protein.

[0529] In some embodiments, the reference AAV capsid protein is an AAV5 capsid protein 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 an AAV5 capsid protein 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 an AAV5 capsid protein, and the targeting peptide is between Q574 and T589 of the reference AAV capsid protein.

[0530] In some embodiments, the reference AAV capsid protein is an AAV6 capsid protein 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 an AAV6 capsid protein 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 an AAV6 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0531] In some embodiments, the reference AAV capsid protein is an AAV7 capsid protein 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 an AAV7 capsid protein 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 an AAV7 capsid protein or a modification thereof, and the targeting peptide is between Q586 and T590 of the modified AAV capsid protein.

[0532] In some embodiments, the reference AAV capsid protein is an AAV8 capsid protein, and the targeting peptide is between N590 and T591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is an AAV8 capsid protein 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 an AAV8 capsid protein or a modification thereof, and the targeting peptide is between Q587 and T591 of the modified AAV capsid protein.

[0533] In some embodiments, the reference AAV capsid protein is an AAV9 capsid protein, and the targeting peptide is between Q588 and A589 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is an AAV9 capsid protein 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 an AAV9 capsid protein or a modification thereof, and the targeting peptide is between Q585 and A589 of the modified AAV capsid protein.

[0534] In some embodiments, the reference AAV capsid protein is an AAVrhlO capsid protein 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 an AAVrhlO capsid protein 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 an AAVrhlO capsid protein or a modification thereof, and the targeting peptide is between Q587 and A591 of the modified AAV capsid protein.

[0535] In some embodiments, the reference AAV capsid protein is an AAVpo.1 capsid protein 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 an AAVpo.1 capsid protein 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 an AAVpo.1 capsid protein or a modification thereof, and the targeting peptide is between N564 and S568 of the modified AAV capsid protein.

[0536] In some embodiments, the reference AAV capsid protein is an AAV12 capsid protein 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 an AAV12 capsid protein 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 an AAV12 capsid protein or a modification thereof, and the targeting peptide is between N589 and A593 of the modified AAV capsid protein.

[0537] In some embodiments, the reference AAV capsid protein is an Anc80 capsid protein 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 an Anc80 capsid protein 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 an Anc80 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0538] In some embodiments, the reference AAV capsid protein is an Anc80L65 capsid protein 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 an Anc80L65 capsid protein 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 an Anc80L65 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0539] In some embodiments, the reference AAV capsid protein is an Anc80-55 capsid protein 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 an Anc80-55 capsid protein 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 an Anc80-55 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0540] In some embodiments, the reference AAV capsid protein is an Anc80-129 capsid protein 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 an Anc80-129 capsid protein 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 an Anc80-129 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0541] In some embodiments, the reference AAV capsid protein is an Anc80-156 capsid protein 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 an Anc80-156 capsid protein 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 an Anc80-156 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0542] In some embodiments, the reference AAV capsid protein is an Anc80-751 capsid protein 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 an Anc80-751 capsid protein 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 an Anc80-751 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0543] In some embodiments, the reference AAV capsid protein is an Anc80-1029 capsid protein 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 an Anc80-1029 capsid protein 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 an Anc80-1029 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein.

[0544] In some embodiments, the reference AAV capsid protein is an Anc80-1712 capsid protein 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 an Anc80-1712 capsid protein 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 an Anc80-1712 capsid protein or a modification thereof, and the targeting peptide is between Q585 and T589 of the modified AAV capsid protein. 6.2.3. Various Embodiments of Modified AAV Capsid Proteins

[0545] The present disclosure provides modified AAV capsid proteins, including a reference AAV capsid protein having one or more modifications to include a targeting peptide at a site within VR VIII of the reference AAV capsid protein, wherein the targeting peptide has the sequence X1X2X3RGDX7X8X9X 10 In some embodiments, the modified AAV capsid protein comprises a targeting peptide selected from SEQ ID NOs: 238-44858 introduced into 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 an amino acid sequence selected from SEQ ID NOs: 238-44858 introduced into VR VIII.

