Recombinant AAVs with improved tropism and specificity

Abstract

The present disclosure provides a modified AAV capsid protein containing a targeting peptide in variable region VIII (VR VIII). The modified AAV capsid protein can form a rAAV with preferred tropism, specificity, or biodistribution in vivo or in vitro. The rAAV of the present disclosure can be used for gene therapy targeted to specific tissues. The targeting peptide has the sequence X1X2X3X4X5X6X7X8X9, where X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently selected from any amino acid residue.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 380,170, filed October 19, 2022, No. 63 / 482,874, filed February 2, 2023, and U.S. Provisional Application No. 63 / 502,871, filed May 17, 2023, each of which is incorporated herein by reference in its entirety.

[0002] 2. Sequence Listing This application contains a Sequence Listing with 55,921 sequences, which has been submitted via the Patent Center and is incorporated herein by reference in its entirety. The XML copy created on October 16, 2023 is named 53023WO_CRF_sequencelisting.xml and is 48,736,195 bytes in size.

[0003] This application also incorporates by reference in its entirety the appendices entitled "Appendix A" and "Appendix B," and the sequences set forth therein. Appendices A and B are being filed concurrently herewith. [Background technology]

[0004] 3.Background Adeno-associated virus (AAV) has become the vector system of choice for in vivo gene therapy. An increasing variety of recombinant AAVs (rAAVs) engineered to deliver therapeutic nucleic acids have been developed and tested in non-human primates and humans, and the FDA recently approved two rAAV gene therapy products for commercialization.

[0005] Although AAV vectors are safer and less inflammatory than other viruses, toxicity has been observed 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.

[0006] 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 differ 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 is the insertion of small peptides into an exposed loop of the capsid protein called variable region VIII (VRVIII). In some situations, inserting novel peptides into the wild-type capsid alters the tropism of the variants.

[0007] However, to date, there is little understanding of how changes in the AAV capsid alter their biological properties, and AAV vectors with the desired tropism and specificity for therapeutic targets such as the central nervous system (CNS) are not yet available. Species-specific differences in AAV tropism between mice and nonhuman primates (NHPs), for example, make it difficult to develop AAV vectors with the desired tropism in humans.

[0008] The treatment of CNS disease remains a difficult problem.Currently, the therapeutic agents for CNS disease are limited because many of them do not cross the blood-brain barrier when delivered intravenously, or do not distribute widely when delivered directly to brain.Therefore, there is a need for the AAV vector that has good and specific tropism to CNS for the treatment of such CNS disease. Summary of the Invention

[0009] 4. Summary of the Invention The present disclosure provides modified AAV capsid proteins capable of forming rAAVs with preferred therapeutic target tropism and specificity. In some embodiments, the modified adeno-associated virus (AAV) capsid protein with preferred tropism comprises (i) a targeting peptide at a site within variable region VIII (VR VIII), the targeting peptide having a sequence of X1X2X3X4X5X6X7X8X9, where X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently selected from any amino acid residue. In some embodiments, the modified capsid protein comprising a targeting peptide at a site within variable region VIII (VR VIII) having a sequence of X1X2X3X4X5X6X7X8X9 comprises a deletion of amino acid residues (e.g., A587 and Q588 of the AAV9 capsid).

[0010] Applicant has previously demonstrated that a single injection of Anc80L65, a rationally designed synthetic vector (the vector described in WO 2015 / 054653, which is incorporated by reference in its entirety), into the CSF of adult cynomolgus monkeys resulted in efficient transduction of large areas of the CNS, significantly exceeding the ability of AAV9 to target the cortex and deep brain nuclei (PCT Application No. PCT / US2022 / 024262, which is incorporated by reference in its entirety).

[0011] Applicant reports herein that modified AAV capsid proteins (e.g., Anc80L65, AAV9, and other AAV capsid proteins) containing a targeting peptide within variable region VIII (VR VIII) provide a synergistic effect on the specific targeting of rAAV to target tissues (e.g., the CNS). Thus, modified AAV capsid proteins of the present disclosure (e.g., modified AAV capsid proteins containing a targeting peptide described herein inserted into an insertion site described herein) can alter the tropism, specificity, and / or biodistribution of AAVs containing the modified AAV capsid proteins.

[0012] Overall, rAAVs containing modified AAV capsid proteins with a targeting peptide at a site within variable region VIII (VR VIII) demonstrate better targeting due to more specific expression of the transgene in target tissues, such as the brain, compared to AAV capsid proteins without a targeting peptide at a site within variable region VIII.

[0013] In another aspect, the disclosure features a modified adeno-associated virus (AAV) capsid protein including a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide has a sequence of X1X2X3X4X5X6X7X8X9, where X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently selected from any amino acid residue. (i) X1 is independently selected from proline (P) and glycine (G); (ii) X2 is independently selected from lysine (L), threonine (T), serine (S), alanine (A), valine (V), and isoleucine (I); (iii) X3 is independently selected from asparagine (N), glutamine (Q), and proline (P); (iv) X4 is independently selected from glycine (G) and alanine (A); (v) X5 is independently selected from alanine (A), threonine (T), serine (S), valine (V), and glycine (G); (vi) X6 is independently selected from valine (V), leucine (L), alanine (A), isoleucine (I), glycine (G), serine (S), and threonine (T); (vii) X7 is independently selected from histidine (H), arginine (R), and lysine (K); (viii) X8 is independently selected from leucine (L) and valine (V); and (ix) X9 is independently selected from tyrosine (Y), arginine (R), histidine (H), lysine (K), and phenylalanine (F).

[0014] In some embodiments, the targeting peptide in VR VIII has a sequence selected from SEQ ID NOs: 620-55819.

[0015] In some embodiments, the targeting peptide has a sequence of PX2X3GAVX7LY (SEQ ID NO: 2), where X2, X3, and X7 are independently selected from any amino acid residue. (i) X2 is independently selected from lysine (L), isoleucine (I), valine (V), and alanine (A); (ii) X3 is asparagine (N) or glutamine (Q), and (iii) X7 is independently selected from histidine (H), arginine (R), and lysine (K).

[0016] In some embodiments, the targeting peptide is (i) PLQGAVHLY (SEQ ID NO: 3); (ii) PLQGAVRLY (SEQ ID NO: 4); (iii) PLQGAVKLY (SEQ ID NO: 5); (iv) PINGAVHLY (SEQ ID NO: 6); (v) PVNGAVHLY (SEQ ID NO: 7); (vi) PANGAVHLY (SEQ ID NO: 8); or (vii) PLNGAVHLY (SEQ ID NO: 9) is.

[0017] In some embodiments, the targeting peptide is inserted between S586 and A589 of the AAV9 capsid protein, thereby replacing A587 and Q588 of the AAV9 capsid protein.

[0018] In some embodiments, the modified AAV capsid protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an AAV9 capsid protein.

[0019] In some embodiments, the targeting peptide is (i) inserted between S586 and T589 of the Anc80L65 capsid protein, thereby replacing A587 and N588 of the Anc80L65 capsid protein; (ii) inserted between Q585 and N588 of the Anc80L65 capsid protein, thereby replacing S586 and A587 of the Anc80L65 capsid protein; (iii) inserted between L584 and A587 of the Anc80L65 capsid protein, thereby replacing Q585 and S586 of the Anc80L65 capsid protein; (iv) inserted between A587 and A590 of the Anc80L65 capsid protein, thereby replacing N588 and T589 of the Anc80L65 capsid protein; or (v) Anc80L65 is inserted between S586 and A587 of the capsid protein.

[0020] In some embodiments, the targeting peptide comprises PLNGAVHLY (SEQ ID NO: 9).

[0021] In some embodiments, the modified AAV capsid protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the Anc80L65 capsid protein.

[0022] In some embodiments, the targeting peptide has a sequence of PX2X3GX5X6X7LY (SEQ ID NO: 10), where X2, X3, X5, X6, and X7 are independently selected from any amino acid residue. (i) X2 is independently selected from leucine (L), threonine (T), or serine (S); (ii) X3 is independently selected from asparagine (N) and glutamine (Q); (iii) X5 is independently selected from alanine (A) and threonine (T); (iv) X6 is independently selected from valine (V) and leucine (L); and (v) X7 is independently selected from histidine (H), arginine (R), and lysine (K).

[0023] In some embodiments, the targeting peptide is (i) PTNGTVRLY (SEQ ID NO: 11); (ii) PTNGTVHLY (SEQ ID NO: 12); (iii) PTNGTVKLY (SEQ ID NO: 13); (iv) PSNGTLRLY (SEQ ID NO: 14); (v) PSNGTLHLY ​​(SEQ ID NO: 15); (vi) PSNGTLKLY (SEQ ID NO: 16); (vii) PTNGTLRLY (SEQ ID NO: 17); (viii) PTNGTLHLY ​​(SEQ ID NO: 18) or (ix) PTNGTLKLY (SEQ ID NO: 19) is.

[0024] In some embodiments, the targeting peptide has a sequence of PX2X3GAVX7X8X9 (SEQ ID NO: 20), where X2, X3, X5, X6, and X7 are independently selected from any amino acid residue. (i) X2 is independently selected from leucine (L), threonine (T), or serine (S); (ii) X3 is independently selected from asparagine (N) and glutamine (Q); (iii) X7 is independently selected from histidine (H) and threonine (T); (iv) X8 is independently selected from valine (V) and leucine (L); and (v) X9 is independently selected from tyrosine (Y) and arginine (R).

[0025] In some embodiments, the targeting peptide is (i) PTQGAVTVR (SEQ ID NO: 21); (ii) PLQGAVTVR (SEQ ID NO: 22); (iii) PLQGAVHVR (SEQ ID NO: 23); (iv) PLQGAVHVY (SEQ ID NO: 24); (v) PSQGAVTLR (SEQ ID NO: 25); (vi) PLQGAVTLR (SEQ ID NO: 26); (vii) PLQGAVHLR (SEQ ID NO: 27); or (viii) PTQGAVTLR (SEQ ID NO: 28) is.

[0026] In some embodiments, the targeting peptide does not include PLNGAVHLY (SEQ ID NO: 9).

[0027] In some embodiments, the targeting peptide is inserted between S586 and A589 of the AAV9 capsid protein, thereby replacing A587 and Q588 of the AAV9 capsid protein.

[0028] In another aspect, the disclosure features a modified adeno-associated virus (AAV) capsid protein that includes a targeting peptide within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 160-619.

[0029] In some embodiments, the modified AAV capsid protein comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to the sequence of a reference AAV capsid protein.

[0030] In some embodiments, the reference AAV capsid protein is selected from VP1, VP2, and VP3.

[0031] In some embodiments, the reference AAV capsid protein is an AAV capsid protein selected from the group consisting of: AAV9; Anc80L65; Anc80-55, Anc80-129, Anc80-156, Anc80-751, Anc80-1029, Anc80-1712, AAV2; AAV1; AAV6; AAV3; AAVLK03; AAV7; AAV8; AAVhu.37; AAVrh.10; AAVhu.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.

[0032] In some embodiments, the reference AAV capsid protein is a capsid protein having a sequence selected from SEQ ID NOs: 54-158, or a fragment thereof.

[0033] In some embodiments, the modified AAV capsid protein has one or more modifications, including amino acid insertions, deletions, substitutions, or combinations thereof, compared to a reference AAV capsid protein.

[0034] In some embodiments, the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 61, or a fragment thereof.

[0035] In some embodiments, the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 142, or a fragment thereof.

[0036] In some embodiments, the targeting peptide is located at 576-601 within VR VIII of the modified AAV capsid protein.

[0037] In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region N-terminal to the targeting peptide.

[0038] In some embodiments, the N-terminal flanking region comprises at least four consecutive amino acid residues from amino acids 576 to 585 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0039] In some embodiments, the N-terminal flanking region has the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), wherein B1, B2, and B3 are each independently selected from any amino acid residue. In some embodiments, B1 is selected from glutamate (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H).

[0040] In some embodiments, the N-terminal flanking region has the sequence SYGQVATNHQS (SEQ ID NO: 55848).

[0041] In some embodiments, the N-terminal flanking region replaces the N-terminal reference sequence of a reference AAV capsid protein, wherein the N-terminal reference sequence has at least 60% sequence identity with the N-terminal flanking region and is located N-terminal to the insertion site of the targeting peptide within the reference AAV capsid protein. In some embodiments, the N-terminal flanking region has the sequence SYGQVATNHQS (SEQ ID NO: 55848).

[0042] In some embodiments, the modified AAV capsid protein further comprises a C-terminal flanking region C-terminal to the targeting peptide.

[0043] In some embodiments, the C-terminal flanking region comprises at least four contiguous amino acids from amino acid residues 589 to 602 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0044] In some embodiments, the C-terminal flanking region has a sequence of AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851), wherein Z1, Z2, Z3, and Z4 are each independently selected from any amino acid residue. In some embodiments, Z1 is selected from threonine (T) or tryptophan (W), Z2 is selected from asparagine (N) or glutamine (Q), Z3 is selected from serine (S) or asparagine (N), and Z4 is selected from alanine (A) or isoleucine (I).

[0045] In some embodiments, the C-terminal flanking region has the sequence AQAQTGWVQNQGI (SEQ ID NO: 55850).

[0046] In some embodiments, the C-terminal flanking region replaces the C-terminal reference sequence of a reference AAV capsid protein, wherein the C-terminal reference sequence has at least 60% sequence identity with the C-terminal flanking region and is located C-terminal to the insertion site of the targeting peptide within the reference AAV capsid protein. In some embodiments, the C-terminal reference sequence has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851).

[0047] In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region having the sequence of B1YGB2VATNB3QS (SEQ ID NO: 55849; B1, B2, and B3 are each independently selected from any amino acid residue), and a C-terminal flanking region having the sequence of AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851; Z1, Z2, Z3, and Z4 are each independently selected from any amino acid residue).

[0048] In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region having the sequence of B1YGB2VATNB3QS (SEQ ID NO: 55860, where B1 is selected from glutamate (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H)), and a C-terminal flanking region having the sequence of AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55861, where Z1 is selected from threonine (T) or tryptophan (W), Z2 is selected from asparagine (N) or glutamine (Q), Z3 is selected from serine (S) or asparagine (N), and Z4 is selected from alanine (A) or isoleucine (I)).

[0049] In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region having the sequence SYGQVATNHQS (SEQ ID NO: 55848) and a C-terminal flanking region having the sequence AQAQTGWVQNQGI (SEQ ID NO: 55850).

[0050] In some embodiments, the modified AAV capsid protein comprises the amino acid sequence of B1YGB2VATNB3QSPLMGAVHLYAQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55852), wherein B1, B2, B3, Z1, Z2, Z3, and Z4 are each independently selected from any amino acid residue. In some embodiments, B1 is selected from glutamate (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), B3 is selected from leucine (L) or histidine (H), Z1 is selected from threonine (T) or tryptophan (W), Z2 is selected from asparagine (N) or glutamine (Q), Z3 is selected from serine (S) or asparagine (N), and Z4 is selected from alanine (A) or isoleucine (I).

[0051] In some embodiments, the targeting peptide is a peptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues that differ from the sequence of SEQ ID NO: 29, and the differing amino acids include insertions, deletions, or substitutions.

[0052] In some embodiments, the targeting peptide is SEQ ID NO:29.

[0053] In some embodiments, the one or more modifications comprise an amino acid insertion, deletion, substitution, or a combination thereof to introduce a targeting peptide into VR VIII of the reference AAV capsid protein.

[0054] In some embodiments, the one or more modifications comprise amino acid modifications outside VR VIII of the reference AAV capsid protein.

[0055] In some embodiments, the one or more modifications outside VR VIII of the reference AAV capsid protein include one or more modifications of VR I, VR II, VR III, VR IV, VR V, VR VI, or VR VII.

[0056] In some embodiments, the one or more modifications outside VR VIII of the reference AAV capsid protein include one or more modifications of VR IV and VR V.

[0057] In some embodiments, one or more modifications of VR IV result in the introduction of the sequence of SEQ ID NO:30.

[0058] In some embodiments, one or more modifications of VR V result in the introduction of the sequence of SEQ ID NO:31.

[0059] In some modifications, the one or more modifications include a deletion of one or more amino acids within VR VIII.

[0060] In some modifications, the deletion of one or more amino acids comprises deletion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII.

[0061] In some modifications, the one or more amino acid deletions comprise deletion of at least one amino acid residue at a position selected from 584, 585, 586, 587, or 588, or a combination thereof, relative to a reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some modifications, the one or more amino acid deletions comprise deletion of the amino acid residue at position 587 relative to a reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some modifications, the one or more amino acid deletions comprise deletion of the amino acid residue at position 588 relative to a reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein.

[0062] In some modifications, the amino acid deletion comprises deletion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some modifications, the amino acid deletion comprises deletion of amino acid residues at positions 589, 590, or 591, or a combination thereof, relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein.

[0063] In some modifications, the amino acid insertion involves the insertion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII.

[0064] In some modifications, the inserted amino acid residues are independently selected from any amino acid residue.

[0065] In some modifications, the inserted amino acid residue is identical to the deleted amino acid residue adjacent to the N- or C-terminus of the targeting peptide in VR VIII.

[0066] In some modifications, the inserted amino acid residues are alanine (A) or asparagine (N). In some modifications, the inserted amino acids are alanine (A) at the position immediately adjacent to the C-terminus of the targeting peptide and asparagine (N) at the next position, thereby having an amino acid sequence of A, N immediately adjacent to the C-terminus of the targeting peptide.

[0067] In some modifications, the targeting peptide is (i) PLNGAVHLYN (SEQ ID NO: 32), or (ii) PLNGAVHLYAN (SEQ ID NO: 33) is.

[0068] In some embodiments, (i) the reference AAV capsid protein is an AAV1 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and S588 of the reference capsid protein; (ii) the reference AAV capsid protein is an AAV2 capsid protein or a modification thereof, and the targeting peptide is between R585 and R588 of the reference AAV capsid protein, thereby replacing residues G586 and N587 of the reference capsid protein; (iii) the reference AAV capsid protein is an AAV3 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and N588 of the reference capsid protein; (iv) the reference AAV capsid protein is an AAV4 capsid protein or a modification thereof, and the targeting peptide is between D582 and N585 of the reference AAV capsid protein, thereby replacing residues Q583 and S584 of the reference capsid protein; (v) the reference AAV capsid protein is an AAV5 capsid protein or a modification thereof, and the targeting peptide is between S575 and T578 of the reference AAV capsid protein, thereby replacing residues S576 and S577 of the reference capsid protein; (vi) the reference AAV capsid protein is an AAV6 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and S588 of the reference capsid protein; (vii) the reference AAV capsid protein is an AAV7 capsid protein or a modification thereof, and the targeting peptide is between A587 and T590 of the reference AAV capsid protein, thereby replacing residues A588 and N589 of the reference capsid protein; (viii) the reference AAV capsid protein is an AAV8 capsid protein or a modification thereof, and the targeting peptide is between Q588 and A591 of the modified AAV capsid protein, thereby replacing residues Q589 and N590 of the reference capsid protein; (ix) the reference AAV capsid protein is an AAV9 capsid protein or a modification thereof, and the targeting peptide is between S586 and A589 of the reference AAV capsid protein, thereby replacing residues A587 and Q588 of the reference capsid protein; (x) the reference AAV capsid protein is the capsid protein of AAVrh10 or a modification thereof, and the targeting peptide is between Q588 and A591 of the reference AAV capsid protein, thereby replacing residues Q589 and N590 of the reference capsid protein; (xi) the reference AAV capsid protein is the capsid protein of AAVpo.1 or a modification thereof, and the targeting peptide is between N565 and S568 of the reference AAV capsid protein, thereby replacing residues Q566 and N567 of the reference capsid protein; (xii) the reference AAV capsid protein is an AAV12 capsid protein or a modification thereof, and the targeting peptide is between N590 and A593 of the reference AAV capsid protein, thereby replacing residues Q591 and N592 of the reference capsid protein; (xiii) the reference AAV capsid protein is an Anc80 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and N588 of the reference capsid protein; (xiv) the reference AAV capsid protein is an Anc80-55 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and N588 of the reference capsid protein; (xv) the reference AAV capsid protein is an Anc80-129 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein; (xvi) the reference AAV capsid protein is the Anc80-156 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein; (xvii) the reference AAV capsid protein is the capsid protein of Anc80-751 or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein; (xviii) the reference AAV capsid protein is the Anc80-1029 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein; or (xix) The reference AAV capsid protein is the capsid protein of Anc80-1712 or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein.

[0069] In some embodiments, (i) 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; (ii) the reference AAV capsid protein is an AAV2 capsid protein or a modification thereof, and the targeting peptide is between R588 and Q589 or between N587 and R588 of the reference AAV capsid protein; (iii) 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; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) 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; (ix) the reference AAV capsid protein is an AAV9 capsid protein or a modification thereof, and the targeting peptide is between Q588 and A589 of the reference AAV capsid protein; (x) the reference AAV capsid protein is an AAVrh10 capsid protein or a modification thereof, and the targeting peptide is between N590 and A591 of the reference AAV capsid protein; (xi) 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; (xii) 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; (xiii) the reference AAV capsid protein is an Anc80 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590, between N588 and T589, or between N587 and T588 of the reference AAV capsid protein; (xiv) 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 reference AAV capsid protein; (xv) 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 reference AAV capsid protein; (xvi) 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 reference AAV capsid protein; (xvii) 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; (xviii) 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 (xix) The reference AAV capsid protein is the Anc80-1712 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.

[0070] In another aspect, the disclosure features a polynucleotide encoding any of the modified AAV capsid proteins described herein.

[0071] In another aspect, the disclosure features a vector including any of the polynucleotides described herein. In some embodiments, the vector includes a promoter operably linked to the polynucleotide.

[0072] 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.

[0073] In another aspect, the disclosure features a recombinant AAV virion (rAAV) that includes any of the modified AAV capsid proteins described herein.

[0074] In some embodiments, the rAAV 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. In some embodiments, the therapeutic protein is used to treat and / or prevent a disease of the central nervous system (CNS).

[0075] In some embodiments, the AAV virion, when administered to a subject in a therapeutically effective amount, has increased specificity for central nervous system (CNS) tissue relative to a reference AAV virion comprising a reference AAV capsid protein that does not comprise a targeting peptide.

[0076] In some embodiments, the AAV virion, when administered to a subject in a therapeutically effective amount, has increased transduction efficiency in the CNS relative to a reference AAV virion comprising a reference AAV capsid protein that does not comprise a targeting peptide.

[0077] In some embodiments, the AAV virion, when administered to a subject in a therapeutically effective amount, has increased blood-brain barrier permeability in a subject relative to a reference AAV virion comprising a reference capsid protein that does not comprise a targeting peptide.

[0078] In another aspect, the disclosure features a pharmaceutical composition including any of the AAV virions described herein.

[0079] In another aspect, the disclosure features a method of treating or ameliorating or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of any AAV virion described herein or any pharmaceutical composition described herein.

[0080] In some embodiments, the disease is a disease of the central nervous system (CNS).

[0081] In some embodiments, the CNS disease is a lysosomal storage disease (LSD).

[0082] In some embodiments, the CNS disease is a leukodystrophy.

[0083] In some embodiments, the CNS disease is metachromatic leukodystrophy (MLD).

[0084] In some embodiments, the CNS disease is Krabbe's disease.

[0085] In some embodiments, the CNS disease is cancer, hi some embodiments, the CNS disease is metastatic breast cancer.

[0086] In some embodiments, any of the modified adeno-associated virus (AAV) capsid proteins described herein are used to treat and / or prevent diseases of the central nervous system (CNS).

[0087] In another aspect, the disclosure features an AAV virion comprising any of the modified AAV capsid proteins described herein or any of the AAV virions described herein used to treat and / or prevent diseases of the central nervous system (CNS).

[0088] In another aspect, the disclosure features any of the modified AAV capsid proteins described herein and / or any of the AAV virions described herein used to treat and / or prevent diseases of the central nervous system (CNS).

[0089] In another aspect, the disclosure features a method for introducing an exogenous polynucleotide into the central nervous system (CNS), comprising administering any of the AAV virions described herein to a subject. In some embodiments, the administration results in introduction of the exogenous polynucleotide in the CNS at a CNS:liver infection ratio of greater than 1, as measured by genome copies of the AAV virion. In some embodiments, the administration results in expression of the exogenous polynucleotide in the CNS at a CNS:liver expression ratio of greater than 10. In some embodiments, the CNS:liver expression ratio of greater than 10, as measured by protein expression.

[0090] In another aspect, the disclosure features a use of any of the AAV capsid proteins described herein, and / or any of the AAV virions described herein, for introducing an exogenous polynucleotide into the central nervous system. In some embodiments, the use is a non-therapeutic use.

[0091] The modified rAAV of the present disclosure is expected to provide an improved therapeutic index due to specific target tissue tropism and greater expression of therapeutic proteins compared to control rAAVs with unmodified capsid proteins.

[0092] 5. Brief description of some drawings 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]

[0093] [Figure 1] FIG. 1 summarizes the NHP study design described in the Examples.

[0094] [Figure 2] Figures 2A-2D show immunohistochemistry (IHC) images of brain sections obtained from NHPs administered with (i) Anc80L65-CAG-GFP or (ii) AAV9-CAG-GFP by intracisternal injection (ICM) or lumbar puncture (LP). Brown staining = GFP expression (arrows). The inset of Anc80L65-LP (Figure 2B) shows primarily neuronal staining. Figure 2A shows GFP expression after administration of Anc80L65 by ICM injection. Figure 2B shows GFP expression after administration of Anc80L65 by LP. Figure 2C shows GFP expression after administration of AAV9 by ICM injection. Figure 2D shows GFP expression after administration of AAV by LP.

[0095] [Figure 3] Figures 3A-C show IHC images of brain sections containing the cortex from NHPs administered vehicle (Figure 3A), Anc80L65-CAG-GFP (Figure 3B), or AAV9-CAG-GFP (Figure 3C). Brown staining = GFP expression.

[0096] [Figure 4]Figures 4A-B show IHC images of brain sections containing the ependyma and caudate nucleus from NHPs administered Anc80L65-CAG-GFP via ICM injection. Figure 4B is a magnified image of a portion of Figure 4A. Brown staining = GFP expression.

[0097] [Figure 5] Figures 5A-5B show IHC images of brain sections containing the caudate nucleus obtained from NHPs administered Anc80L65-CAG-GFP via ICM injection. Figure 5B is a magnified image of a portion of Figure 5A. Brown staining = GFP expression.

[0098] [Figure 6] Figure 6 shows IHC images of brain sections containing the substantia nigra from NHPs administered Anc80L65-CAG-GFP via ICM injection. Brown staining = GFP expression.

[0099] [Figure 7] Figures 7A and 7B show IHC images of brain sections containing perivascular cells obtained from NHPs administered Anc80L65-CAG-GFP via ICM injection. Figure 7B is a magnified image of a portion of Figure 7A. Brown staining = GFP expression.

[0100] [Figure 8] Figures 8A and 8B are IHC images of a brain section containing the cortex from an NHP administered Anc80L65-CAG-GFP via ICM injection. Figure 8B is a magnified image of a portion of Figure 8A. Brown staining = GFP expression.

[0101] [Figure 9] Figure 9 shows IHC images of brain sections containing the cortex obtained from NHPs administered Anc80L65-CAG-GFP via lumbar puncture (LP). Brown staining = GFP expression.

[0102] [Figure 10-1]Figures 10A-10C provide a centralized analysis of transgene expression, as determined by measurement of eGFP mRNA transcripts, calculated according to the formula: %eGFP expression = (eGFP cp / uL ÷ RPP30 cp / uL) × 100, in various brain regions of animals administered AAV9-CAG-GFP by ICM injection or Anc80L65-CAG-GFP by LP. Figure 10A provides data for the frontal cortex, Figure 10B provides data for the motor cortex, and Figure 10C provides data for the parietal lobe of the cortex. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0103] [Figure 11-1] Figures 11A-11B provide a unified analysis of transgene expression, as determined by measurement of eGFP mRNA transcripts, calculated according to the formula: %eGFP expression = (eGFP cp / uL ÷ RPP30 cp / uL) × 100, in various brain regions administered AAV9-CAG-GFP by ICM injection or Anc80L65-CAG-GFP by LP. Figure 11A provides data for the caudate nucleus, and Figure 11B provides data for the globus pallidus. [Figure 11-2] Same as above.

[0104] [Figure 12-1] Figures 12A-12B provide a centralized analysis of transgene expression, as determined by measurement of eGFP mRNA transcripts, calculated according to the formula: %eGFP expression = (eGFP cp / uL ÷ RPP30 cp / uL) × 100, in various brain regions administered AAV9-CAG-GFP by ICM injection or Anc80L65-CAG-GFP by LP. Figure 12A provides data for the putamen, and Figure 12B provides data for the substantia nigra. [Figure 12-2] Same as above.

[0105] [Figure 13]Figures 13A-17 provide a one-way analysis of viral genome (DNA) copies per diploid genome (VGC / DG) determined by measuring genome copy number using ddPCR and calculating the (VGC / DG) value using the formula: VGC / DG = (eGFP cp / uL ÷ RPP30 cp / uL) × 2. Each figure provides data for a different brain region or liver, including the cerebellar cortex (Figure 13A), dorsal root ganglion, cervical region (Figure 13B), dorsal root ganglion, lumbar region (Figure 14A), frontal cortex (Figure 14B), liver (Figure 15A), motor cortex (Figure 15B), spinal cord, cervical region (Figure 16A), spinal cord, lumbar region (Figure 16B), and sciatic nerve (Figure 17). [Figure 14] Figures 13A-17 provide a one-way analysis of viral genome (DNA) copies per diploid genome (VGC / DG) determined by measuring genome copy number using ddPCR and calculating the (VGC / DG) value using the formula: VGC / DG = (eGFP cp / uL ÷ RPP30 cp / uL) × 2. Each figure provides data for a different brain region or liver, including the cerebellar cortex (Figure 13A), dorsal root ganglion, cervical region (Figure 13B), dorsal root ganglion, lumbar region (Figure 14A), frontal cortex (Figure 14B), liver (Figure 15A), motor cortex (Figure 15B), spinal cord, cervical region (Figure 16A), spinal cord, lumbar region (Figure 16B), and sciatic nerve (Figure 17). [Figure 15] Figures 13A-17 provide a one-way analysis of viral genome (DNA) copies per diploid genome (VGC / DG) determined by measuring genome copy number using ddPCR and calculating the (VGC / DG) value using the formula: VGC / DG = (eGFP cp / uL ÷ RPP30 cp / uL) × 2. Each figure provides data for a different brain region or liver, including the cerebellar cortex (Figure 13A), dorsal root ganglion, cervical region (Figure 13B), dorsal root ganglion, lumbar region (Figure 14A), frontal cortex (Figure 14B), liver (Figure 15A), motor cortex (Figure 15B), spinal cord, cervical region (Figure 16A), spinal cord, lumbar region (Figure 16B), and sciatic nerve (Figure 17). [Figure 16]Figures 13A-17 provide a one-way analysis of viral genome (DNA) copies per diploid genome (VGC / DG) determined by measuring genome copy number using ddPCR and calculating the (VGC / DG) value using the formula: VGC / DG = (eGFP cp / uL ÷ RPP30 cp / uL) × 2. Each figure provides data for a different brain region or liver, including the cerebellar cortex (Figure 13A), dorsal root ganglion, cervical region (Figure 13B), dorsal root ganglion, lumbar region (Figure 14A), frontal cortex (Figure 14B), liver (Figure 15A), motor cortex (Figure 15B), spinal cord, cervical region (Figure 16A), spinal cord, lumbar region (Figure 16B), and sciatic nerve (Figure 17). [Figure 17] Figures 13A-17 provide a one-way analysis of viral genome (DNA) copies per diploid genome (VGC / DG) determined by measuring genome copy number using ddPCR and calculating the (VGC / DG) value using the formula: VGC / DG = (eGFP cp / uL ÷ RPP30 cp / uL) × 2. Each figure provides data for a different brain region or liver, including the cerebellar cortex (Figure 13A), dorsal root ganglion, cervical region (Figure 13B), dorsal root ganglion, lumbar region (Figure 14A), frontal cortex (Figure 14B), liver (Figure 15A), motor cortex (Figure 15B), spinal cord, cervical region (Figure 16A), spinal cord, lumbar region (Figure 16B), and sciatic nerve (Figure 17).

[0106] [Figure 18] Figures 18A, 18B, 19A, 20A, 20B, and 21 provide a unified analysis of transgene expression as determined by measurement of eGFP mRNA transcripts, calculated according to the formula: % eGFP expression = (eGFP cp / uL ÷ RPP30 cp / uL) x 100. Each figure provides data for a different brain region, including the caudate nucleus (Figure 18A), frontal cortex (Figure 18B), globus pallidus (Figure 19A), motor cortex (Figure 19B), parietal cortex (Figure 20A), putamen (Figure 20B), and substantia nigra (Figure 21). [Figure 19]Figures 18A, 18B, 19A, 20A, 20B, and 21 provide a unified analysis of transgene expression as determined by measurement of eGFP mRNA transcripts, calculated according to the formula: % eGFP expression = (eGFP cp / uL ÷ RPP30 cp / uL) x 100. Each figure provides data for a different brain region, including the caudate nucleus (Figure 18A), frontal cortex (Figure 18B), globus pallidus (Figure 19A), motor cortex (Figure 19B), parietal cortex (Figure 20A), putamen (Figure 20B), and substantia nigra (Figure 21). [Figure 20] Figures 18A, 18B, 19A, 20A, 20B, and 21 provide a unified analysis of transgene expression as determined by measurement of eGFP mRNA transcripts, calculated according to the formula: % eGFP expression = (eGFP cp / uL ÷ RPP30 cp / uL) x 100. Each figure provides data for a different brain region, including the caudate nucleus (Figure 18A), frontal cortex (Figure 18B), globus pallidus (Figure 19A), motor cortex (Figure 19B), parietal cortex (Figure 20A), putamen (Figure 20B), and substantia nigra (Figure 21). [Figure 21] Figures 18A, 18B, 19A, 20A, 20B, and 21 provide a unified analysis of transgene expression as determined by measurement of eGFP mRNA transcripts, calculated according to the formula: % eGFP expression = (eGFP cp / uL ÷ RPP30 cp / uL) x 100. Each figure provides data for a different brain region, including the caudate nucleus (Figure 18A), frontal cortex (Figure 18B), globus pallidus (Figure 19A), motor cortex (Figure 19B), parietal cortex (Figure 20A), putamen (Figure 20B), and substantia nigra (Figure 21).

[0107] [Figure 22]Figures 22A-22D are immunohistochemistry (IHC) images of brain sections obtained from NHPs administered with Anc80L65-CAG-GFP or AAV9-CAG-GFP via intracisternal injection. Brown staining = GFP expression. Figure 22A shows GFP expression in the cortex after administration of Anc80L65-CAG-GFP. Figure 22B shows GFP expression in the caudate nucleus after administration of Anc80L65-CAG-GFP. Figure 22C shows GFP expression in the cortex after administration of AAV9-CAG-GFP. Figure 22D shows GFP expression in the caudate nucleus after administration of AAV9-CAG-GFP.

[0108] [Figure 23] Figures 23 and 24 show GFP mRNA expression measured by ddPCR in the NHP brain and spinal cord 2 weeks after ICM or LP delivery of AAV9-CAG-GFP or Anc80L65-CAG-GFP. Figure 23 provides % GFP expression in the frontal cortex, motor cortex, and parietal cortex. Figure 24 provides % GFP expression in the caudate nucleus, globus pallidus, putamen, and substantia nigra. [Figure 24] Figures 23 and 24 show GFP mRNA expression measured by ddPCR in the NHP brain and spinal cord 2 weeks after ICM or LP delivery of AAV9-CAG-GFP or Anc80L65-CAG-GFP. Figure 23 provides % GFP expression in the frontal cortex, motor cortex, and parietal cortex. Figure 24 provides % GFP expression in the caudate nucleus, globus pallidus, putamen, and substantia nigra.

[0109] [Figure 25] Figure 25 shows vector genome copy analysis by qPCR. LP or ICM injection provides VGC per cell (VGC / DG presented as average vector genome copies per diploid genome) in NHPs injected with Anc80L65-CAG-GFP and AAV9-CAG-GFP.

[0110] [Figure 26]Figures 26A-26F show double immunofluorescence (IF) staining images of brain sections administered with Anc80L65-CAG-GFP (Figures 26A, 26B, and 26C) or AAV9-CAG-GFP (Figures 26D, 26E, and 26F). Transgene expression from the AAV was detected by staining for GFP, and cell types were detected by staining for cell-type-specific markers, including NeuN for neurons (Figures 26A and 26D), GFAP for astrocytes (Figures 26B and 26E), and Iba1 for microglial cells (Figures 26C and 26F). Examples were imaged from the motor cortex. In all cases, GFP+ cells are shown in red, cell-specific markers are shown in green, and merged images are shown with double-labeled cells in yellow / orange (arrows for double-labeled cells).

[0111] [Figure 27] Figures 27A-27F show double immunofluorescence (IF) staining images of brain sections from NHPs administered Anc80L65-CAG-GFP via LP (Figures 27A, 27B, and 27C) or via ICM (Figures 27D, 27E, and 27F). Examples were imaged from the motor cortex. Transgene expression from Anc80L65 was detected by staining for GFP, and oligodendrocytes were detected by staining for the oligodendrocyte-specific marker OLIG2, shown in green (Figures 27A and 27D). GFP+ cells are shown (Figures 27B and 27E). Merged images are indicated by arrows pointing to double-labeled cells (Figures 27C and 27F).