[0546] In some embodiments, the modified AAV capsid protein is an AAV9 capsid protein containing a targeting peptide, RGDLLLS (SEQ ID NO: 1), inserted in VR VIII. In one embodiment, the modified AAV capsid protein has the sequence of SEQ ID NO: 158. In some embodiments, the modified AAV capsid protein has amino acids 138-736 of SEQ ID NO: 158. In some embodiments, the modified AAV capsid protein has amino acids 203-736 of SEQ ID NO: 158. In some embodiments, the modified AAV capsid protein has an 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. In some embodiments, the modified AAV capsid protein has a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 44900-44909.

[0547] In some embodiments, the modified AAV capsid protein has a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:158.

[0548] In another aspect, the 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 a liver-toggle mutant.

[0549] In one embodiment, the modified AAV capsid protein is an AAV9 capsid protein containing a targeting peptide, RGDLLLS (SEQ ID NO: 1), inserted into VR VIII. The modified AAV capsid protein may include one or more additional modifications to include: (i) an alanine (A) amino acid residue at the amino acid position corresponding to position 266 in Anc80, or b) a lysine (K) amino acid residue at the amino acid position corresponding to position 168 in Anc80. In one embodiment, the modified AAV capsid protein has the sequence of SEQ ID NO: 159. In some embodiments, the modified AAV capsid protein has amino acids 138-736 of SEQ ID NO: 159. In some embodiments, the modified AAV capsid protein has amino acids 203-736 of SEQ ID NO: 159.

[0550] In some embodiments, the modified AAV capsid protein has a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:159.

[0551] The modified AAV capsid proteins of the present disclosure can alter the tropism, specificity, and / or biodistribution of AAVs comprising the modified AAV capsid proteins. In preferred embodiments, AAVs comprising the modified AAV capsid proteins have increased targeting to target cells, tissues, or organs when administered to a subject. In some embodiments, AAVs comprising the modified AAV capsid proteins have reduced distribution outside of target cells, tissues, or organs when administered to a subject. In some embodiments, the tropism of modified AAV capsid proteins 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:

[0552] Tissue enrichment score = log2 fold change = log2 MN 組織 -log2MN 試験品

[0553] The formula for scaled log2 fold change is provided below:

[0554] Scaled log2 fold change = (log2 fold change - Min(log2 fold change)) / (Max(log2 fold change) - Min(log2FC))

[0555] 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

[0556] 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, or may be a VP1, VP2, or VP3 capsid protein of a naturally occurring or non-naturally occurring AAV variant.

[0557] Non-naturally occurring VP1, VP2, or VP3 capsid proteins include capsid proteins generated by biological or chemical modification or computer design, or variants of naturally occurring AAV capsid proteins. Thus, reference AAV capsid proteins include, but are not limited to, capsid proteins of various AAV serotypes (e.g., AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9) or variants thereof. Non-naturally occurring VP1, VP2, or VP3 capsid proteins also include artificial capsid proteins created by computer design or synthesis. Artificial capsid proteins include, but are not limited to, the 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 by reference in their entireties.

[0558] In some embodiments, the reference AAV capsid protein is selected from the group consisting 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: AAN03857.1), AAV11 (Genbank Ace. No: AAN03857.1), AAV12 (Genbank Ace. No: AAN03857.1), AAV13 (Genbank Ace. No: AAN03857.1), AAV14 (Genbank Ace. No: AAN03857.1), AAV15 (Genbank Ace. No: AAN03857.1), AAV16 (Genbank Ace. No: AAN03857.1), AAV17 (Genbank Ace. No: AAN03857.1), AAV18 (Genbank Ace. No: AAN03857.1), AAV19 (Genbank Ace. No: AAN03756.1), AAV20 (Genbank Ace. No: AAN03756.1), AAV21 (Genbank Ace. No: AAN03857.1), AAV22 (Genbank Ace. No: AAN 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) capsid protein. In certain embodiments, the AAV capsid protein is the capsid protein of AAV9 (Genbank accession number AAS99264.1).

[0559] Reference AAV capsid proteins are 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.49 (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 The reference AAV capsid protein may be a VP1 capsid protein having a sequence selected from the group consisting of AAV9 VP1 (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 may be a VP2 or VP3 protein having a portion of one of the sequences. For example, the VP2 protein may have a sequence corresponding to amino acids 138-736 of AAV9 VP1, and the VP3 protein may have a sequence corresponding to amino acids 138-736 of AAV9 VP1.