[0112] [Figure 28] Figure 28 illustrates the structure of the AAV VP1 protein, highlighting specific variable regions (VR I, VR IV, VR V, and VR VIII). The location of the targeting peptide insertion site in VR VIII is indicated.

[0113] [Figure 29-1]Figures 29A-29C provide sequence alignments of the VP1 sequences of specific AAV variants using AAV2 VP1 as a reference. The location of the insertion site for the targeting peptide (Figure 29B) is indicated. Figure 29 discloses, in order of appearance, SEQ ID NO:55, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:89, SEQ ID NO:111, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:62, and SEQ ID NO:57, respectively. [Figure 29-2] Same as above. [Figure 29-3] Same as above.

[0114] [Figure 30-1] Figures 30A-30D provide sequence alignments of ancestral AAV VP1 sequences using AAV2 as a reference. The location of the insertion site for the targeting peptide (Figure 30C) is indicated. One or more representative member sequences for each of the Anc80, Anc81, Anc82, Anc83, Anc84, Anc94, Ac110, Anc113, Anc126, and Anc127 libraries were used for the alignment. Figures 30A-30D disclose, in order of appearance, SEQ ID NOs: 55, 55911, 143, 147, 144, 146, 145, 148, 149-150, 142, and 55912-55921, respectively. [Figure 30-2] Same as above. [Figure 30-3] Same as above. [Figure 30-4] Same as above.

[0115] [Figure 31] Figure 31 provides the sequences of the window in VR VIII of a modified AAV capsid protein (e.g., Anc80L65) bearing a targeting peptide at various positions in VR VIII. The underlined sequence represents the amino acid residues of the targeting peptide. Figure 31 discloses SEQ ID NOs: 55875-55881, respectively, in order of appearance.

[0116] [Figure 32-1]Figure 32A shows the structure of the AAV VP1 capsid protein, focusing on variable regions (VR) IV, VR V, and VR VIII.

[0117] Figure 32B provides partial sequence information for three AAV capsid protein variants with various combinations of the VR IV, VR V, and VR VIII regions of Anc80L65 or the AAV9 capsid protein.

[0118] [Figure 32-2] Figure 32C provides a sequence alignment of amino acid residues 540-640 of SEQ ID NOs: 61 (AAV9 capsid protein), 142 (Anc80L65 capsid protein), and 55853. The box indicates amino acid positions 576-601, which correspond to positions in AAV9 without a targeting peptide inserted. Figure 32C discloses SEQ ID NOs: 55853 and 55882-55883, respectively, in the order listed.

[0119] [Figure 32-3] Figure 32D provides a sequence alignment of amino acid residues 540-640 of AAV9 (SEQ ID NO: 61), AAV9 (SEQ ID NO: 61) with residues 588-589 inserted (586 and 587 removed), and a targeting peptide (SEQ ID NO: 9). AFT-6 has an Anc80L65 capsid protein backbone with an N-terminal flanking region from AAV9 (SEQ ID NO: 55848), a targeting peptide of SEQ ID NO: 9, and a C-terminal flanking region from AAV9 (SEQ ID NO: 55850). Boxes indicate amino acid positions 559-635. Figure 32D discloses SEQ ID NOs: 55884-55885, 55884, and 55886, respectively, in the order listed.

[0120] [Figure 33]Figure 33 provides the sequence of the VR VIII window of a modified AAV capsid protein (e.g., AAV9) with the targeting peptide shown as underlined amino acid residues and blank spaces at positions 587 and 588, corresponding to the deletion of A587 and Q588 from the modified AAV9 capsid protein. Each of AFT-9 through AFT-31 is inserted into the AAV9 capsid protein as described. Figure 33 discloses SEQ ID NOs: 55875, and 55887 through 55909, respectively, in order of appearance.

[0121] [Figure 34] Figure 34 shows a plot of gene transfer efficacy data (RNA logMN_fold change) averaged across all brain tissues for each of the rAAVs in the AAV-mini library. ** indicates technical replicates for AFT-6 (SEQ ID NO: 55820).

[0122] [Figure 35] Figure 35 shows a plot of tissue enrichment scores (log10 scale of expression) for the indicated tissues. Capsids tested included AAV9 and the Anc80L65 capsid containing the N2 targeting peptide (SEQ ID NO: 9) located in VR VIII (the complete capsid sequence is referred to as AFT-6 (SEQ ID NO: 55820)). "On-target" CNS tissues analyzed included the frontal lobe, motor cortex, parietal lobe, occipital lobe, temporal lobe, cerebellum, putamen, thalamus, globus pallidus, caudate nucleus, and substantia nigra. "Off-target" tissues analyzed included dorsal root ganglia, such as cervical, lumbar, thoracic, and liver. Higher tissue enrichment score values ​​represent higher targeting.

[0123] [Figure 36]Figure 36 shows a plot of tissue enrichment scores (log10 scale of expression) for the indicated tissues. Capsids tested included AAV9, AAV9 containing an N3 targeting peptide (PLNGSVHLY (SEQ ID NO: 3603 in Appendix B)) located in VR VIII between amino acid residues 586 and 589, replacing amino acids A587 and Q588, and AAV9 containing an N4 targeting peptide (PLNGTVHLY (SEQ ID NO: 1232 in Appendix B)) located in VR VIII between amino acid residues 586 and 589, replacing amino acids A587 and Q588. CNS tissues analyzed included the frontal cortex, temporal lobe, putamen, thalamus, and globus pallidus. Higher tissue enrichment score values ​​represent higher tropism.

[0124] [Figure 37] Figure 37 shows a plot of tissue enrichment scores (loglO scale of expression) for the indicated tissues. Capsids tested included AAV9 containing an N1 targeting peptide (SEQ ID NO:9) located in VR VIII of AAV9, Anc80L65 capsid containing an N2 targeting peptide (SEQ ID NO:9) located in VR VIII (the complete capsid sequence is referred to as AFT-6 (SEQ ID NO:55820)), AAV9 containing an N3 targeting peptide (PLNGSVHLY (SEQ ID NO:3603)) located in VR VIII between amino acid residues 586 and 589, replacing amino acids A587 and Q588, and AAV9 containing an N4 targeting peptide (PLNGTVHLY (SEQ ID NO:1232)) located in VR VIII between amino acid residues 586 and 589, replacing amino acids A587 and Q588. Tissues analyzed included the frontal lobe, motor cortex, parietal lobe, occipital lobe, cerebellum, putamen, thalamus, globus pallidus, caudate nucleus, substantia nigra, liver, DRG-cervical, DRG-lumbar, and DRG-thoracic. Higher tissue enrichment score values ​​represent higher orientation.

[0125] [Figure 38]Figure 38 provides sequences of the VR VIII window of a modified AAV capsid library having targeting peptides (e.g., X1X2X3X4X5X6X7X8X9) in which residues X1 through X9 are selected from the amino acid residues in the corresponding column. In the library, blanks at positions 587 and 588 correspond to deletions of A587 and Q588 from the modified AAV capsid protein. Figure 38 discloses SEQ ID NOs: 55875 through 55910, respectively, in order of appearance. DETAILED DESCRIPTION OF THE INVENTION

[0126] 6. Detailed Description of the Invention 6.1.Definition "AAV" is an adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term encompasses all subtypes, serotypes and pseudotypes, and both natural and recombinant forms, except where otherwise required.

[0127] The term "AAV capsid protein" or simply "capsid protein" refers to the VP1, VP2, or VP3 capsid protein of AAV. In some embodiments, the AAV capsid protein is the wild-type or modified capsid protein of AAV9; AAV2; AAV1; AAV6; AAV3; AAVLK03; AAV7; AAV8; AAVhu.37; AAVrh.10; AAVhu.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.

[0128] The term "modified AAV capsid protein" or simply "modified capsid protein" refers to a capsid protein that has been modified relative to a natural or synthetic / artificial capsid protein, such as that referred to as a "reference AAV capsid protein" or "reference capsid protein." As used herein, a reference AAV capsid protein may be a VP1, VP2, or VP3 capsid protein of a naturally occurring AAV variant, or a non-natural VP1, VP2, or VP3 capsid protein known in the art.

[0129] As used herein, the term "targeting peptide" refers to an amino acid sequence ranging from 5 to 16 amino acids in length within variable region VIII (VRVIII) of a modified AAV capsid protein introduced by one or more modifications described herein. AAV containing a modified capsid protein with a targeting peptide may have different localization and distribution in target cells, tissues, or organs than AAV having a capsid protein that does not contain a targeting peptide.

[0130] The term "amino acid position" in the AAV capsid protein herein refers to the position of the 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.

[0131] For the avoidance of doubt, as used herein, a reference to an insertion site 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 amino acid at position X and before the amino acid at position X+1.

[0132] 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 primer-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 mutants thereof.

[0133] The term "modification" when combined with an amino acid residue, amino acid residues, or modified sequence refers to an insertion, deletion, substitution, or combination thereof.

[0134] The terms "operably linked" and "operably linked" refer to the functional relationship between a nucleic acid sequence and a regulatory sequence of nucleotides, such as a promoter, enhancer, transcription and translation stop site, and other signal sequence, indicating that two or more DNA segments are linked to each other so that they function in concert for their intended purpose. For example, operably linking a nucleic acid sequence, typically DNA, to a regulatory sequence or promoter region refers to the physical and functional relationship between the DNA and the regulatory sequence or promoter such that 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.

[0135] The term "parenteral" administration of a composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0136] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0137] The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, excipients, stabilizers and adjuvants. For examples of carriers, excipients, stabilizers and adjuvants, see Remington: The Science and Practice of Pharmacy, 22nd Revised Ed., Pharmaceutical Press, 2012.

[0138] The abbreviation "rAAV" refers to a recombinant adeno-associated virus particle composed of at least one AAV capsid protein and an encapsidated polynucleotide, sometimes referred to herein as a "genome." rAAV can include a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome), e.g., a heterologous polynucleotide encoding a gene to be delivered to a mammalian cell, e.g., a genome including a sequence encoding a therapeutic protein.

[0139] Terms such as "percent sequence identity" (% sequence identity), "percent identical" (% identical), and the like refer to the percent sequence identity between two nucleotide sequences or two amino acid sequences, calculated by aligning the two sequences, determining the number of nucleotide or amino acid residue matches between the two sequences, dividing the number of matches by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues), and multiplying by 100 to arrive at a percent sequence identity value. To calculate percent sequence identity (% sequence identity), two or more sequences are aligned using the EMBOSS Needle Pairwise Sequence Alignment software tool, which is based on the Needleman and Wunsch algorithm (available at www.ebi.ac.uk / Tools / psa / emboss_needle), with the following parameters: matrix: BLOSUM62 (for protein sequences) or DNA complete (for DNA sequences); gap open: 10; gap extension: 0.5; end gap penalty: false; end gap open: 10; and end gap extension: 0.5.

[0140] Methods for aligning comparative nucleotide and amino acid sequences are well known in the art. The local homology algorithm (BESTFIT) of Smith and Waterman (1981) Adv. Appl. Math 2:482, the homology alignment algorithm (GAP) of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the search for similarity method (Tfasta and Fasta) of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, and computerized implementations of these algorithms can enable optimal alignment of the compared sequences. Examples of suitable PC / Gene programs include, but are not limited to, CLUSTAL from the Wisconsin Genetics Software Package, Version 8 (available from the Genetics Computer Group (GCG™ program (Accelrys, Inc., San Diego, Calif.)), Intelligenetics, Mountain View, Calif.), GAP, BESTFIT, BLAST, FASTA, and TFASTA. The CLUSTAL program is described in detail in Higgins and Sharp (1988) Gene 73:237-244; Higgins and Sharp (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-10890; Huang et al. (1992) Computer Applications in the Biosciences 8:155-165; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. An example of a good program to use for optimal global alignment of multiple sequences is PileUp (Feng and Doolittle (1987) J. Mol. Evol. 25:351-260), which is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-153 (incorporated herein by reference). The BLAST family of programs that can be used for database similarity searching includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See Current Protocols in Molecular Biology, Chapter 19, edited by Ausubel et al.See Greene Publishing and Wiley-Interscience, New York (1995). Updated versions of the BLAST family of programs include the BLAST+ family (Camacho, C., et al. (2009 December 15) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0141] As used herein, the term "tissue-specific" promoter or expression control element (ERE) refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0142] The terms "treatment," "treating," and the like are generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in that a disease, condition, or symptom thereof is completely or partially prevented, and / or can be therapeutic, in that a disease or condition is partially or completely cured and / or an adverse effect caused by the disease or condition. As used herein, "treatment" 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 (e.g., halting its progression); or (c) alleviating the disease or condition (e.g., causing regression of the disease or condition and providing improvement in one or more symptoms).

[0143] The terms "vector," "AAV vector," and "rAAV vector" refer to a rAAV that contains a heterologous polynucleotide, e.g., a transgene.

[0144] As used herein, the term "variable region" or "VR" refers to one or more of the nine sequence-variable regions (e.g., VRI to VRIX) of 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, such as receptor binding, transduction efficiency, and antigen reactivation. The relative locations of VR I, VR IV, VR V, and VR VIIII are shown in Figure 28, although the specific locations of the variable regions within the capsid protein may vary depending on the capsid protein and / or sequence alignment method.

[0145] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the method and composition belong. Methods and materials similar or equivalent to those described herein can be used to carry out or test the method and composition, and suitable methods and materials are described below. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0146] 6.2. Modified AAV Capsid Proteins One aspect of the present disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide has a sequence of X1X2X3X4X5X6X7X8X9, where X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently selected from any amino acid residue.

[0147] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a targeting peptide within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 620-55819.

[0148] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide has a sequence of PX2X3GAVX7LY (SEQ ID NO: 2), where X2, X3, and X7 are independently selected from any amino acid residue.

[0149] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide has a sequence of PX2X3GX5X6X7LY (SEQ ID NO: 10), where X2, X3, X5, X6, and X7 are independently selected from any amino acid residue.

[0150] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a targeting peptide within variable region VIII (VR VIII), wherein the targeting peptide has a sequence of PX2X3GAVX7X8X9 (SEQ ID NO: 20), wherein X2, X3, X5, X6, and X7 are independently selected from any amino acid residue.

[0151] In some embodiments, the modified AAV capsid protein has one or more amino acid insertions, deletions, substitutions, or combinations thereof compared to a reference AAV capsid protein. Some or all of the amino acid insertions, deletions, substitutions, or combinations thereof are for introducing a targeting peptide into the reference AAV capsid protein. In some embodiments, all of the differences between the modified AAV capsid protein and the reference AAV capsid protein are within variable region VIII (VR VIII). In some embodiments, all of the differences between the modified AAV capsid protein and the reference AAV capsid protein are within the targeting peptide.

[0152] In some embodiments, the modified AAV capsid protein further comprises one or more modifications comprising amino acid insertions, deletions, substitutions, or combinations thereof outside of the introduction of the targeting peptide into VR VIII of the reference AAV capsid protein.

[0153] In some embodiments, the modified capsid protein further comprises one or more modifications outside of VR VIII of the reference AAV capsid protein.

[0154] In some embodiments, the modified AAV capsid protein comprises one or more modifications including amino acid insertions, deletions, substitutions, or combinations thereof to introduce a targeting peptide within VR VIII (e.g., VR VIII corresponds to the amino acids between positions 565 and 595 of a reference AAV capsid protein).

[0155] In some embodiments, the reference AAV capsid protein is an AAV9 capsid protein. In some embodiments, the modified AAV capsid protein has a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the AAV9 capsid protein. In some embodiments, the modified AAV capsid protein has a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid residues that differ from the AAV9 capsid protein (e.g., by insertion, deletion, or substitution). In some embodiments, the modified AAV capsid protein has a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an AAV9 capsid protein having a targeting peptide (e.g., any targeting peptide described herein) in VR VIII. In some embodiments, the modified AAV capsid protein has a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from an AAV9 capsid protein having a targeting peptide (e.g., any targeting peptide described herein) in VR VIII.

[0156] In some embodiments, the reference AAV capsid protein is an Anc80L65 capsid protein. In some embodiments, the modified AAV capsid protein has a sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the Anc80L65 capsid protein. In some embodiments, the modified AAV capsid protein has a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid residues that differ from the Anc80L65 capsid protein (e.g., by insertion, deletion, or substitution). In some embodiments, the modified AAV capsid protein has a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an Anc80L65 capsid protein having a targeting peptide (e.g., any targeting peptide described herein) in VR VIII. In some embodiments, the modified AAV capsid protein has a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from an Anc80L65 capsid protein having a targeting peptide (e.g., any targeting peptide described herein) in VR VIII.

[0157] In some embodiments, the modified AAV capsid protein has a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 34-38 or 55820-55847. In some embodiments, the modified AAV capsid protein has a sequence selected from SEQ ID NOs: 34-38 or 55820-55847. In some embodiments, the modified AAV capsid protein has a sequence selected from SEQ ID NOs: 34-39.

[0158] In some embodiments, the AAV virions comprising the modified AAV capsid protein, when administered to a subject in a therapeutically effective amount, have increased specificity for central nervous system (CNS) tissue relative to AAV virions comprising a reference capsid protein that does not contain a targeting peptide.

[0159] In some embodiments, AAV virions comprising the modified AAV capsid protein, when administered to a subject in a therapeutically effective amount, have increased transduction efficiency in the CNS relative to AAV virions comprising a reference capsid protein that does not contain a targeting peptide.

[0160] In some embodiments, the AAV virions comprising the modified AAV capsid protein, when administered to a subject in a therapeutically effective amount, have increased blood-brain barrier permeability in a subject relative to AAV virions comprising a reference capsid protein that does not contain a targeting peptide.

[0161] In some embodiments, the AAV virions comprising the modified AAV capsid protein, when administered to a subject in a therapeutically effective amount, have reduced specificity for central nervous system (CNS) tissue relative to AAV virions comprising a reference capsid protein that does not contain a targeting peptide.

[0162] In some embodiments, the AAV virions comprising the modified AAV capsid protein, when administered to a subject in a therapeutically effective amount, have reduced transduction efficiency in the CNS relative to AAV virions comprising a reference capsid protein that does not contain a targeting peptide.

[0163] In some embodiments, the AAV virions comprising the modified AAV capsid protein, when administered to a subject in a therapeutically effective amount, have reduced blood-brain barrier permeability in a subject relative to AAV virions comprising a reference capsid protein that does not contain a targeting peptide.

[0164] 6.2.1. Targeting Peptides In some embodiments, the targeting peptide within variable region VR VIII has a sequence of X1X2X3X4X5X6X7X8X9, where X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently selected from any amino acid residue. In some embodiments, the modified capsid protein comprises a targeting peptide having a sequence of X1X2X3X4X5X6X7X8X9 at a site within variable region VIII (VR VIII), where the targeting peptide is inserted between S586 and A589 of the AAV9 capsid protein, thereby replacing A587 and Q588 of the AAV9 capsid protein. In some embodiments, (i) X1 is independently selected from proline (P) and glycine (G); (ii) X2 is independently selected from lysine (L), threonine (T), serine (S), alanine (A), valine (V), and isoleucine (I); (iii) X3 is independently selected from asparagine (N), glutamine (Q), and proline (P); (iv) X4 is independently selected from glycine (G) and alanine (A); (v) X5 is independently selected from alanine (A), threonine (T), serine (S), valine (V), and glycine (G); (vi) X6 is independently selected from valine (V), leucine (L), alanine (A), isoleucine (I), glycine (G), serine (S), and threonine (T); (vii) X7 is independently selected from histidine (H), arginine (R), and lysine (K); (viii) X8 is independently selected from leucine (L) and valine (V); and (ix) X9 is independently selected from tyrosine (Y), arginine (R), histidine (H), lysine (K), and phenylalanine (F).

[0165] 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: 620-55819 introduced into VRVIII. In some embodiments, the modified AAV capsid protein comprises a targeting peptide, wherein the peptide has a sequence that differs from a reference peptide by 0, 1, 2, 3, or 4 residue substitutions relative to an amino acid sequence selected from SEQ ID NOs: 620-55819 introduced into VR VIII.

[0166] In some embodiments, the targeting peptide has a sequence of PX2X3GAVX7LY (SEQ ID NO: 2), where X2, X3, and X7 are independently selected from any amino acid residue. In some embodiments, the modified capsid protein comprises a targeting peptide having a sequence of PX2X3GAVX7LY (SEQ ID NO: 2), where the targeting peptide insertion replaces amino acid residues A587 and Q588. In some embodiments, (i) X2 is independently selected from lysine (L), isoleucine (I), valine (V), and alanine (A); (ii) X3 is asparagine (N) or glutamine (Q), and (iii) X7 is independently selected from histidine (H), arginine (R), and lysine (K).

[0167] In some embodiments, the targeting peptide is selected from the following: (i) PLQGAVHLY (SEQ ID NO: 3); (ii) PLQGAVRLY (SEQ ID NO: 4); (iii) PLQGAVKLY (SEQ ID NO: 5); (iv) PINGAVHLY (SEQ ID NO: 6); (v) PVNGAVHLY (SEQ ID NO: 7); (vi) PANGAVHLY (SEQ ID NO: 8); or (vii) PLNGAVHLY (SEQ ID NO: 9).

[0168] In some embodiments, the targeting peptide is PLNGAVHLY (SEQ ID NO: 9). In some embodiments, the targeting peptide is not PLNGAVHLY (SEQ ID NO: 9).

[0169] In some embodiments, the targeting peptide has a sequence selected from SEQ ID NOs: 620-55819.

[0170] In some embodiments, the targeting peptide has a sequence of PX2X3GX5X6X7LY (SEQ ID NO: 10), where X2, X3, X5, X6, and X7 are independently selected from any amino acid residue. In some embodiments, the modified capsid protein comprises a targeting peptide having a sequence of PX2X3GX5X6X7LY (SEQ ID NO: 10), where the targeting peptide insertion replaces amino acid residues A587 and Q588. In some embodiments, (i) X2 is independently selected from leucine (L), threonine (T), or serine (S); (ii) X3 is independently selected from asparagine (N) and glutamine (Q); (iii) X5 is independently selected from alanine (A) and threonine (T); (iv) X6 is independently selected from valine (V) and leucine (L); and (v) X7 is independently selected from histidine (H), arginine (R), and lysine (K).

[0171] In some embodiments, the targeting peptide is selected from the following: (i) PTNGTVRLY (SEQ ID NO: 11); (ii) PTNGTVHLY (SEQ ID NO: 12); (iii) PTNGTVKLY (SEQ ID NO: 13); (iv) PSNGTLRLY (SEQ ID NO: 14); (v) PSNGTLHLY ​​(SEQ ID NO: 15); (vi) PSNGTLKLY (SEQ ID NO: 16); (vii) PTNGTLRLY (SEQ ID NO: 17); (viii) PTNGTLHLY ​​(SEQ ID NO: 18); or (ix) PTNGTLKLY (sequence number 19).

[0172] In some embodiments, the targeting peptide has a sequence of PX2X3GAVX7X8X9 (SEQ ID NO: 20), where X2, X3, X5, X6, and X7 are independently selected from any amino acid residue. In some embodiments, the modified capsid protein comprises a targeting peptide having a sequence of PX2X3GAVX7X8X9 (SEQ ID NO: 20), where the targeting peptide insertion replaces amino acid residues A587 and Q588. In some embodiments, (i) X2 is independently selected from leucine (L), threonine (T), or serine (S); (ii) X3 is independently selected from asparagine (N) and glutamine (Q); (iii) X7 is independently selected from histidine (H) and threonine (T); (iv) X8 is independently selected from valine (V) and leucine (L); and (v) X9 is independently selected from tyrosine (Y) and arginine (R).

[0173] In some embodiments, the targeting peptide is selected from the following: (i) PTQGAVTVR (SEQ ID NO: 21); (ii) PLQGAVTVR (SEQ ID NO: 22); (iii) PLQGAVHVR (SEQ ID NO: 23); (iv) PLQGAVHVY (SEQ ID NO: 24); (v) PSQGAVTLR (SEQ ID NO: 25); (vi) PLQGAVTLR (SEQ ID NO: 26); (vii) PLQGAVHLR (SEQ ID NO: 27); or (viii) PTQGAVTLR (sequence number 28).

[0174] In some embodiments, the targeting peptide is PLNGSVHLY (SEQ ID NO: 3603).

[0175] In some embodiments, the modified AAV capsid protein comprises a modified AAV9 capsid protein that includes a targeting peptide having the sequence PLNGSVHLY (SEQ ID NO: 3603) located in VR VIII between amino acid residues 586 and 589, replacing amino acids A587 and Q588.

[0176] In some embodiments, the targeting peptide is PLNGTVHLY (SEQ ID NO: 1232).

[0177] In some embodiments, the modified AAV capsid protein comprises a modified AAV9 capsid protein that includes a targeting peptide having a sequence of PLNGTVHLY (SEQ ID NO: 1232) located in VR VIII between amino acid residues 586 and 589, replacing amino acids A587 and Q588.

[0178] In some embodiments, the targeting peptide is any selected from Appendix B.

[0179] In some embodiments, the targeting peptide does not include the sequence of SEQ ID NO:9.

[0180] In some embodiments, the targeting peptide is in VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 160 to 619. In some embodiments, the targeting peptide is inserted into an AAV9 or Anc80L65 backbone.

[0181] 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: 160-619 introduced into VRVIII. In some embodiments, the modified AAV capsid protein comprises a targeting peptide, wherein the peptide has a sequence that differs from a reference peptide by 0, 1, 2, 3, or 4 residue substitutions relative to an amino acid sequence selected from SEQ ID NOs: 160-619 introduced into VR VIII.

[0182] In some embodiments, the modified AAV capsid protein comprises a targeting peptide having a sequence selected from SEQ ID NOs: 518, 396, 393, 186, 368, 493, 179, 217, 614, 475, 523, 411, 443, 192, 403, 266, 416, 579, 619, 589, 247, 367, 263, 410, 615, and 597. In some embodiments, the modified AAV capsid protein comprises a targeting peptide having a sequence selected from SEQ ID NOs: 518, 396, 393, 186, 368, 493, 179, 217, 614, 475, 523, 411, 443, 192, 403, 266, and 416. In some embodiments, the modified AAV capsid protein comprises a targeting peptide having a sequence selected from SEQ ID NOs: 518, 396, 393, 186 and 368. In some embodiments, the modified AAV capsid protein comprises a targeting peptide having a sequence selected from Appendix A.

[0183] In some embodiments, the targeting peptide is a targeting peptide disclosed in US 2017 / 0166926, which is incorporated by reference in its entirety.

[0184] In some embodiments, the targeting peptide is not a targeting peptide disclosed in WO 2020 / 210655, WO 2021 / 222831, WO 2021 / 202651, WO 2021 / 211753, WO 2021 / 226167, WO 2021 / 230987, or WO 2022 / 040527, the entireties of which are incorporated by reference herein.

[0185] In some embodiments, the targeting peptide does not have a sequence specified in variable region VR VIII of the reference AAV capsid protein.

[0186] 6.2.2. Targeting Peptide Sites The modified AAV capsid protein of the present disclosure contains a targeting peptide within VR VIII of the reference AAV capsid protein (Figure 28).

[0187] Preferably, the targeting peptide is at an externally exposed site of the capsid, preferably based on structural predictions and / or experimental data, and more preferably, the targeting peptide is at an externally exposed site of the AAV capsid so as not to interfere with the activity of the protein in capsid assembly.

[0188] In some embodiments, the one or more modifications comprise an amino acid insertion, deletion, substitution, or a combination thereof to introduce a targeting peptide into VR VIII of the reference AAV capsid protein.

[0189] In some embodiments, the one or more modifications comprise deletion of one or more amino acids within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of one amino acid residue immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of two amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of three amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of four amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII.

[0190] In some embodiments, the one or more amino acid deletions comprise deletion of at least one amino acid residue at a position selected from 584, 585, 586, 587, or 588, or a combination thereof, relative to a reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the one or more amino acid deletions comprise deletion of the amino acid residue at position 587 relative to a reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the one or more amino acid deletions comprise deletion of the amino acid residue at position 588 relative to a reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the one or more amino acid deletions comprise deletion of amino acid residues at positions 587 and 588 relative to a reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the one or more amino acid deletions comprise deletion of amino acid residues at positions 586, 587, and 588 relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the one or more amino acid deletions comprise deletion of amino acid residues at positions 585, 586, 587, and 588 relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein.

[0191] In some embodiments, the one or more amino acid deletions comprise deletion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of one amino acid residue immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of two amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of three amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid deletions comprise deletion of four amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII.

[0192] In some embodiments, the amino acid deletion comprises deletion of the amino acid residue at position 589, 590, or 591, or a combination thereof, relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the amino acid deletion comprises deletion of the amino acid residue at position 589 relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the amino acid deletion comprises deletion of the amino acid residue at position 590 relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the amino acid deletion comprises deletion of the amino acid residue at position 591 relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the amino acid deletion comprises deletion of the amino acid residues at positions 589 and 590 relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the amino acid deletions comprise deletion of amino acid residues at positions 5890 and 591 relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein. In some embodiments, the amino acid deletions comprise deletion of amino acid residues at positions 589, 590, and 591 relative to the reference sequence numbered according to the amino acid sequence of a reference AAV capsid protein.

[0193] In some embodiments, the one or more modifications comprise an amino acid insertion to introduce a targeting peptide into VR VIII of the reference AAV capsid protein. In some embodiments, the amino acid insertion comprises insertion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of one amino acid residue immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of two amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of three amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of four amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of five amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII.

[0194] In some embodiments, the one or more modifications comprise an amino acid insertion to introduce a targeting peptide into VR VIII of the reference AAV capsid protein. In some embodiments, the amino acid insertion comprises insertion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of one amino acid residue immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of two amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of three amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of four amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII. In some embodiments, the one or more amino acid insertions comprise insertion of five amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII.

[0195] In some embodiments, the inserted amino acid residues are independently selected from any amino acid residue. In some embodiments, where VR VIII contains insertions and deletions relative to the amino acid sequence of a reference AAV capsid protein, the inserted amino acid residues are identical to the deleted amino acid residues adjacent to the N- or C-terminus of the targeting peptide in VR VIII. In some embodiments, the inserted amino acid residue is alanine (A) or asparagine (N). In some embodiments, the inserted amino acid residue is alanine (A). In some embodiments, the inserted amino acid residue is or asparagine (N). In some embodiments, the inserted amino acids are alanine (A) at the position immediately adjacent to the C-terminus of the targeting peptide and asparagine (N) at the next position, thereby resulting in an amino acid sequence of A, N, immediately adjacent to the C-terminus of the targeting peptide.

[0196] In some embodiments, in which the modified capsid protein comprises an amino acid insertion in VR VIII, the targeting peptide comprises (i) PLNGAVHLYN (SEQ ID NO: 32), or (ii) PLNGAVHLYAN (SEQ ID NO: 33).

[0197] In some embodiments, the one or more modifications comprise an amino acid insertion, deletion, and / or substitution to introduce a targeting peptide into VR VIII of the reference AAV capsid protein. In one embodiment, the modified capsid protein comprises an insertion of two amino acid residues immediately adjacent to the C-terminus of the targeting peptide and a deletion of four amino acid residues immediately adjacent to the N-terminus of the targeting peptide. In another embodiment, the modified capsid protein comprises an insertion of one amino acid residue immediately adjacent to the C-terminus of the targeting peptide and a deletion of three amino acid residues immediately adjacent to the N-terminus of the targeting peptide. In yet another embodiment, the modified capsid protein comprises an insertion of two amino acid residues immediately adjacent to the C-terminus of the targeting peptide and a deletion of two amino acid residues immediately adjacent to the N-terminus of the targeting peptide. In yet another embodiment, the modified capsid protein does not comprise an insertion of an amino acid residue immediately adjacent to the C-terminus of the targeting peptide, but comprises a deletion of one amino acid residue immediately adjacent to the N-terminus of the targeting peptide.

[0198] In some embodiments, insertion sites for targeting peptides are provided in Figures 29A-29C and 30A-30D. In some embodiments, insertion sites for targeting peptides are provided in Figure 29B. In some embodiments, insertion sites for targeting peptides are provided in Figure 30C.

[0199] In some embodiments, insertion sites for targeting peptides are provided in Table 1. In Table 1, preferred sites are indicated by a "-" compared to the wild-type VP1 capsid polypeptide. In some embodiments, Table 1 includes exemplary insertion sites for targeting peptides selected from SEQ ID NOs: 620-55819. In some embodiments, Table 1 includes exemplary insertion sites for targeting peptides selected from SEQ ID NOs: 160-619. [Table 1-1] [Table 1-2]

[0200] Three exemplary insertion sites are shown in Table 1. 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, two amino acids are deleted before insertion of the targeting peptide. In such cases, "[ ]" represents the deleted amino acid residue / position. For example, for AAV9 capsid protein insertion site 3, the targeting peptide is inserted between positions S586 and A589 after deletion of amino acid "AQ", located between A586 and A589 (A587 and Q588), and is indicated as "S[ ][ ]-A".

[0201] In some embodiments, the targeting peptide is between 560 and 610 within VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is between 565 and 605 within VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is between 570 and 600 within VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is between 575 and 595 within VR VIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is between 580 and 590 within VR VIII of the modified AAV capsid protein.

[0202] 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 S586 and T589 of the reference AAV capsid protein, and amino acid residues S587 and S588 are deleted.

[0203] 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 R585 and R588, thereby replacing amino acid residues G586 and N587.

[0204] 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues S587 and N588.

[0205] 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 D582 and N585 of the reference AAV capsid protein, thereby replacing amino acid residues Q583 and S584.

[0206] 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 S575 and T578 of the reference AAV capsid protein, thereby replacing S576 and S577.

[0207] 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 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues S587 and S588.

[0208] 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 A587 and T590 of the reference AAV capsid protein, thereby replacing amino acid residues A588 and N589.

[0209] 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 Q588 and T591 of the modified AAV capsid protein, thereby replacing amino acid residues Q589 and N590.

[0210] 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 S586 and A589 of the reference AAV capsid protein, thereby replacing amino acid residues A587 and Q588.

[0211] 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 Q588 and A591 of the reference AAV capsid protein, thereby replacing amino acid residues Q589 and N590.

[0212] 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 N565 and S568 of the reference AAV capsid protein, thereby replacing amino acid residues Q566 and N567.

[0213] 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 N590 and A593 of the reference AAV capsid protein, thereby replacing amino acid residues Q591 and N592.

[0214] 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, between N588 and T589, or between S587 and N588 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues S587 and N588.

[0215] 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, between N588 and T589, or between A587 and N588 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.

[0216] In some embodiments, the reference capsid protein is an Anc80L65 capsid protein or a modification thereof, and the targeting peptide is (i) between S586 and T589 of the Anc80L65 capsid protein, thereby replacing A587 and N588 of the Anc80L65 capsid protein, or (ii) between Q585 and N588 of the Anc80L65 capsid protein, thereby replacing S586 and N588 of the Anc80L65 capsid protein. and A587 of the Anc80L65 capsid protein, thereby substituting Q585 and S586 of the Anc80L65 capsid protein; (iv) between A587 and A590 of the Anc80L65 capsid protein, thereby substituting N588 and T589 of the Anc80L65 capsid protein; or (v) between S586 and A587 of the Anc80L65 capsid protein.

[0217] 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 S587 and N588 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues S587 and N588.

[0218] 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 A587 and N588 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues A587 and N588.

[0219] 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 A587 and N588 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues A587 and N588.

[0220] 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 A587 and N588 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues A587 and N588.

[0221] 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 A587 and N588 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues A587 and N588.

[0222] 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 A587 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 S586 and T589 of the reference AAV capsid protein, thereby replacing amino acid residues A587 and N588.

[0223] 6.2.3. MODIFIED AAV CAPSID PROTEINS WITH FLANKING REGIONS In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region on the N-terminus of the targeting peptide, a C-terminal flanking region on the C-terminus of the targeting peptide, or both.

[0224] In some embodiments, the N-terminal flanking region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues. In a non-limiting example, the N-terminal flanking region comprises 11 amino acids, wherein the amino acids are selected from amino acid residues 578-588 of AAV9, 577-587 of AAV9, or 576-586 of AAV9.

[0225] In some embodiments, the N-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 558-589 (e.g., 558-588, 558-587, 558-586, and 558-585) of a reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the N-terminal flanking region comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from amino acids 558 to 589 of a reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0226] In some embodiments, the N-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 576-585 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the N-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 576-586 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the N-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 576-587 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the N-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids of a reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the N-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 576 to 589 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0227] In some embodiments, the N-terminal flanking region has the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), where B1, B2, and B3 are each independently selected from any amino acid residue. In some embodiments, the N-terminal flanking region has the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), where B1 is selected from glutamate (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H).

[0228] In some embodiments, the N-terminal flanking region has a sequence of SYGQVATNHQS (SEQ ID NO: 55848). In some embodiments, the N-terminal flanking region comprises a sequence having 1, 2, 3, 4, 5 or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from the sequence of SYGQVATNHQS (SEQ ID NO: 55848).

[0229] In some embodiments, the N-terminal flanking region replaces the N-terminal reference sequence of a reference AAV capsid protein, wherein the N-terminal reference sequence has at least 60% (e.g., at least 70%, at least 80%, at least 90%, or at least 95%) sequence identity with the N-terminal flanking region and is located N-terminal to the insertion site of the targeting peptide within the reference AAV capsid protein. The modified AAV capsid protein of claim 34, wherein the N-terminal reference sequence has the sequence of B1YGB2VATNB3QS (SEQ ID NO: 55849).