[0560] The reference AAV capsid protein can be a VP1 capsid protein having any member sequence of an 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 portion of one of the sequences. For example, a VP2 protein can have a sequence corresponding to amino acids 138-736 of AAV9 VP1, and a VP3 protein can have a sequence corresponding to amino acids 138-736 of AAV9 VP1. When a SEQ ID NO of a library sequence is used in this disclosure, it refers to the sequence of any one member of the library.

[0561] 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.

[0562] 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; 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 the capsid protein of any member protein of an ancestral AAV library selected from Anc80, Anc81, Anc82, Anc83, Anc84, Anc94, Anc113, Anc126 and Anc127.

[0563] 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 138-736 of AAV9 VP1) or VP3 portion (corresponding to amino acids 138-736 of AAV9 VP1) of a protein having a sequence selected from SEQ ID NOs: 54-131 and 143-152.

[0564] In some embodiments, the reference AAV capsid protein is a capsid protein of an AAV variant modified to include one or more liver-toggle mutations described in WO2019 / 217911. In some embodiments, the reference AAV capsid protein includes (1) an alanine (A) or glycine (G) amino acid residue at the amino acid position corresponding to position 266 in Anc80 VP1 and / or (2) a lysine (K) or arginine (R) amino acid residue at the amino acid position corresponding to position 168 in Anc80 VP1. In some embodiments, the reference AAV capsid protein includes (i) an alanine (A) amino acid residue at the amino acid position corresponding to position 266 in Anc80 VP1 and / or (b) a lysine (K) amino acid residue at the 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 the amino acid position corresponding to position 267 in the AAV9 VP1 protein and / or a threonine (T) amino acid residue at the amino acid position corresponding to position 269 in AAV9 VP1.

[0565] 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 having at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 44885-44898, 44916-44917, or a fragment thereof.

[0566] In some embodiments, the reference AAV capsid protein comprises one or more modifications described in WO2019 / 217911 or WO2021 / 050614, both of which are incorporated by reference in their entireties. In some embodiments, the reference AAV capsid protein comprises one or more modifications described in PCT Application No. PCT / US2022 / 015842, which is incorporated by reference in its entirety. 6.2.5. Peptide Segments

[0567] 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 peptide segment within VR I that alters its tropism. In some embodiments, the modified AAV capsid protein comprises a 12 amino acid peptide segment (P1P2P3P4P5P6P7P8P9P) within VR I that differs from a reference AAV capsid protein. 10 P 11 P 12 In some embodiments, only one amino acid of the 12 amino acids differs from the amino acid at the corresponding position in a reference AAV capsid protein. In some embodiments, more than one amino acid residue in the peptide segment differs from the amino acid residue at the corresponding position in a reference AAV capsid protein.

[0568] In some embodiments, the peptide segment comprises 12 amino acids located between about amino acid 259 and 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 located between positions 261 and 274 in a reference AAV capsid protein or between positions 260 and 273 in a reference AAV capsid protein (P1P2P3P4P5P6P7P8P9P 10 P 11 P 12 In some embodiments, the peptide segment comprises 11 amino acids (P2P3P4P5P6P7P8P9P) between about amino acid 259 and about amino acid 275 in a reference AAV capsid protein (e.g., any of the reference AAV capsid proteins described herein). 10 P 11 P 12 In some embodiments, the peptide segment comprises 10 amino acids between about amino acid 259 and about amino acid 275 in a reference AAV capsid protein (e.g., any of the reference AAV capsid proteins described herein).

[0569] In some embodiments, P1P2P3P4P5P6P7P8P9P 10 P 11 P 12 One or more amino acids in are identical to one or more amino acids at corresponding positions in a reference AAV capsid protein.

[0570] 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.

[0571] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment is P1P2P3P4P5P6P7P8NDNP 12 and P1, P2, P3, P4, P5, P6, P7, P8 and P 12 are independently selected from any amino acid residue.

[0572] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), the peptide segment being P1P2P3P4P5P6P7P8NDNP 12 and P1, P2, P3, P4, P5, P6, P7, P8 and P 12 are independently selected from any amino acid residue.

[0573] In some embodiments, the modified AAV capsid protein is P1P2P3P4P5P6P7P8NDNP 12 and (i) P1 is independently selected from asparagine (N), serine (S), or threonine (T); (ii) P2 is independently selected from serine (S) or glycine (G); (iii) P3 is independently selected from threonine (T), glutamine (Q), alanine (A), or glutamic acid (E); (iv) P4 is independently selected from serine (S), threonine (T), or alanine (A); (v) P5 is independently selected from glycine (G) or alanine (A); (vi) P6 is independently selected from glycine (G) or alanine (A); (vii) P7 is independently selected from alanine (A) or serine (S); (viii) P8 is independently selected from serine (S) or threonine (T); (ix)P 12 are independently selected from histidine (H), threonine (T) or alanine (A).

[0574] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment is 10 P 11 P 12 and having the amino acid sequence 10 and P 11 or combinations thereof are unaltered compared to a reference AAV capsid protein.

[0575] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a peptide segment within variable region I (VR I), the peptide segment being P1P2P3P4P5P6P7P8P9P 10 P 11 P 12 and having the amino acid sequence 10 and P 11 or combinations thereof are unaltered compared to a reference AAV capsid protein.

[0576] In another aspect, the disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has the amino acid sequence P1P2P3P4GGP7P8NDNP 12 (SEQ ID NO: 44921), and 12 are independently selected from any amino acid residue.

[0577] 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), the peptide segment having the amino acid sequence P1P2P3P4GGP7P8NDNP 12 (SEQ ID NO: 44921), and 12 are independently selected from any amino acid residue.

[0578] In some embodiments, the modified AAV capsid protein is P1P2P3P4GGP7P8NDNP 12 (SEQ ID NO: 44921), (i) P1 is independently selected from asparagine (N) or serine (S); (ii) P2 is independently selected from serine (S) or glycine (G); (iii) P3 is independently selected from threonine (T) or glutamine (Q); (iv) P4 is independently selected from serine (S), threonine (T), or alanine (A); (v) P7 is independently selected from alanine (A) or serine (S); (vi) P8 is independently selected from serine (S) or threonine (T); (vii)P 12 are independently selected from histidine (H), threonine (T) or alanine (A).

[0579] In some embodiments, the modified AAV capsid protein is P1P2TP4GGP7P8NDNP 12 (SEQ ID NO: 44922), and may include peptide segments having the sequence P1, P2, P4, P7, P8 and P 12 are independently selected from any amino acid residue.

[0580] In some embodiments, the modified AAV capsid protein is P1P2QP4GGP7P8NDNP 12(SEQ ID NO: 44923), and may include peptide segments P1, P2, P4, P7, P8, and P 12 are independently selected from any amino acid residue.

[0581] In some embodiments, the modified AAV capsid protein is P1P2TP4GGP7TNDNP 12 (SEQ ID NO: 44924), and may include peptide segments P1, P2, P4, P7, and P 12 are independently selected from any amino acid residue.

[0582] In some embodiments, the modified AAV capsid protein is NP2TP4GGP7P8NDNP 12 (SEQ ID NO: 44925), and may include peptide segments P2, P4, P7, P8, and P 12 are independently selected from any amino acid residue.

[0583] In some embodiments, the modified AAV capsid protein is SP2TP4GGP7P8NDNP 12 (SEQ ID NO: 44926), and may include peptide segments P2, P4, P7, P8, and P 12 are independently selected from any amino acid residue.

[0584] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment is 10 P 11 P 12wherein positions P5, P6, or a combination thereof are not modified compared to a reference AAV capsid protein. In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment is 10 P 11 P 12 and the amino acid sequence is 10 and P 11 or combinations thereof are unaltered compared to a reference AAV capsid protein.

[0585] In another aspect, the disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has the amino acid sequence of NSTSGGP7P8NDNH (SEQ ID NO: 44927), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or 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 the amino acid sequence of NSTSGGP7P8NDNH (SEQ ID NO: 44927), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T). In some embodiments, the peptide segment comprises (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 alterations (e.g., insertions, deletions, mutations, and / or substitutions).

[0586] In another aspect, the disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has the amino acid sequence of NSTTGGP7P8NDNH (SEQ ID NO:44928), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or 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 the amino acid sequence of NSTTGGP7P8NDNH (SEQ ID NO:44928), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T). In some embodiments, the peptide segment comprises (i) NSTTGGASNDNH (SEQ ID NO: 46073), (ii) NSTTGGATNDNH (SEQ ID NO: 46076), (iii) NSTTGGSSNDNH (SEQ ID NO: 46079), (iv) NSTTGGSTNDNH (SEQ ID NO: 46082), or a corresponding sequence having one or more modifications (e.g., insertions, deletions, mutations, and / or substitutions).