[0230] In some embodiments, the modified AAV capsid protein further comprises a C-terminal flanking region C-terminal to the targeting peptide.

[0231] In some embodiments, the C-terminal flanking region has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues. In a non-limiting example, the C-terminal flanking region comprises 13 amino acids, wherein the amino acids are selected from amino acid residues 589-602 of AAV9, 588-601 of AAV9, or 587-602 of AAV9.

[0232] In some embodiments, the C-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 589-635 of a reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the C-terminal flanking region comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from amino acid residues 589-635, where residues 589-635 relative to the reference sequence are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0233] In some embodiments, the C-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 589-601 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the C-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 590-601 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the C-terminal flanking region comprises at least four (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10) contiguous amino acid residues from amino acids 591-601 of the reference AAV capsid protein, where the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0234] In some embodiments, the C-terminal flanking region has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851), wherein Z1, Z2, Z3, and Z4 are each independently selected from any amino acid residue. In some embodiments, the C-terminal flanking region has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55861), wherein Z1 is selected from threonine (T) or tryptophan (W), Z2 is selected from asparagine (N) or glutamine (Q), Z3 is selected from serine (S) or asparagine (N), and Z4 is selected from alanine (A) or isoleucine (I).

[0235] In some embodiments, the C-terminal flanking region has a sequence of AQAQTGWVQNQGI (SEQ ID NO: 55850). In some embodiments, the C-terminal flanking region comprises a sequence having 1, 2, 3, 4, 5 or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from the sequence of AQAQTGWVQNQGI (SEQ ID NO: 55850).

[0236] In some embodiments, the C-terminal flanking region replaces the C-terminal reference sequence of a reference AAV capsid protein, and the C-terminal reference sequence has at least 60% (e.g., at least 70%, at least 80%, at least 90%, or at least 95%) sequence identity with the C-terminal flanking region and is located C-terminal to the insertion site of the targeting peptide within the reference AAV capsid protein. In some embodiments, the C-terminal reference sequence has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851).

[0237] In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region having the sequence of B1YGB2VATNB3QS (SEQ ID NO: 55849; B1, B2, and B3 are each independently selected from any amino acid residue), and a C-terminal flanking region having the sequence of AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851; Z1, Z2, Z3, and Z4 are each independently selected from any amino acid residue).

[0238] In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region having the sequence of B1YGB2VATNB3QS (SEQ ID NO: 55860, where B1 is selected from glutamate (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H)), and a C-terminal flanking region having the sequence of AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55861, where Z1 is selected from threonine (T) or tryptophan (W), Z2 is selected from asparagine (N) or glutamine (Q), Z3 is selected from serine (S) or asparagine (N), and Z4 is selected from alanine (A) or isoleucine (I)).

[0239] In one embodiment, the modified AAV capsid protein further comprises an N-terminal flanking region having the sequence SYGQVATNHQS (SEQ ID NO: 55848) and a C-terminal flanking region having the sequence AQAQTGWVQNQGI (SEQ ID NO: 55850).

[0240] In some embodiments, the modified AAV capsid protein further comprises the amino acid sequence of B1YGB2VATNB3QSPLMGAVHLYAQAQTGZ1VZ2Z3QGZ4 (sequence number 55852), wherein B1, B2, B3, Z1, Z2, Z3, and Z4 are each independently selected from any amino acid residue. In some embodiments, the modified AAV capsid protein further comprises the amino acid sequence of B1YGB2VATNB3QSPLMGAVHLYAQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55862), wherein B1 is selected from glutamate (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), B3 is selected from leucine (L) or histidine (H), Z1 is selected from threonine (T) or tryptophan (W), Z2 is selected from asparagine (N) or glutamine (Q), Z3 is selected from serine (S) or asparagine (N), and Z4 is selected from alanine (A) or isoleucine (I).

[0241] In one embodiment, the targeting peptide for VR VIII is SEQ ID NO: 29. In some embodiments, the targeting peptide is SEQ ID NO: 29 and, when inserted into VR VIII, one or more residues are replaced by the insertion.

[0242] In some embodiments, the targeting peptide is a peptide having at least 90% (e.g., 92%, 94%, 96%, or 98%) sequence identity to the sequence of SEQ ID NO: 29. In some embodiments, the targeting peptide is a peptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from the sequence of SEQ ID NO: 29.

[0243] 6.2.4. Further Modifications of Modified AAV Capsid Proteins In some embodiments, the modified AAV capsid protein has one or more modifications, including amino acid insertions, deletions, substitutions, or combinations thereof, compared to a reference AAV capsid protein.

[0244] In some embodiments, the one or more modifications comprise an amino acid insertion, deletion, substitution, or a combination thereof to introduce a targeting peptide into VR VIII of the reference AAV capsid protein.

[0245] In some embodiments, the one or more modifications comprise amino acid modifications outside VR VIII of the reference AAV capsid protein.

[0246] In some embodiments, the one or more modifications outside VR VIII of the reference AAV capsid protein include one or more modifications of VR I, VR II, VR III, VR IV, VR V, VR VI, or VR VII.

[0247] In some embodiments, variable region I (VR I) corresponds to the sequence between about position 259 and about position 275 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR I corresponds to the sequence between about position 262 and about position 272 of an AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0248] In some embodiments, variable region II (VR II) corresponds to the sequence between about position 329 and about position 336 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR II corresponds to the sequence between about position 330 and about position 335 of an AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0249] In some embodiments, variable region III (VR III) corresponds to the sequence between about position 378 and about position 400 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR III corresponds to the sequence between about position 385 and about position 394 of an AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0250] In some embodiments, variable region IV (VR IV) corresponds to the sequence between about position 438 and about position 480 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR IV corresponds to the sequence between about position 456 and about position 476 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR IV corresponds to the sequence between about position 449 and about position 468 of an AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0251] In some embodiments, the variable region V (VR V) corresponds to the sequence between about position 483 and about position 518 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, the VR V corresponds to the sequence between about position 494 and about position 512 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, the VR V corresponds to the sequence between about position 487 and about position 504 of an AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0252] In some embodiments, variable region VI (VR VI) corresponds to the sequence between about position 531 and about position 549 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR VI corresponds to the sequence between about position 533 and about position 545 of an AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0253] In some embodiments, variable region VII (VR VII) corresponds to the sequence between about position 551 and about position 567 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR VII corresponds to the sequence between about position 553 and about position 563 of an AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0254] In some embodiments, variable region VIII (VR VIII) corresponds to the sequence between about position 570 and about position 605 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, variable region VIII (VR VIII) corresponds to the sequence between about position 576 and about position 601 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, variable region VIII (VR VIII) corresponds to the sequence between about position 576 and about position 608 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, variable region VIII (VR VIII) corresponds to the sequence between about position 579 and about position 594 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR VIII corresponds to a sequence between about position 585 and about position 591 of an AAV capsid protein (eg, a reference capsid or a modified capsid).

[0255] In some embodiments, variable region IX (VR IX) corresponds to a sequence between about position 709 and about position 736 of an AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR IX corresponds to a sequence between about position 714 and about position 721 of an AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0256] In some embodiments, the variable region of the AAV capsid is selected from Padron et al., J. Virology, 79(8):5047-5058(2005), doi.org / 10.1128 / JVI.79.8.5047-5058.2005; Dimattia et al., J. Virology, 86(12):6947-6958(2012), doi.org / 10.1128 / JVI.07232-11; Meyer et al., eLIFE;8:e44707.DOI:doi.org / 10.7554 / eLife.44707(2019); Goertsen et al., Nat Neurosci., 25(1):106-115. doi:10.1038 / s41593-021-00969(2022), each of which is incorporated herein by reference in its entirety.

[0257] In some embodiments, the variable region of an AAV capsid is defined by the presence of amino acids in surface-exposed loops. In some embodiments, the variable region of an AAV capsid is defined by the presence of amino acids in surface-exposed loops, which contribute to tropism (i.e., contribute to binding of the AAV virion to a receptor). In some embodiments, the presence of a targeting peptide in the modified capsid protein can alter the amino acid residues present in the surface-exposed loops.

[0258] In some embodiments, the one or more modifications outside VR VIII of the reference AAV capsid protein include one or more modifications of VR IV and VR V.

[0259] In some embodiments, the one or more modifications in VR IV include modifications that result in the introduction of the sequence of SEQ ID NO: 30. In some embodiments, the one or more modifications in VR VI result in the introduction of a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of SEQ ID NO: 30. In some embodiments, the one or more modifications in VR VI result in the introduction of a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from the sequence of SEQ ID NO: 30.

[0260] In some embodiments, the one or more modifications in VR V include modifications that result in the introduction of the sequence of SEQ ID NO: 31. In some embodiments, the one or more modifications in VR VI result in the introduction of a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of SEQ ID NO: 31. In some embodiments, the one or more modifications in VR VI result in the introduction of a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from the sequence of SEQ ID NO: 31.

[0261] In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR I derived from a second reference capsid protein (e.g., any of the reference capsid proteins described herein). In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR II derived from the second reference capsid protein (e.g., any of the reference capsid proteins described herein). In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR III derived from a second reference capsid protein (e.g., any of the reference capsid proteins described herein). In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR IV derived from the second reference capsid protein (e.g., any of the reference capsid proteins described herein). In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR V from a second reference capsid protein (e.g., any of the reference capsid proteins described herein).In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR VI derived from a second reference capsid protein (e.g., any of the reference capsid proteins described herein). In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR VII derived from the second reference capsid protein (e.g., any of the reference capsid proteins described herein). In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR VIII derived from a second reference capsid protein (e.g., any of the reference capsid proteins described herein). In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR IX derived from the second reference capsid protein (e.g., any of the reference capsid proteins described herein).

[0262] In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) sequence identity to SEQ ID NO: 142 and comprises VR I from AAV9. In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) sequence identity to SEQ ID NO: 142 and comprises VR IV from AAV9. In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) sequence identity to SEQ ID NO: 142 and comprises VR V from AAV9. In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) sequence identity to SEQ ID NO: 142 and comprises VR VIII from AAV9.

[0263] In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises two or more variable regions (VR I, II, III, IV, V, VI, VII, VIII, or IX) from a second reference capsid protein (e.g., any of the reference capsid proteins described herein). In some embodiments, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to the sequence of a reference AAV capsid protein (e.g., any of the reference capsid proteins described herein) and comprises a VR IV from a second reference capsid protein (e.g., any of the reference capsid proteins described herein) and a VR V from a second reference capsid protein (e.g., any of the reference capsid proteins described herein). In one embodiment, the modified capsid protein has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to SEQ ID NO: 142 and comprises a VR IV from AAV9 and a VR V from AAV9.

[0264] 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. In preferred embodiments, AAVs containing the modified AAV capsid proteins have increased targeting to target cells, tissues, or organs when administered to a subject. In some embodiments, AAVs containing 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 the 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.

number

[0265] The formula for scaled log2 fold change is provided below.

number

[0266] 6.2.5. Reference AAV Capsid Proteins The reference AAV capsid protein used in various embodiments of the present disclosure is the VP1, VP2, or VP3 capsid protein of AAV known in the art. It can be the VP1, VP2, or VP3 capsid protein of a naturally occurring AAV variant or a non-naturally occurring AAV variant. The reference AAV capsid protein does not contain the targeting peptide disclosed herein in VR VIII.

[0267] Non-naturally occurring VP1, VP2, or VP3 capsid proteins include capsid proteins generated by biological or chemical modification, in silico design, or mutation 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 further include artificial capsid proteins created by in silico 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.

[0268] In some embodiments, the reference AAV capsid protein is selected from the group consisting of AAV9 (Genbank Accession Number: AAS99264.1), AAV1 (Genbank Accession Number: AAD27757.1), AAV2 (Genbank Accession Number: AAC03780.1), AAV3 (Genbank Accession Number: AAC55049.1), AAV3b (Genbank Accession Number: AF028705.1), AAV4 (Genbank Accession Number: AAC58045.1), AAV5 (Genbank Accession Number: AAD13756.1), AAV6 (Genbank Accession Number: AF028704.1), AAV7 (Genbank Accession Number: AAN03855.1), AAV In certain embodiments, the capsid protein is that of AAV8 (Genbank accession number: AAN03857.1), AAV10 (Genbank accession number: AAT46337.1), AAVrh10 (Genbank accession number: AY243015.1), AAV11 (Genbank accession number: AAT46339.1), AAV12 (Genbank accession number: ABI16639.1), or AAV13 (Genbank accession number: ABZ10812.1), AAVpol (Genbank accession number: FJ688147.1). In certain embodiments, the AAV capsid protein is that of AAV9 (Genbank accession number: AAS99264.1).

[0269] 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 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 to 736 of AAV9 VP1, and the VP3 protein may have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1.

[0270] Reference AAV capsid proteins are 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 The reference AAV capsid protein may be a VP1 capsid protein having the sequence of any member of an ancestral AAV library selected from SEQ ID NO:142 (Anc80L65 (AKU89595)), Anc127 (AKU89603), and Anc80L65 (AKU89595). 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 protein. When a SEQ ID NO for a library sequence is used in this disclosure, it refers to the sequence of any one member of the library.

[0271] In some embodiments, the reference AAV capsid protein is a liver toggle mutant described in WO 2019 / 217911, which is incorporated by reference in its entirety.

[0272] AAV 2, AAV 1, AAV 6, AAV 3, AAV, and 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.43-B. p.63-B、hu.56-B、hu.45-B、rh.57-B、rh.35-B、rh.58-B、rh.2 8-B、rh.51-B、rh.19-B、rh.49-B、rh.52-B、rh.13-B、AAV2-B、r p.20-B、rh.24-B、rh.64-B、hu.27-B、hu.21-B、hu.22-B、hu.2 3-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、r h.53-E、hu.37-E、rh.43-E、rh.50-E、rh.49-E、rh.61-E、hu.4 1-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、Anc80-50、Anc80L59、Anc80L60、Anc80L59、Anc80L60、Anc80L12 80-156、Anc80-751、Anc80-1029、Anc80-1712、Anc110、およびAn c80DI, VP1, VP2, 3, VP1, VP1, 100,000.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.

[0273] In some embodiments, the reference AAV capsid protein is a protein having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 54-131 and 143-158. In some embodiments, the reference AAV capsid protein is a protein having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues that differ (e.g., by insertion, deletion, or substitution) from a sequence selected from SEQ ID NOs: 54-131 and 143-158. In some embodiments, the reference AAV capsid protein is a protein having a sequence selected from SEQ ID NOs: 54-131 and 143-158. 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 143-158.

[0274] 6.2.6. Other Modifications In some embodiments, the modified AAV capsid protein comprises one or more additional modifications compared to a reference AAV capsid protein, which may be insertions, deletions, substitutions, or combinations thereof, and may be located within and / or outside VRVIII.

[0275] In some embodiments, the modified AAV capsid protein differs from the reference AAV capsid protein by having one or more amino acid substitutions in a 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 28).

[0276] 6.3. Polynucleotides Encoding Modified AAV Capsid Proteins, Vectors, and Host Cells In another aspect, the disclosure provides a polynucleotide encoding a modified AAV capsid protein described herein. In some embodiments, the polynucleotide is codon-optimized for expression in bacterial or mammalian cells.

[0277] In some embodiments, the polynucleotide is inserted into an expression vector. In some embodiments, the polynucleotide is operably linked to a promoter or a sequence that directs expression of a protein from the polynucleotide. The present disclosure provides a vector comprising a polynucleotide encoding a modified AAV capsid protein. The vector can be used to produce the modified AAV capsid protein. In some embodiments, the vector is used to generate AAV virions comprising the modified AAV capsid protein. In some embodiments, the vector further comprises an AAV rep protein or a fragment thereof. In some embodiments, the modified AAV capsid protein and the reference capsid protein for the rep protein are derived from the same clade of AAV. In some embodiments, the modified AAV capsid protein and the reference capsid protein for the rep protein are derived from different clades of AAV.

[0278] In some embodiments, the polynucleotide is transfected into a host cell. The present disclosure provides a host cell comprising a polynucleotide encoding a modified AAV capsid protein. The host cell can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a mammalian cell or a yeast cell.

[0279] In some embodiments, the host cell further comprises another polynucleotide encoding an AAV protein. In some embodiments, the host cell comprises a functional rep gene. A recombinant nucleic acid vector comprising an AAV inverted terminal repeat (ITR) and an expressible polynucleotide; and helper functions sufficient to allow packaging of the recombinant nucleic acid vector into a modified AAV capsid protein.

[0280] In some embodiments, the components necessary for a host cell to package a recombinant nucleic acid vector into a modified AAV capsid protein are provided in trans to the host cell. In some embodiments, any one or more of the necessary components (e.g., the recombinant nucleic acid vector, the rep sequence, the cap sequence, and / or helper functions) are provided by a stable host cell that has been engineered to contain one or more of the necessary components using methods known to those of skill in the art. In some embodiments, such a stable host cell contains the necessary components under the control of an inducible promoter. In some embodiments, the necessary components are under the control of a constitutive promoter.

[0281] 6.4. Modified AAV virions The present disclosure further provides modified recombinant AAV (rAAV) virions comprising the modified AAV capsid proteins described herein. In some embodiments, the modified rAAV comprises a modified AAV capsid protein and a recombinant nucleic acid vector.

[0282] In some embodiments, the modified rAAV comprising the modified AAV capsid protein achieves higher infection of the target after administration to a mammalian subject compared to an rAAV comprising a corresponding reference AAV capsid protein, hi some embodiments, the modified rAAV achieves higher expression in the target of the expressible polynucleotide in the recombinant nucleic acid vector after administration to a mammalian subject compared to the expression of the expressible polynucleotide administered with an rAAV comprising the corresponding reference AAV capsid protein.

[0283] In some embodiments, a modified rAAV comprising a modified AAV capsid protein achieves lower off-target infection after administration to a mammalian subject compared to a rAAV comprising a corresponding reference AAV capsid protein. In some aspects, a modified rAAV achieves lower off-target expression of an expressible polynucleotide in a recombinant nucleic acid vector after administration to a mammalian subject compared to expression of an expressible polynucleotide administered with a rAAV comprising a corresponding reference AAV capsid protein. In some embodiments, the corresponding reference AAV capsid protein is the same capsid protein as the modified AAV capsid protein, except that it does not contain the targeting peptide described above.

[0284] In some embodiments, the target is the brain, muscle, spinal cord, eye, or other organ. In some embodiments, the non-target tissue is muscle, liver, or other organ. In one embodiment, the target is the CNS.

[0285] In some embodiments, the modified rAAV has lower hepatotoxicity than a rAAV containing a corresponding reference AAV capsid protein administered by the same route of administration and at the same dose. In some embodiments, the lower hepatotoxicity is due to detargeting of the modified rAAV to the liver.