[0587] In another aspect, the disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein having one or more modifications to include 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), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or 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), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T). In some embodiments, the peptide segment comprises (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., insertions, deletions, and / or substitutions).

[0588] In another aspect, the disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein having one or more modifications to include 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), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or 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), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T). In some embodiments, the peptide segment has the sequence of (i) SGTAGGASNDNT (SEQ ID NO: 46554), (ii) SGTAGGSSNDNT (SEQ ID NO: 46560), or a corresponding sequence having one or more modifications (e.g., insertions, deletions, and / or substitutions). In some embodiments, the peptide segment does not comprise SGTAGGATNDNT (SEQ ID NO: 46557) or SGTAGGSTNDNT (SEQ ID NO: 46563).

[0589] In another aspect, the disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has the amino acid sequence of SGTSGGP7P8NDNA (SEQ ID NO:44931), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or 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 the amino acid sequence of SGTSGGP7P8NDNA (SEQ ID NO:44931), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T). In some embodiments, the peptide segment comprises (i) SGTSGGASN DNA (SEQ ID NO: 46600), (ii) SGTSGGATN DNA (SEQ ID NO: 46600), (iii) SGTSGSSN DNA (SEQ ID NO: 46603), (iv) SGTSGSTN DNA (SEQ ID NO: 46609), or a corresponding sequence having one or more modifications (e.g., insertions, deletions, and / or substitutions).

[0590] In another aspect, the disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein having one or more modifications to include 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), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or 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), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T). In some embodiments, the peptide segment comprises (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., insertions, deletions, and / or substitutions). In some embodiments, the peptide segment is SGTTGGSSNDNT (SEQ ID NO: 46656).

[0591] In another aspect, the disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein, wherein the peptide segment has the amino acid sequence of SSTAGGP7P8NDNA (SEQ ID NO: 44933), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or 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 the amino acid sequence of SSTAGGP7P8NDNA (SEQ ID NO: 44933), and wherein P7 and P8 are independently selected from any amino acid residue. In some embodiments, P7 is independently selected from alanine (A) or serine (S), and P8 is independently selected from serine (S) or threonine (T). In some embodiments, the peptide segment comprises (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., insertions, deletions, and / or substitutions). In some embodiments, the peptide segment is SSTAGGASNDNA (SEQ ID NO: 47128). In some embodiments, the peptide segment is SSTAGGATNDNA (SEQ ID NO: 47131).

[0592] In some embodiments, the peptide segment is NSTSGASTNDNA (SEQ ID NO: 48390).

[0593] In some embodiments, the peptide segment is a selected peptide segment shown in Table 20.

[0594] In some embodiments, the modified AAV capsid proteins of the present disclosure can alter the tropism, specificity, and / or biodistribution of an AAV comprising the modified AAV capsid protein. In some embodiments, the AAV comprising the modified AAV capsid protein has increased targeting to target cells, tissues, or organs when administered to a subject. In some embodiments, the AAV comprising the modified AAV capsid protein has decreased distribution outside of target cells, tissues, or organs when administered to a subject.

[0595] In some embodiments, the 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 is P1P2P3P4P5P6P7P8P9P 10 P 11 P 12 Figure 52A provides an exemplary enrichment score formula, which is reproduced below. Enrichment score = a0 + a1X1T + a2X1S + a3X2S + a4X3E + a5X3Q + a5X3T +... + a 13 X 12 H+a_ 14 X 12 T where X1, for example, refers to amino acid position 1 (i.e., P1), a refers to the calculated index of the assigned amino acid residue at that amino acid position, and "T", "S", "E", "Q", "H", and "T" refer to the amino acid residue at the X amino acid position. Variable Region I (VR I)

[0596] In some embodiments, variable region I (VR I) corresponds to a sequence between about position 259 and about 275 in a modified AAV capsid protein. In some embodiments, variable region I (VR I) corresponds to a sequence between about position 259 and about 275 in a reference AAV capsid protein (e.g., any of the reference AAV capsid proteins described herein (see, e.g., Section 4.2.4)).

[0597] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a reference AAV capsid protein having one or more modifications to include a peptide segment within variable region I (VR I) of the reference AAV capsid protein.