[0286] 6.5. Methods for producing rAAV The rAAV of the present disclosure comprises a recombinant nucleic acid vector containing a heterologous polynucleotide. In some embodiments, the heterologous polynucleotide comprises an expressible polynucleotide operably linked to an ERE. The expressible polynucleotide and ERE optionally replace the AAV genome coding region (e.g., replacing the AAV rep gene and cap gene). The expressible polynucleotide and ERE are generally flanked on both sides by AAV inverted terminal repeat (ITR) regions, although a single ITR may be sufficient to perform the functions normally associated with a structure containing two ITRs (see, for example, WO 94 / 13788), and therefore, a vector construct with only one ITR may be used in conjunction with the rAAV of the present disclosure.

[0287] In some embodiments, the rAAV of the present disclosure comprises a therapeutic protein coding sequence operably linked to an ERE, where the therapeutic protein coding sequence and ERE optionally replace the AAV genome coding region (e.g., replacing the AAV rep and cap genes).

[0288] To replicate and package the vector, the missing functions are complemented by a packaging gene or genes that together encode the necessary functions for the products of the various missing rep and / or cap genes. The packaging gene or gene cassette, in one embodiment, is not flanked by AAV ITRs and, in one embodiment, does not share substantial homology with the rAAV genome.

[0289] The rAAV vector constructs and complementary packaging gene constructs can be implemented in several different forms: viral particles, plasmids, and stably transformed host cells can all be used to introduce such constructs into packaging cells, either transiently or stably.

[0290] In certain embodiments of the present invention, the AAV vector and complementary packaging genes, if present, are provided in the form of bacterial plasmids, AAV particles, or any combination thereof.In other embodiments, the AAV vector sequence, packaging genes, or both, are provided in the form of genetically modified (preferably genetically modified) eukaryotic cells.The development of genetically modified host cells to express the AAV vector sequence, AAV packaging genes, or both, provides a reliable source of expressed material.

[0291] Thus, various different genetically modified cells can be used in accordance with the present invention. Illustratively, mammalian host cells can be used with at least one intact copy of a stably integrated rAAV vector. An AAV packaging plasmid containing at least the AAV rep gene operably linked to a promoter can be used to provide replication functions (as described in U.S. Pat. No. 5,658,776). Alternatively, a stable mammalian cell line with an AAV rep gene operably linked to a promoter can be used to provide replication functions (see, e.g., WO 95 / 13392, WO 98 / 23018, and U.S. Pat. No. 5,656,785). The AAV cap gene, which provides the encapsidation proteins described above, can be provided together with or separately from the AAV rep gene (see, e.g., the above patent documents and WO 98 / 27204).

[0292] Thus, the rAAV of the present disclosure can be assembled, for example, by expressing its components in a packaging host cell. The components of the viral particle (e.g., rep sequence, cap sequence, inverted terminal repeat (ITR) sequence) can be introduced into the packaging host cell using one or more viral vectors.

[0293] Once assembled, the rAAV particles can be purified, if desired, using standard methods. As used herein, "purified" viral particles refer to viral particles that have been removed from components of the mixture from which they were made, including, but not limited to, viral components (e.g., rep sequences, cap sequences), packaging host cells, and partially or incompletely assembled viral particles.

[0294] 6.6. Pharmaceutical Compositions Comprising Modified rAAV In one aspect, the present disclosure provides a pharmaceutical composition comprising a modified AAV capsid protein or modified rAAV of the present disclosure and a pharmaceutically acceptable carrier. The modified rAAV can comprise a recombinant nucleic acid vector containing the modified AAV capsid protein described herein and an expressible polynucleotide.

[0295] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising an rAAV whose genome comprises a therapeutic protein used to treat and / or prevent a disease of the central nervous system, wherein the coding sequence for the therapeutic protein is operably linked to an expression regulatory element (ERE).

[0296] Pharmaceutical compositions can be formulated using one or more carriers, excipients, stabilizers, and adjuvants to, for example, (1) enhance stability, (2) increase cell transfection or transduction, (3) allow for sustained or delayed release, (4) alter biodistribution (e.g., targeting rAAV particles to specific tissues or cell types), (5) increase translation of the encoded protein in vivo, and / or (6) alter the release profile of the encoded protein in vivo.

[0297] Formulations of the pharmaceutical compositions provided herein can include, but are not limited to, saline, which can be formulated with various buffers (e.g., phosphate buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, water, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, nanoparticle mimetics, and combinations thereof.

[0298] Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparative methods include bringing into association the active ingredient with the carrier and / or one or more other accessory ingredients (e.g., excipients, stabilizers, and adjuvants).

[0299] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.As used herein, a unit dose refers to a discrete amount of pharmaceutical composition containing a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dosage, for example, 1 / 2 or 1 / 3 of such a dosage.

[0300] The relative amounts of the active ingredient (e.g., rAAV), pharmaceutically acceptable carrier, and / or any additional components of a pharmaceutical composition according to the present disclosure may vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered.

[0301] A variety of carriers, excipients, stabilizers and adjuvants for formulating pharmaceutical compositions, and the techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 22nd Revised Edition, Pharmaceutical Press, 2012, its entirety is incorporated herein by reference).The use of suitable conventional carriers, excipients, stabilizers and adjuvants is contemplated within the scope of the present disclosure.

[0302] In some embodiments, the pharmaceutical composition contains about 1×10 1 ~Approx. 1×10 16 Genome copies (GC) / ml of rAAV (e.g., approximately 1 x 10 3 ~Approx. 1×10 14 It is in the form of a solution containing 100 mg of HCl (a solution containing 100 mg of HCl / ml of HCl).

[0303] 6.6.1. Route of Administration The present disclosure provides methods for administering rAAV to transfer polynucleotides into the CNS. In some embodiments, the rAAV is administered locally or systemically.

[0304] In certain embodiments, the rAAV is administered locally to the CNS. In some embodiments, the rAAV is administered to the cerebrospinal fluid (CSF) of the subject. In some embodiments, the rAAV is administered to the cisterna magna, intraventricular space, ventricle, subarachnoid space, intrathecal space, and / or ependyma of the subject.

[0305] In some embodiments, the rAAV is administered intrathecally, intracranially, intracerebroventricularly (ICV), or intraparenchymally or into the lateral ventricles of the brain.

[0306] In some embodiments, the rAAV is administered by lumbar injection (e.g., into the lumbar cisterna magna) and / or intracisternal (ICM) injection.

[0307] In some embodiments, the rAAV is administered to the ventricular system, ie, to the rostral lateral ventricle, and / or to the caudal lateral ventricle, and / or to the right lateral ventricle, and / or to the left lateral ventricle, and / or to the right rostral lateral ventricle, and / or to the left rostral lateral ventricle, and / or to the right caudal lateral ventricle, and / or to the left caudal lateral ventricle.

[0308] In some embodiments, the rAAV is administered such that the rAAV contacts ependymal cells of the subject, such that the encoded polypeptide is expressed by the cells, and optionally, the polypeptide is expressed by the cells.

[0309] In some embodiments, the polypeptide is expressed and / or distributed in the lateral ventricles, CSF, and / or brain (eg, striatum, thalamus, medulla, cerebellum, occipital cortex, and / or prefrontal cortex).

[0310] In some embodiments, the rAAV is administered intravenously or systemically.

[0311] In some embodiments, the rAAV is administered interdigitally.

[0312] To specifically deliver rAAV to specific regions of the CNS, particularly the brain, it can be administered via stereotactic microinjection. For example, on the day of surgery, the patient may have a stereotactic frame base fixed in place (screwed into the skull). The brain, with its fiducial marks and MRI compatibility, can be imaged using high-resolution MRI. The MRI images can then be transferred to a computer running stereotactic software. A series of coronal, sagittal, and axial images can be used to determine the target site and trajectory of vector injection. The software directly converts the trajectory into 3D coordinates appropriate for the stereotactic frame. A burr hole can be drilled above the entry site, and the stereotactic device can be localized with a needle implanted at a given depth. The vector can then be injected in a pharmaceutically acceptable carrier. The AAV vector can then be administered by direct injection into the primary target site and retrogradely transported via axons to distal target sites. Additional administration routes can be used, such as superficial cortical application under direct visualization, or other non-stereotactic applications.

[0313] In some embodiments, the rAAV is delivered by a pump. The pump may be implantable. Another convenient method for administering the rAAV is to use a cannula or catheter.

[0314] In some embodiments, rAAV is administered by convection-enhanced delivery (CED) (Nguyen et al., (2003) J. Neurosurg. 98:584-590), which has been clinically used for gene therapy (AAV2-hAADC) of Parkinson's disease (Fiandaca et al., (2008) Exp. Neurol. 209:51-57). The principle underlying CED involves pumping the injectate into the brain parenchyma under sufficient pressure to overcome the hydrostatic pressure of the interstitial fluid, thereby bringing the injected particles into intimate contact with the dense perivascular parts of the brain. The pulsation of these blood vessels acts as a pump, distributing particles over large distances throughout the parenchyma (Hadaczek et al., (2006) Hum. Gene Ther. 17:291-302). To enhance the safety and efficacy of CED, reflux-resistant cannulas (Krauze et al., (2009) Methods Enzymol. 465:349-362) can be used in conjunction with real-time MRI-based monitoring of delivery. Monitoring of delivery allows for quantification and control of abnormal events such as cannula reflux and leakage of infusate into the ventricle (Eberling et al., (2008) Neurology 70:1980-1983; Fiandaca et al., (2009) Neuroimage 47 Suppl. 2:T27-35; Saito et al., (2011) Journal of Neurosurgery Pediatrics 7:522-526). US 20190111157A1 provides an improved procedure for achieving widespread expression of AAV vectors in the cortex and / or striatum.

[0315] In some embodiments, rAAV is administered to the striatum.In some embodiments, rAAV is administered to at least the putamen and caudate nucleus of the striatum.In some embodiments, rAAV is administered to at least the putamen and caudate nucleus of each hemisphere of the striatum.In some embodiments, rAAV is administered to at least one site in the caudate nucleus and two sites in the putamen.

[0316] In some embodiments, rAAV is delivered by intraparenchymal administration to specific regions of the brain, ie, the putamen, striatum, basal forebrain, substantia nigra, and / or ventral tegmental area.

[0317] In some embodiments of the above aspects and embodiments, the rAAV is delivered by stereotactic delivery. In some embodiments, the rAAV is delivered by convection-enhanced delivery (CED). In some embodiments, the rAAV is delivered using a CED delivery system. In some embodiments, the CED system includes a cannula. In some embodiments, the cannula is a reflux-resistant cannula or a stepped cannula. In some embodiments, the CED system includes a pump. In some embodiments, the pump is a manual pump. In some embodiments, the pump is an osmotic pump. In some embodiments, the pump is an infusion pump.

[0318] The modified rAAV of the present disclosure can be administered to a subject (e.g., a human or non-human mammal) in a suitable carrier. Suitable carriers include saline, which can be formulated with various buffers (e.g., phosphate-buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, and water. The modified rAAV is typically administered in an amount sufficient to transduce or infect the desired cells and provide a sufficient level of gene transfer and expression to provide a therapeutic benefit without undue adverse effects. Conventional pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to organs such as muscle, liver, or lung, oral, intranasal, intratracheal, intrathecal, intravenous, intramuscular, intraocular, subcutaneous, intradermal, or other routes of administration. If desired, routes of administration can be combined.

[0319] 6.6.2.Target The present disclosure provides a method for transferring a polynucleotide into the central nervous system (CNS) of a subject, such as a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has a CNS disease. In some embodiments, the subject has a genetic defect associated with a CNS disease or disorder.

[0320] In some embodiments, the CNS disease or disorder is adrenoleukodystrophy, Alexander disease, Alzheimer's disease, amyotrophic lateral sclerosis, Angelman syndrome, ataxia-telangiectasia, Canavan disease, Charcot-Marie-Tooth syndrome, Cockayne syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), hearing loss, Duchenne muscular dystrophy, epilepsy, essential tremor, Fragile X syndrome, Friedreich's ataxia, Gaucher disease, GM1 gangliosidosis, GM2 gangliosidosis, Huntington's disease, frontotemporal glial cell carcinoma, glauc ... and / or encephalopathy syndrome (FTD), Lesch-Nyhan syndrome, maple syrup urine disease, Menkes syndrome, metachromatic leukodystrophy (MLD), myotonic dystrophy, multiple sclerosis, narcolepsy, neurofibromatosis, Niemann-Pick disease, Parkinson's disease, phenylketonuria, Prader-Willi syndrome, Refsum disease, Rett syndrome, spinal muscular atrophy, spinocerebellar ataxia, Tangier disease, Tay-Sachs disease, tuberous sclerosis, von Hippel-Lindau syndrome, Williams syndrome, Wilson's disease, and Zellweger syndrome.

[0321] In some embodiments, the CNS disease or disorder is a demyelinating or white matter disease. In some embodiments, the subject has a monogenic defect. In some embodiments, the subject has a genetic defect in a protein expressed in the CNS. In some embodiments, the subject has a monogenic defect in a protein expressed in the CNS.

[0322] In some embodiments, the subject has a lysosomal storage disease (LDS). In some embodiments, the subject has mucopolysaccharidosis type I, such as Hurler syndrome and variant Scheie syndrome and Hurler-Scheie syndrome, Hunter syndrome, mucopolysaccharidosis type III, such as Sanfilippo syndrome, mucopolysaccharidosis type IV, such as Morquio syndrome, mucopolysaccharidosis type VI, such as Maroteaux-Lamy syndrome, mucopolysaccharidosis type II, mucopolysaccharidosis type III, mucopolysaccharidosis type IV, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type VIII, mucopolysaccharidosis type IX, Tay-Sachs syndrome ... IX, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type VIII, mucopolysaccharidosis type IX, Tay-Sachs syndrome, mucopolysaccharidosis type IX, mucopolysaccharidosis type VI, mucopolysaccharidosis type VII, mucopolysaccharidosis type VIII, mucopolysaccharidosis type IX, mucopolysaccharidosis type IX, mucopolysaccharidosis type IX, mucopolysaccharidosis type IX, mucopolysaccharidosis type IX, mucopolysaccharidosis type IX, mucopolysaccharidosis type IX, mucopolysaccharidosis type IX, mucopolysaccharidosis The patient has a disorder selected from the group consisting of: fibromyalgia, Sandhoff disease, GM1 gangliosidosis, Fabry disease, Krabbe disease, leukodystrophy, metachromatic leukodystrophy, Pompe disease, fucosidosis deficiency, alpha-mannosidosis deficiency, beta-mannosidosis deficiency, Gaucher disease, infantile Batten disease, classic late-infantile Batten disease, juvenile Batten disease, Batten disease, other forms of Niemann-Pick disease, Niemann-Pick disease without sphingomyelinase deficiency, and Wolman disease.

[0323] In some embodiments, the subject has brain cancer. In some embodiments, the subject has brain metastases from cancer. In some embodiments, the subject has brain metastases from breast cancer. In some embodiments, the subject has brain metastases from HER2-positive breast cancer.

[0324] Dosage The dose of viral vector administered to a subject depends primarily on factors such as the condition being treated and the subject's age, weight, and health. For example, a therapeutically effective amount of viral vector administered to a human subject generally ranges from about 0.1 ml to about 10 ml of a solution containing a concentration of about 1E1 to about 1E16 genome copies (GC) per ml of virus (e.g., a solution containing a concentration of about 1E3 to about 1E14 GC / ml). In some embodiments, the total dose of rAAV administered to a subject is less than 3E14 GC, e.g., 1E14 GC or less, 5E13 GC or less, 1E13 GC or less, 5E12 GC or less, or 1E12 GC or less.

[0325] In another embodiment, a therapeutically effective amount of a viral vector administered to a human subject generally ranges from about 0.1 ml to about 10 ml of solution containing virus at a concentration of about 1E1 to 1E12 genome copies (e.g., about 1E3 to 1E9 GC). Transgene transduction and / or expression can be monitored at various time points after administration by DNA, RNA, or protein assays. In some cases, transgene expression levels can be monitored to determine the frequency and / or amount of administration. Dosage regimens similar to those described for therapeutic purposes can also be utilized for immunization.

[0326] In some embodiments, an effective amount is 1E10 to 1E16 genome copies (GC) of rAAV per subject. In some embodiments, an effective dose for a human patient corresponds to a monkey dose of 1E12 to 1E15 GC of rAAV. In some embodiments, an effective dose for a human patient corresponds to a monkey dose of 1E13 to 1E14 GC of rAAV. In some embodiments, an effective dose for a human patient corresponds to a monkey dose of about 4E13 GC of rAAV.

[0327] In some embodiments, an effective amount is 1E11 to 1E15 GC of rAAV per gram brain mass. In some embodiments, an effective amount is 1E11 to 1E13 GC of rAAV per gram brain mass. In some embodiments, an effective amount is 1E11 to 1E12 GC of rAAV per gram brain mass. In some embodiments, an effective amount is 1E12 to 1E14 GC of rAAV per gram brain mass. In some embodiments, an effective amount is about 5E11 GC of rAAV per gram brain mass. In some embodiments, an effective amount is about 2.5E11 GC of rAAV per gram brain mass. In some embodiments, an effective amount is about 5E10 GC of rAAV per gram brain mass. In some embodiments, an effective amount is about 2.5E10 GC of rAAV per gram brain mass.

[0328] In some embodiments, an effective amount is 1E10 to 1E16 genome copies (GC) of rAAV per kg of body weight. In some embodiments, an effective amount is 1E11 to 1E15 genome copies (GC) of rAAV per kg of body weight. In some embodiments, an effective amount is 1E12 to 5E14 genome copies (GC) of rAAV per kg of body weight. In some embodiments, an effective amount is 0.5E13 to 2E14 genome copies (GC) of rAAV per kg of body weight.

[0329] The transduction and / or expression of the transgene can be monitored at various times after administration by DNA, RNA or protein assay.In some cases, the expression level of the transgene can be monitored to determine the frequency and / or amount of administration.Similar administration regimens as described for therapeutic purposes can also be used for immunization.

[0330] In one aspect, the present invention provides a unit dose of an rAAV provided herein. The unit dose comprises about 0.1 ml to about 10 ml of a solution containing a concentration of about 1E9 to 1E17 genome copies (GC) per ml of an rAAV described herein. In some embodiments, the unit dose comprises about 1E10 to 1E16 genome copies (GC) per ml of an rAAV described herein. In some embodiments, the unit dose comprises about 1E11 to 1E15 genome copies (GC) per ml of an rAAV described herein. In some embodiments, the unit dose comprises about 1E12 to 1E14 genome copies (GC) per ml of an rAAV described herein. In some embodiments, the unit dose comprises about 2E13 genome copies (GC) per ml of an rAAV described herein.