[0598] In some embodiments, the peptide segment is located between S261 and Y274 of the AAV9 capsid protein (SEQ ID NO: 61).

[0599] In some embodiments, the peptide segment is at position c between S260 and Y273 of the Anc80 capsid protein (SEQ ID NO: 132).

[0600] In some embodiments, the peptide segment is located between S260 and Y273 of the Anc80L65 capsid protein (SEQ ID NO: 142).

[0601] In some embodiments, the peptide segments are at positions S260 and Y273 of the AAV2 capsid protein (SEQ ID NO: 55). 6.2.7. Other Modifications

[0602] In some embodiments, the modified AAV capsid protein comprises one or more additional modifications compared to a reference AAV capsid protein, which may be an insertion, deletion, substitution, or a combination thereof, and may be located within or outside of VRVIII.

[0603] In some embodiments, the modified AAV capsid protein differs from the reference AAV capsid protein by having one or more amino acid substitutions in the variable region of the reference AAV capsid protein, hi some embodiments, the one or more amino acid substitutions are in the variable region, VR I, of the reference AAV capsid protein (Figure 1).

[0604] Modified AAV capsid proteins can be biologically or chemically produced. Modified AAV capsid proteins can have different tropism, specificity or localization than reference AAV capsid proteins, particularly in the liver, when administered to mammalian subjects. Mammalian subjects can be humans, non-human primates (NHPs), mice, rats, birds, rabbits, guinea pigs, hamsters, livestock (including pigs and sheep), dogs or cats.

[0605] In some embodiments, the modified AAV capsid protein comprises a sequence that differs 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.

[0606] In some embodiments, the modified AAV capsid protein comprises a sequence that differs 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 that differs 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.

[0607] In some embodiments, the modified AAV capsid protein comprises a sequence that differs from a reference AAV capsid protein by having a glycine (G) amino acid residue at the amino acid position corresponding to position 266 in Anc80 or an arginine (R) amino acid residue at the amino acid position corresponding to position 168 in Anc80. In some embodiments, the modified AAV capsid protein comprises a sequence that differs from a reference AAV capsid protein by having a glycine (G) amino acid residue at the amino acid position corresponding to position 266 in Anc80 and an arginine (R) amino acid residue at the amino acid position corresponding to position 168 in Anc80.

[0608] The amino acid positions corresponding to positions 266 in Anc80 VP1 and 168 in Anc80 VP1 in the various VP1 protein sequences are indicated by boxes in Figures 3A-C and 4A-D.

[0609] In some embodiments, the modified AAV capsid protein differs from the reference AAV capsid protein, other than by the targeting peptide, 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. In some embodiments, the modified AAV capsid protein differs from the reference AAV capsid protein, other than by the targeting peptide, only at two amino acid positions—the amino acid position corresponding to position 266 in Anc80 VP1 and the amino acid position corresponding to position 168 in Anc80 VP1. In some embodiments, the modified AAV capsid protein differs from the reference AAV capsid protein by more than two amino acid substitutions.

[0610] In some embodiments, the modified AAV capsid protein comprises a sequence that differs from a reference AAV capsid protein by having an alanine (A) amino acid residue at the amino acid position corresponding to position 267 in the AAV9 VP1 protein or a threonine (T) amino acid residue at the amino acid position corresponding to position 269 in AAV9 VP1. In some embodiments, the modified AAV capsid protein comprises a sequence that differs from a reference AAV capsid protein by having an alanine (A) amino acid residue at the amino acid position corresponding to position 267 in the AAV9 VP1 protein and a threonine (T) amino acid residue at the amino acid position corresponding to position 269 in AAV9 VP1. In some embodiments, the modified AAV capsid protein differs from the reference AAV capsid protein only at the amino acid position corresponding to position 267 in the AAV9 VP1 protein or the amino acid position corresponding to position 269 in AAV9 VP1, other than the targeting peptide. In some embodiments, the modified AAV capsid protein differs from the reference AAV capsid protein at only two amino acid positions other than the targeting peptide - the amino acid position corresponding to position 267 in the AAV9 VP1 protein and the amino acid position corresponding to position 269 in AAV9 VP1.

[0611] In some embodiments, the reference AAV capsid is the AAV capsid protein disclosed in WO2019 / 217911, which is incorporated by reference in its entirety.