[0331] In some embodiments, the unit dose comprises about 1E10 to 1E16 genome copies (GC) of an rAAV described herein. In some embodiments, the unit dose comprises about 1E11 to 1E15 genome copies (GC) of an rAAV described herein. In some embodiments, the unit dose comprises about 1E12 to 1E15 genome copies (GC) of an rAAV described herein. In some embodiments, the unit dose comprises about 1E13 to 1E15 genome copies (GC) of an rAAV described herein.

[0332] The unit dose further comprises a pharmaceutically acceptable excipient.

[0333] 6.6.4. Targeting The pharmaceutical composition can be used to deliver recombinant nucleic acid vectors to targets in mammalian subjects.When the pharmaceutical composition is administered, the modified rAAV can achieve higher infection of target cells after administration to mammalian subjects compared with the rAAV containing the corresponding reference AAV capsid protein administered by the same administration route and at the same dose.In some embodiments, the modified rAAV achieves higher expression of the expressible polynucleotide in the recombinant nucleic acid genome in target cells after administration to mammalian subjects compared with the expressible polynucleotide administered by the rAAV containing the corresponding reference AAV capsid protein administered by the same administration route and at the same dose.

[0334] The targeting of rAAV can be tested in experimental animals by measuring rAAV infection or polynucleotide expression. In some embodiments, targeting is measured in non-human primates (NHPs), mice, rats, birds, rabbits, guinea pigs, hamsters, livestock (including pigs and sheep), dogs or cats.

[0335] rAAV targeting can be measured after systemic or local administration of rAAV. In some embodiments, rAAV targeting is measured after intravenous infusion or local administration to the CNS. In certain embodiments, targeting is measured after administration to the CNS by lumbar puncture (LP), via intracisternal injection (e.g., approximately L3-L4) or intracisternal administration (ICM).

[0336] In some embodiments, the targeting of the modified rAAV is measured by measuring the copy number ratio of the transgene transcript to the housekeeping gene (e.g., RPP30, actin, GAPDH, or ubiquitin) transcript. In certain embodiments, the transcript is measured by RT-ddPCR. In some embodiments, the ratio is measured after initial administration to a mammal, such as a primate, e.g., a monkey (e.g., a cynomolgus or rhesus monkey) or a mouse.

[0337] In some embodiments, the rAAV of the present disclosure provides at least a 10-fold, at least a 20-fold, at least a 30-fold, at least a 40-fold, at least a 50-fold, at least a 60-fold, at least a 70-fold, at least a 80-fold, at least a 90-fold, at least a 100-fold, at least a 150-fold, at least a 200-fold, at least a 500-fold, at least a 1000-fold ratio of infection (i.e., expression) in the brain (or target regions of the brain) or other tissues (or non-target regions of the brain) compared to AAV9.

[0338] In some embodiments, the brain:control tissue infection ratio is determined by the ratio of each test rAAV 試験 It is measured by comparing the ratio between the copy numbers of the transgene transcript and the housekeeping gene (e.g., RPP30) transcript in the same organ (e.g., brain) or the same tissue (e.g., caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, substantia nigra) in two individuals or two groups of animals administered AAV (e.g., rAAV containing modified AAV capsid proteins) or AAV9.

number

[0339] In some embodiments, rAAV 試験 achieves an infection ratio of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 20, at least 30, at least 40, or at least 50 compared to AAV9 in the brain. 試験 achieves an infection ratio of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or at least 50 compared to AAV9 in one of the target tissues, the caudate nucleus, the frontal cortex, the globus pallidus, the motor cortex, the parietal cortex, the putamen, and the substantia nigra.

[0340] In some embodiments, the targeting of the modified rAAV is measured by measuring the ratio between the copy number of the transgene DNA genome and the copy number of the host gene or locus (e.g., RPP30). In certain embodiments, the genome is measured by RT-ddPCR. In some embodiments, the ratio is measured after the first administration to a mammal, such as a mouse, or a non-human primate, such as a marmoset or rhesus monkey.

[0341] In some embodiments, the brain:control tissue infection ratio (DNA) is determined for each test rAAV. 試験 It is measured by comparing the ratio between the copy numbers of the transgene DNA genome and the housekeeping gene (e.g., RPP30) genome in the same organ (e.g., brain) or the same tissue (e.g., caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, substantia nigra) in two individuals or two groups of animals administered AAV (e.g., rAAV containing modified AAV capsid proteins) or AAV9.

number

[0342] In some embodiments, rAAV試験 achieves an infection ratio of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 20, at least 30, at least 40, or at least 50 compared to AAV9 in the brain. 試験 achieves an infection ratio of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or at least 50 compared to AAV9 in one of the target tissues, the caudate nucleus, the frontal cortex, the globus pallidus, the motor cortex, the parietal cortex, the putamen, and the substantia nigra.

[0343] In certain embodiments, when the (transgene genome / housekeeping genome) in the control tissue is below the limit of detection, the brain:control tissue infection ratio is reported by convention as >10,000.

[0344] In some embodiments, the modified rAAV of the present disclosure provides a brain:comparison tissue infection ratio (DNA) of at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 500, at least 1,000, or at least 10,000. In some embodiments, the muscle is the triceps surae, biceps, heart, or quadriceps.

[0345] In some embodiments, the modified rAAV of the present disclosure provides a brain:comparative tissue infection ratio (DNA) in the range of 0.5-1, 0.5-5, 0.5-10, 1-10, 1-100, 2-8, 5-10, 10-20, 20-80, 10-50, 10-100, 50-80, 100-500, 100-1000, or 500-1000. In some embodiments, the muscle is triceps surae, biceps, heart, or quadriceps. In some embodiments, the modified rAAV achieves a brain:comparative tissue infection ratio (DNA) of at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 500, or at least 1000. In some embodiments, the modified rAAV achieves an infection ratio of brain:control tissue of 0.1 to 1, 1 to 5, 1 to 10, 1 to 20, 1 to 50, 1 to 100, 1 to 200, 1 to 300, 100 to 500, 250 to 750, or 500 to 1000.

[0346] 6.6.4.1 RNA Data - Brain:Liver Infection Rates In some embodiments, the targeting of modified rAAV is measured by measuring the copy number ratio of transgene transcripts to housekeeping gene (e.g., RPP30) transcripts. In certain embodiments, the transcripts are measured by RT-ddPCR. In some embodiments, the ratio is measured after the first administration to mammals, such as mice, or non-human primates, such as marmosets or rhesus monkeys.

[0347] In some embodiments, the brain:liver infection ratio (RNA) is measured by comparing the ratio between the copy numbers of the transgene transcript and the housekeeping gene (e.g., RPP30) transcript in two different organs (e.g., brain vs. liver).

number

[0348] In some embodiments, the modified rAAV of the present disclosure provides a (transgene transcript / housekeeping transcript) ratio in the liver of less than 1000, less than 900, less than 800, less than 700, less than 600, less than 500, less than 400, less than 300, less than 200, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10.

[0349] In certain embodiments, if the (transgene transcript / housekeeping transcript) in the liver is below the limit of detection, the brain:liver infection ratio is reported by convention as >10,000.

[0350] In some embodiments, the modified rAAV of the present disclosure provides a brain:liver infection ratio (RNA) of at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 150, at least 200, at least 500, or at least 1000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra.

[0351] In some embodiments, the modified rAAV of the present disclosure provides a brain:liver infection ratio (RNA) of 1 to 10, 1 to 100, 10 to 20, 10 to 50, 10 to 80, 10 to 100, 20 to 100, 100 to 500, 100 to 1000, or 500 to 1000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra.

[0352] 6.6.4.2 DNA Data - Brain:Liver Infection Ratio In some embodiments, the targeting of the modified rAAV is measured by measuring the ratio between the copy number of the transgene DNA genome and the copy number of the host gene or locus (e.g., RPP30). In certain embodiments, the genome is measured by RT-ddPCR. In some embodiments, the ratio is measured after the first administration to a mammal, such as a mouse, or a non-human primate, such as a marmoset or rhesus monkey.

[0353] In some embodiments, the brain:liver infection ratio (DNA) is measured by comparing the ratio between the copy numbers of the transgene DNA genome and the housekeeping gene (e.g., RPP30) genome in two different organs (e.g., brain vs. liver).

number

[0354] In some embodiments, the modified rAAV of the present disclosure provides a (transgene genome / housekeeping genome) ratio in the liver of less than 1, or in the range of 1 to 10, 1 to 5, 1 to 2, 0.1 to 1, 0 to 1, 0.01 to 0.1, 0.01 to 0.5, or 0.01 to 0.05.

[0355] In certain embodiments, if the (transgene genome / housekeeping genome) in the liver is below the limit of detection, the brain:liver infection ratio is reported by convention as >10,000.

[0356] In some embodiments, the modified rAAV of the present disclosure provides a brain:liver infection ratio (DNA) of at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 500, at least 1,000, or at least 10,000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra.

[0357] In some embodiments, the modified rAAV of the present disclosure provides a brain:liver infection ratio (DNA) in the range of 0.5-1, 0.5-5, 0.5-10, 1-10, 1-100, 2-8, 5-10, 10-20, 20-80, 10-50, 10-100, 50-80, 100-500, 100-1000, or 500-1000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra. In some embodiments, the modified rAAV achieves a brain:liver infection ratio (DNA) of at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 500, or at least 1000. In some embodiments, the modified rAAV achieves a brain:liver infection ratio of 0.1 to 1, 1 to 5, 1 to 10, 1 to 20, 1 to 50, 1 to 100, 1 to 200, 1 to 300, 100 to 500, 250 to 750, or 500 to 1000.

[0358] 6.6.4.3 IHC Data Brain:Liver Infection Ratio In some embodiments, the targeting of the modified rAAV is calculated using the percentage of cells that are successfully transduced and express the transgene in the tissue (e.g., eGFP). In certain embodiments, the expression of the transgene is measured by immunohistochemistry. In some embodiments, the percentage is measured after the first administration to a mammal, such as a mouse, or a non-human primate, such as a marmoset or rhesus monkey.

[0359] In some embodiments, the brain:liver infection ratio (IHC) is measured by comparing the ratio between transgene %GFP+ cells and housekeeping gene (e.g., RPP30) %GFP+ cells in two different organs (e.g., brain vs. liver).

number

[0360] In some embodiments, the modified rAAV of the present disclosure provides a (transgene % GFP / housekeeping % GFP) ratio in the liver of less than 1, less than 5, less than 10, or in the range of 1-10, 1-5, 1-2, 0.1-1, 0-1, 0.01-0.1, 0.01-0.5, or 0.01-0.05.

[0361] In certain embodiments, if the (transgene % GFP / housekeeping % GFP) in the liver is below the limit of detection, the brain:liver infection ratio is reported by convention as >10,000.

[0362] In some embodiments, the modified rAAV of the present disclosure provides a brain:liver infection ratio (IHC) of at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 500, or at least 1,000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra.

[0363] In some embodiments, the modified rAAV of the present disclosure provides a brain:liver infection ratio (IHC) of 1 to 5, 1 to 10, 1 to 100, 2 to 8, 10 to 20, 20 to 30, 10 to 50, 10 to 100, 20 to 80, 50 to 80, 100 to 500, 100 to 1000, or 500 to 1000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra.

[0364] 6.7.How to use The modified rAAV described herein can be used for research and / or therapeutic applications. In some embodiments, the modified rAAV is used for genetic modification of cells in vitro or in vivo. In some embodiments, the modified rAAV is used for gene therapy or vaccination in humans or animals. More specifically, the modified rAAV can be used for gene addition of hepatocytes or non-hepatocytes, gene enhancement, gene delivery of polypeptide therapeutics, gene vaccination, gene silencing, genome editing, gene therapy, RNAi delivery, cDNA delivery, mRNA delivery, miRNA delivery, miRNA sponging, genetic immunization, optogenetic gene therapy, genetic recombination, DNA vaccination, or DNA immunization.

[0365] In some embodiments, the modified rAAV of the present disclosure is used for treatment to treat, ameliorate, or prevent a disease or symptom in a subject. In some embodiments, the disease is a disease of the central nervous system (CNS).

[0366] In some embodiments, the modified rAAV of the present disclosure is used to transfer exogenous polynucleotides into the central nervous system (CNS). In some embodiments, transfer of the exogenous polynucleotide into the CNS results in a CNS:liver infection ratio of greater than 1, as measured by AAV virion genome copies. In some embodiments, transfer of the exogenous polynucleotide into the CNS results in expression of the exogenous polynucleotide in the CNS at a CNS:liver expression ratio of greater than 10. In some embodiments, transfer of the exogenous polynucleotide into the CNS results in expression of the exogenous polynucleotide in the CNS at a CNS:liver expression ratio of greater than 10, as measured by protein expression.

[0367] The modified rAAV of the present disclosure can be administered to a subject in a suitable pharmaceutical carrier.

[0368] The rAAV of the present disclosure is typically administered in an amount sufficient to transduce or infect the desired cells and provide a sufficient level of gene transfer and expression to provide a therapeutic benefit to a subject suffering from a disease. In certain embodiments, the rAAV is administered in an amount sufficient to provide a therapeutic benefit to a subject suffering from a disease of the central nervous system (CNS).

[0369] Conventional pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to an organ such as the brain, intracisternal (ICM), interdigital, intravenous, oral, intranasal, intratracheal, intrathecal, intramuscular, intraocular, subcutaneous, intradermal, or other routes of administration. Routes of administration may be combined, if desired.

[0370] Transduction and / or expression of the transgene can be monitored at various time points after administration by DNA, RNA or protein assays.

[0371] Thus, the present disclosure provides methods of treating and / or preventing muscle diseases and / or muscle degeneration by administering the modified rAAV described herein. [Example]

[0372] 7. Working Example 7.1. Example 1: Evaluation of the CNS tropism of Anc80L65 compared to AAV9 Applicants evaluated the distribution of AAV9 and Anc80L65 vectors encoding an EGFP reporter 14 days after injection in adult cynomolgus monkeys by either lumbar puncture (LP) injection into the lumbar cisterna magna (approximately L3-L4) or intracisternal magna (ICM) injection (4E13 gc / animal; 2E13 vg / ml). Applicants demonstrated that a single injection of Anc80L65 into the CSF of adult cynomolgus monkeys resulted in efficient transduction of a wide area of ​​the CNS.

[0373] After ICM injection, Anc80L65 was more widely distributed throughout the cortex and within deep brain nuclei compared with AAV9. After LP injection, Anc80L65 distribution throughout the cortex was comparable to that seen with ICM delivery and superior to that seen with AAV9 via ICM delivery. AAV9 showed limited transduction in the cortex after LP delivery. AAV9 and Anc80L65 efficiently transduced spinal anterior horn motor neurons with both administration routes.

[0374] Specifically, Anc80L65 transduced both neurons and astrocytes. Rare oligodendrocyte transduction was also observed in cortical regions with Anc80L65, but microglial cells were not found to be transduced using the microglial marker Iba1. AAV9 exhibited similar tropism to Anc80L65 in the nonhuman primate CNS, transducing primarily neurons and astrocytes. As with Anc80L65, microglial double labeling was not observed. Oligodendrocyte transduction was not observed with AAV9, but transduction was less throughout the CNS compared with Anc80L65, making comparisons difficult.

[0375] This study demonstrates the ability of Anc80L65 to target broad regions of the CNS following CSF delivery, surpassing the distribution of AAV9 in targeting cortical and deep brain regions. The ability of Anc80L65 to mediate efficient gene transfer and expression in neurons and astrocytes throughout the NHP brain and spinal cord supports the use of Anc80L65 vectors for the treatment of a wide range of neurological disorders.

[0376] 7.1.1 Experimental Procedure 7.1.1.1 Lumbar puncture (LP) injection The animal was injected with anesthesia and placed in a lateral recumbent position. A 22-gauge Gerti Marx spinal needle was inserted percutaneously into the lumbar cistern (approximately L3-L4). Fluoroscopy was used for guidance, if necessary. Once the needle was in place, the stylet was removed, positive cerebrospinal fluid (CSF) flow was confirmed, and a pre-dose CSF sample was collected. A test substance syringe was then attached to the needle, and the test substance was slowly injected manually as a slow bolus over approximately 120 ± 5 seconds. After completion of the injection, the needle was removed, and gentle manual pressure was applied to the injection site. The animal was then placed in the Trendelenburg position (30°, head down) for a minimum of approximately 10 minutes. The animal was then allowed to recover naturally from anesthesia. Lumbar puncture is an intrathecal injection.

[0377] 7.1.1.2 Intracisternal (ICM) injection The animals were injected with anesthesia and placed in a recumbent position. A 22-gauge spinal needle was advanced percutaneously into the cisterna magna. Correct needle placement was verified by the presence of positive cerebrospinal fluid (CSF) flow, and pre-administration CSF was collected. The appropriate test substance syringe was then connected to the spinal needle, and the test substance was manually administered via a slow bolus injection (120 ± 5 seconds). After completion of the injection, the syringe was removed and brief manual pressure was applied. The animals were then placed in the Trendelenburg position (30°, head down) for a minimum of approximately 10 minutes. The animals were then allowed to recover naturally from anesthesia.

[0378] 7.1.1.3 Immunohistochemistry (IHC) Two weeks after injection, tissue samples were collected and stored in 10% neutral buffered formalin (NBF) for 48–72 hours, then transferred to 70% ethanol. Brains were placed in pre-chilled brain matrices, sliced ​​into 4 mm sections, and then hemisectioned. Even-numbered hemisectioned slabs were stored in 10% NBF and used for immunohistochemistry (IHC). Odd-numbered hemisectioned brain slabs were frozen on dry ice and stored at -60 to -90 °C until use in ddPCR analysis.

[0379] To detect GFP expression, slides were incubated with an antibody against GFP (GeneTex, GTX20290) diluted 1:1,000 in Monet Blue Diluent (Biocare Medical, PD901). Slides were washed with Valent Wash Buffer (Biocare Medical, VLT8013MX) and incubated with Farma HRP-conjugated anti-rabbit antibody (Biocare Medical, BRR4009) for 30 minutes. Slides were washed, then reacted with Betazoid DAB for 5 minutes (Biocare Medical, BDB2004) and counterstained with Mayer's hematoxylin for 5 minutes (StatLab, HXMMHPT). After reaction with Betazoid DAB or Mayer's hematoxylin, slides were washed with Aqua Rinse (Biocare Medical, VLT8012MX).

[0380] GFP staining with 3,3'-diaminobenzidine (DAB): Sections (3 per 6 mm block, 2 mm separation) were washed three times with PBST and then treated with 1% HO. Sections were stained with a primary anti-GFP antibody diluted 1:1000 in Da Vinci Green Diluent as previously described (Lluis Samaranch, Ernesto A. Salegio, Waldy San Sebastian, Adrian P. Kells, John R. Bringas, John Forsayeth, and Krystof S. Bankiewicz Human Gene Therapy. Volume: 24 Issue 5: March 20, 2013, incorporated herein by reference).