[0612] In particular, the AAV capsid proteins described herein for generating "liver-off" ("liver-detargeted") AAV can be used in embodiments herein. In other embodiments, the AAV capsid proteins described herein for generating "liver-on" ("liver-targeted") AAV can be used herein.

[0613] In some emb...

Claims

1. A modified adeno-associated virus (AAV) capsid protein, (i) Targeted peptide within VR VIII, (ii) Peptide segment within VR I Includes, The targeted peptide has the sequence X1 X2 X3 RGDX7 X8 X9 X10, and the peptide segment has the amino acid sequence P1 P2 P3 P4 P5 P6 P7 P8 NDNP12. X1 is S, A, or E, X2 is A, S, or N, X3 is R, Q, K, or S, X7 is R, F, or Y, X 8 is G, T, or N, X 9 is S, N, or Q, X 10 is I, L, or M, P1 is N or S, P2 is S or G, P3 is T, P4 is either S or T, P5 is G, P6 is G or A, P7 is S, P8 is either S or T, A modified adeno-associated virus (AAV) capsid protein in which P12 is either T or A.

2. The modified AAV capsid protein according to claim 1, having at least 90% sequence identity with respect to the sequence of a reference AAV capsid protein.

3. The modified AAV capsid protein according to claim 1, wherein the AAV capsid protein is selected from VP1, VP2, and VP3. **Claim 4**: The reference AAV capsid protein according to claim 2, which 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.6l-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; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc110; and Anc80DI.

5. The modified AAV capsid protein according to claim 2, wherein the reference AAV capsid protein is a capsid protein having the sequence or fragment thereof of Sequence ID No.

61.

6. The modified AAV capsid protein according to claim 1, wherein X1, X2, X3 or any combination thereof are the same amino acids as the amino acids at the corresponding positions of the reference AAV capsid protein.

7. The modified AAV capsid protein according to claim 1, wherein X1, X2, and X3 are ENR, SAQ, ASS, or ENK.

8. The modified AAV capsid protein according to claim 1, wherein X7 X8 X9 X10 is YSTM, FNNL, or RGQI.

9. The modified AAV capsid protein according to claim 2, wherein the reference AAV capsid protein is the capsid protein of AAV9 or a variant thereof, and the targeted peptide is inserted between amino acids 585 and 589 such that the amino acids at positions 586, 587, and 588 are replaced by three amino acids from the targeted peptide.

10. The peptide segment is (i) NSTSGGGSSNDNA (Sequence ID 48388), (ii) NSTSGASTNDNA (Sequence ID 48390), or (iii) SGTGGGSNDNT (Sequence ID 46656) A modified AAV capsid protein according to claim 1, comprising:

11. (i) The targeted peptide has the amino acid sequence ENKRGDYTSM (SEQ ID NO: 91413), (ii) The peptide segment has the amino acid sequence NSTSGGGSSNDNA (SEQ ID NO: 48388), The modified AAV capsid protein according to claim 1.

12. (i) The targeted peptide has the amino acid sequence ENRRGDFNNL (SEQ ID NO: 44864), (ii) The peptide segment has the amino acid sequence NSTSGGGSSNDNA (SEQ ID NO: 48388), The modified AAV capsid protein according to claim 1.

13. (i) The targeted peptide has the amino acid sequence SAQRGDRGQI (SEQ ID NO: 44911), (ii) The peptide segment has the amino acid sequence NSTSGASTNDNA (SEQ ID NO: 48390), The modified AAV capsid protein according to claim 1.

14. (i) The targeted peptide has the amino acid sequence ASSRGDFNNL (SEQ ID NO: 81877), (ii) The peptide segment has the amino acid sequence SGTGGGSNDNT (SEQ ID NO: 46656), The modified AAV capsid protein according to claim 1.

15. A polynucleotide encoding the modified AAV capsid protein described in Claim 1.

16. A vector comprising the polynucleotide described in Claim 15.

17. A host cell comprising the modified AAV capsid protein described in Claim 1.

18. Recombinant AAV virion (rAAV) comprising the modified AAV capsid protein described in Claim 1.

19. A pharmaceutical composition comprising the modified AAV capsid protein described in Claim 1.

20. A composition for treating, alleviating, or preventing a disease or condition in a subject, comprising the rAAV virion described in Claim 18.

21. A composition for transferring exogenous polynucleotides into muscle cells, comprising the rAAV virion described in Claim 18.