[0381] For detection of trastuzumab expression, slides were incubated with an antibody against IgG(Fc), which can serve as a surrogate for trastuzumab expression.

[0382] 7.1.1.4 Double immunofluorescence: Fluorescent immunostaining of different cell markers (NeuN, GFAP, Iba1, Olig2+) with GFP as previously described ( San Sebastian et al., 2013 ).

[0383] Sample Collection:

[0384] Tissue samples were collected and stored in 10% neutral buffered formalin (NBF) for 48–72 hours, then transferred to 70% ethanol. Brains were placed in pre-chilled brain matrices, sliced ​​into 4 mm sections, and then hemisectioned. Even-numbered hemisectioned slabs were stored in 10% NBF and used for immunohistochemistry (IHC). Odd-numbered hemisectioned brain slabs were frozen on dry ice and stored at -60 to -90 °C until use in ddPCR analysis.

[0385] Immunohistochemistry protocol for GFP expression: Bake the slides at 55-65°C for 15 minutes to remove the paraffin. 〇 Load the slides onto the Valent Staining Platform (Biocare Medical) 〇 Val DePar 8 minutes (Biocare Medical, VLT8001MM) Low pH AR 98°C for 60 minutes (Biocare Medical, VLT8004MM) 〇 Peroxidized 1 5 minutes (Biocare Medical, PX968) 〇 Background Punisher 5 minutes (Biocare Medical,BP974) 〇 GFP (GeneTex, GTX20290) 1:1,000 in Monet Blue Diluent (Biocare Medical, PD901) 〇 Rabbit on Farma HRP 30 minutes (Biocare Medical, BRR4009) 〇 Betazoid DAB 5 minutes (Biocare Medical, BDB2004) Counterstain with Mayer's hematoxylin for 5 minutes (StatLab, HXMMHPT)

[0386] Valent Wash Buffer (Biocare Medical, VLT8013MX) was used after all steps except for Betazoid DAB and Mayer's hematoxylin, which were followed by Aqua Rinse (Biocare Medical, VLT8012MX).

[0387] Double staining of IBA1, NeuN and GFAP with GFP:

[0388] reagent: ● Monet Blue Diluent (Biocare Medical, PD901) containing GFP (GeneTex, GTX20290) 1:1,000 and GFAP (Cell Signaling, 3670) 1:500 ● Monet Blue Diluent (Biocare Medical, PD901) containing GFP (GeneTex, GTX20290) 1:1,000 and IBA1 (Millipore, MABN92) 1:250 ● Monet Blue Diluent (Biocare Medical, PD901) containing GFP (GeneTex, GTX20290) 1:1,000 and NeuN (Abcam, ab104224) 1:250

[0389] protocol: Bake the slides at 55-65°C for 15 minutes to accelerate the removal of paraffin. 〇 Load the slides onto the Valent Staining Platform (Biocare Medical) 〇 Val DePar 8 minutes (Biocare Medical, VLT8001MM) Low pH AR 98°C for 60 minutes (Biocare Medical, VLT8004MM) 〇 Peroxidized 1 5 minutes (Biocare Medical, PX968) 〇 Background Punisher 10 minutes (Biocare Medical,BP974) Primary antibody cocktail: rabbit 594nm (Invitrogen, A32740) 1:500, mouse 488nm (Invitrogen, A-21202) 1:500, and Da Vinci Green (Biocare Medical, PD900) for 60 minutes. Coverslips with DAPI and Prolong Diamond Antifade reagent Valent Wash Buffer (Biocare Medical, VLT8013MX) was used after all steps.

[0390] 7.1.1.5 ddPCR After euthanasia and exsanguination, the brains were placed in a pre-chilled brain matrix, sliced ​​into 4 mm sections, and then hemisectioned. The odd number of hemisectioned slabs were frozen on dry ice and then stored at -60°C to -90°C until analysis. Prior to nucleic acid isolation, brain regions were isolated using 2 mm or 3 mm diameter tissue punches (Miltex, catalog number: 95039-098, 98PUN6-4).

[0391] Tissues were homogenized in lysis buffer with the Qiagen Dneasy Blood and Tissue Kit or the Qiagen RNeasy Lipid Tissue Mini Kit using the Tissuelyser II (20 rps for 2 minutes) according to standard Qiagen protocols. Samples were eluted in 50 μL of buffer. Prior to analysis, DNA and RNA concentrations and quality were determined using a NanoDrop One with the Nucleic Acid (DNA or RNA) program. DNA samples were analyzed for vector genome biodistribution using a duplex ddPCR method targeting the transgene (eGFP) and a reference gene (RPP30). RNA samples were analyzed for eGFP transgene expression using a duplex one-step RT-ddPCR method and a reference gene (RPP30).

[0392] Olig2 and GFP double staining (performed by StageBio):

[0393] reagent: ● Monet Blue Diluent (Biocare Medical, PD901) containing GFP (GeneTex, GTX20290) 1:1,000 and Olig2 (Millipore, MABN50) 1:250

[0394] protocol: Bake the slides at 55-65°C for 15 minutes to accelerate the removal of paraffin. 〇 Load the slides onto the Valent Staining Platform (Biocare Medical) 〇 Val DePar 8 minutes (Biocare Medical, VLT8001MM) Low pH AR 98°C for 60 minutes (Biocare Medical, VLT8004MM) 〇 Peroxidized 5 minutes (Biocare Medical, PX968) 〇 Background Punisher 10 minutes (Biocare Medical,BP974) Primary antibody cocktail: Biotinylated mouse (Vector Laboratories, BA-9200) 1:500 in Da Vinci Green Diluent Rabbit 594nm (Invitrogen, A32740) 1:500, streptavidin 488nm (Invitrogen, S11223) 1:500, and Da Vinci Green (Biocare Medical, PD900) were mixed together for 60 minutes. o After all steps, coverslips were used with DAPIValent Wash Buffer (Biocare Medical, VLT8013MX) and Prolong Diamond Antifade Reagent.

[0395] DNA analysis:

[0396] For DNA isolation, tissues were homogenized in lysis buffer with the Qiagen DNeasy Blood and Tissue Kit (Part Number 69506) using the Tissuelyser II (20 rps for 2 minutes) according to standard Qiagen protocols. Samples were eluted with 50 μL of AE buffer. Prior to analysis, DNA concentration and quality were determined using a NanoDrop One with the Nucleic Acids (DNA) program.

[0397] DNA samples were analyzed for vector genome biodistribution using duplex ddPCR targeting the transgene (eGFP or trastuzumab) and reference gene (RPP30). Specific primer probe sequences are listed in the table below. [Table 10]

[0398] Samples were analyzed according to the standard Bio-Rad ddPCR protocol for probe-based analysis of DNA biodistribution. Briefly, a reaction mixture containing two primer-probe sets, DNA sample, and Bio-Rad ddPCR Supermix for Probes (without dUTP) (product number 186-3024) was prepared according to the recipe in the table below. [Table 11] * DNA samples were prediluted to 2 ng / μL (liver), 10 ng / μL (DRG, no dilution for samples with concentrations <10 ng / μL), and 20 ng / μL (other samples) with nuclease-free water.

[0399] After droplet generation, the reaction was amplified using the thermal cycling program shown below. [Table 12]

[0400] Data are reported as vector genome copies per diploid genome (VGC / DG). The formula for calculating output is: VGC / DG = (eGFP cp / µL ÷ RPP30 cp / µL) × 2 for eGFP, and VGC / DG = (trastuzumab cp / µL ÷ RPP30 cp / µL) × 2 for trastuzumab.

[0401] RNA analysis:

[0402] To isolate mRNA, tissues were homogenized in 1 ml of Qiazol with a Qiagen Tissuelyser II (20 rps for 1 minute) from the Qiagen RNeasy Lipid Tissue Mini Kit (Part Number 74804) according to standard Qiagen protocols. Samples were eluted in 50 μL of nuclease-free water. Prior to analysis, RNA concentration and quality were determined using a NanoDrop One with the Nucleic Acids (RNA) program.

[0403] DNA samples were analyzed for expression of the eGFP or trastuzumab transgene using a duplex one-step RT-ddPCR method targeting the transgene (eGFP or trastuzumab) and a reference gene (RPP30). Specific primer probe sequences are listed in the table below. [Table 13]

[0404] Samples were analyzed according to the standard Bio-Rad RT-ddPCR protocol for probe-based analysis of RNA expression. Briefly, a reaction mixture containing two primer probe sets, RNA sample, and Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes (product number 186-4021) was prepared according to the recipe in the table below. [Table 14] * RNA samples were prediluted to 20 ng / μL with nuclease-free water.

[0405] After droplet generation, the reaction was amplified using the thermal cycling program shown below. [Table 15]

[0406] Data are reported as % eGFP expression, which is calculated according to the formula: % eGFP expression = (eGFP cp / μL ÷ RPP30 cp / μL) × 100 or % trastuzumab expression = (trastuzumab cp / μL ÷ RPP30 cp / μL) × 100.

[0407] 7.1.2. Broad CNS Penetration and Wider Distribution of Anc80L65 Compared to AAV9 The purpose of this study was to determine the biodistribution and initial feasibility of Anc80L65 vectors compared to AAV9 vectors when administered via a single lumbar puncture or intracisternal administration. Results confirm the broad penetration and wide distribution of Anc80L65 compared to AAV9.

[0408] Two AAV constructs were used in the experiment: (i) Anc80L65-CAG-GFP and (ii) AAV9-CAG-GFP, each containing an AAV genome construct containing the coding sequence for GFP. GFP was used to detect AAV distribution and transgene expression. Cynomolgus monkeys were used as subjects.

[0409] As summarized in Figure 1 and Table 2, a total of 14 animals were divided into six groups. Groups 1 and 4 were control animals administered vehicle. Groups 2 and 5 administered 4E13vg (viral genome or GC) of Anc80L65, and groups 3 and 6 administered 4E13vg of AAV9. Two administration routes were tested: Groups 1–3 administered by ICM, and groups 4–6 administered by LP. Animals were sacrificed on days 14 or 15 after vehicle or AAV administration, and their organ samples were collected for analysis. [Table 2]

[0410] The collected samples were processed for IHC and stained with an antibody against GFP. Images of IHC staining are provided in Figures 2A-9 and 22A-22D. Figures 2A-2D provide immunohistochemistry (IHC) images of cortical tissue from brain sections obtained from NHPs administered Anc80L65 or AAV9 by intracisternal injection or lumbar puncture. Figures 22A-22D provide IHC images of brain sections of the cortex and caudate nucleus obtained from NHPs administered Anc80L65 or AAV9 by intracisternal injection.

[0411] These results demonstrate that both ICM and LP administration of Anc80L65 significantly improved transgene (GFP) expression compared to AAV9. Compared to AAV9, more cells stained for GFP expression in the cortex and caudate nucleus after Anc80L65 administration. Figures 2A-2D further demonstrate that ICM administration provided better results in terms of the breadth of intracerebral distribution than LP administration using both vectors (i.e., Anc80L65 and AAV9).

[0412] IHC results in other parts of the brain, specifically the cortex (Figures 3A-3C, 8A-8C, and 9), ependyma and caudate nucleus (Figures 4A-4B), caudate nucleus (Figure 5A-5B), substantia nigra (Figure 6), and perivascular cells (Figure 7A-7B), are also provided. The results demonstrate the widespread penetration and wide distribution of Anc80L65 compared to AAV9.

[0413] To characterize the cell types expressing GFP after Anc80L65 or AAV9 administration, NHP brain sections were double-stained for GFP and cell-type-specific markers. Figures 26A–26F and 27A–27F provide images of double-staining for GFP and a neuronal marker (NeuN) (Figures 26A and 26D), GFP and an astrocyte marker (Figures 26B and 26E), GFP and a microglial marker (iba1), and GFP and an oligodendrocyte marker (Figures 27A, 27B, 27C) in motor cortex transfected with Anc80L65 or AAV9. In all cases, GFP+ cells are shown in red, cell-specific markers are shown in green, and merged images show double-labeled cells (arrows) in yellow / orange. The staining results demonstrate that Anc80L65 can mediate efficient transgene expression in neurons, astrocytes, and oligodendrocytes across large regions of the NHP brain after a single LP or ICM injection. This suggests that Anc80L65 may be used in clinical applications to treat a wide range of neurological disorders, particularly using relatively non-invasive routes of administration such as LP.

[0414] The transgene transduction and expression capabilities of Anc80L65 and AAV9 administered to NHPs via ICM or LP were also tested by ddPCR by measuring the amount of transgene (eGFP) DNA and mRNA in the NHP brain and spinal cord 2 weeks after ICM or LP delivery. The DNA genome copies and mRNA transcript copies of the transgene (eGFP) were quantified relative to the amount of DNA genome copies or mRNA transcript copies of a housekeeping gene (RPP30), respectively. Specifically, DNA genome copies are reported as vector genome copies per diploid genome (VGC / DG). The formula for calculating output is VGC / DG = (eGFP cp / µL ÷ RPP30 cp / µL) × 2. RNA transcript copies are reported as %eGFP expression, which is calculated according to the formula %eGFP expression = (eGFP cp / µL ÷ RPP30 cp / µL) × 100.

[0415] The viral DNA genome copies (VGC) per diploid genome (i.e., VGC per cell) measured in the experiments are provided in Figures 13A-17. Each figure provides data corresponding to a different brain region or liver, including the cerebellar cortex (Figure 13A), dorsal root ganglia, cervical (Figure 13B), dorsal root ganglia, lumbar (Figure 14A), frontal cortex (Figure 14B), liver (Figure 15A), motor cortex (Figure 15B), spinal cord, cervical (Figure 16A), spinal cord, lumbar (Figure 16B), and sciatic nerve (Figure 17). The VGC data were further analyzed and are summarized in Figure 25.

[0416] The data show that Anc80L65 resulted in more vector genome copies per cell in the frontal cortex, motor cortex and spinal cord (cervical and lumbar) compared to AAV9, regardless of injection route, as shown in Figure 25.

[0417] The RNA transcripts measured from the experiments are provided in Figures 18A, 18B, 19A, 19B, 20A, 20B, and 21. Each figure provides data corresponding to a different brain region, including the caudate nucleus (Figure 18A), frontal cortex (Figure 18B), globus pallidus (Figure 19A), motor cortex (Figure 19B), parietal cortex (Figure 20A), putamen (Figure 20B), and substantia nigra (Figure 21). Administration of Anc80L65 induced higher levels of GFP expression in several brain regions, including the caudate nucleus after ICM administration, the globus pallidus after LP administration, the motor cortex after both ICM and LP administration, the parietal cortex after both ICM and LP administration, and the putamen after LP administration.

[0418] A single-way statistical analysis of the expression data is provided in Figures 10A-12B. The results of the analysis are also tabulated in Figures 23 and 24. Figures 10A-10C and 23 provide analyses of data from the frontal cortex (Figures 10A, 23), motor cortex (Figures 10B, 23), and parietal lobe of the cortex (Figures 10C, 23). The data show significantly higher expression of GFP in the cortex of animals injected with Anc80L65 via ICM or LP compared to AAV9 via ICM or LP. Figures 11A-11B, 12A-12B, and 24 show similar analyses in the caudate nucleus (Figures 11A, 24), globus pallidus (Figures 11B, 24), putamen (Figures 12A, 24), and substantia nigra (Figures 12B, 24). These figures also show significantly higher GFP expression in most brain regions in animals injected with Anc80L65 via the ICM or LP compared with AAV9 via the ICM or LP. These results suggest that both ICM and LP injection of Anc80L65 may be effective methods of delivering and expressing transgenes, superior to ICM administration of AAV9.

[0419] A statistical analysis of the ddPCR data is also provided in Table 3 below. The table provides fold changes and p-value results from the Tukey-Kramer HSD test showing a comparison of GFP transcript (RNA) expression in various tissues between Anc80L65(ICM) vs. AAV9(ICM), Anc80L65(LP) vs. AAV9(ICM), and Anc80L65(LP) vs. AAV9(LP). Positive differences indicate the magnitude of the expression advantage attributable to Anc80L65. Statistically significant p-values ​​are indicated in red (asterisks). This analysis shows that the superiority of Anc80L65 is statistically significant compared to AAV9 in various brain regions. [Table 3]

[0420] 7.2. Example 2: AAV in Non-Human Primates to Identify Capsid Mutants with Enhanced CNS Tropism -Lib460 Analysis of The objective of this study was to identify capsids with VR VIII variants that enhance CNS tropism in non-human primates (NHPs). -Lib460 The objective of this study was to evaluate 460 modified capsid proteins from the Anc80L65 VP1 capsid protein. The 460 targeting peptides included the sequences of SEQ ID NOS: 160-619. The targeting peptides were inserted between Q588 and A589 of the Anc80L65 VP1 capsid protein.

[0421] NHPs were incubated with a library containing 460 unique sequences of the modified Anc80L65 VP1 capsid protein produced as described above (referred to herein as AAV -Lib460The mice were administered a library of 460 modified Anc80L65 VP1 capsid mutants. The targeting peptide was positioned between Q588 and A589 of VR VIII for each of the 460 modified Anc80L65 VP1 capsid mutants. Controls included wild-type Anc80L65, wild-type AAV9, and wild-type AAV9-retro (as described in Tervo et al., Neuron, 92(2):372-382 (2016), doi.org / 10.1016 / j.neuron.2016.09.021 and Lin et al., Molecular Brain, 13:138 (2020), doi.org / 10.1186 / s13041-020-00679-1).

[0422] Experimental Design Three cynomolgus macaques were treated as summarized in Table 4 below. All animals received the indicated amount or concentration of AAV. -Lib460 All three treatments (including 460 modified capsid proteins and controls (Anc80L65 capsid protein, AAV9 capsid protein, and AAV9-Retro capsid protein)) (see Table 4) were received by ICM, IG, or IV. [Table 4]

[0423] 7.2.2. Intracisternal (ICM) injection ICM was performed as described in Example 1.

[0424] 7.2.3. Interdigital injection The animal is injected with anesthesia and placed in a recumbent position. A 25-gauge needle loaded with the appropriate test article syringe is advanced into the pads of the animal's upper extremities. Specifically, the middle and index fingers of the right hand (both connected to the median nerve) are injected with equal volumes of 0.2 mL of test article per site (0.4 mL total).

[0425] Intravenous injection The animals were injected with anesthesia and placed in a recumbent position. The animals were restrained in a position that allowed venous access. The injection site was surgically prepared (shaved and aseptically cleaned). The vein was distended by compressing the vein proximal to the heart than the catheter entry site. Compression was applied manually. The vein was visualized, and the catheter was inserted and advanced into the vein, holding the stylet stationary while slowly advancing the catheter into the vessel until the hub reached the skin puncture site. The dose was administered by syringe through the injection cap. The catheter was flushed with sterile saline after administration of the test article.

[0426] 7.2.5.Analysis The animals were sacrificed 28 days after AAV vector administration, and their tissue samples were collected for analysis, including CNS, liver, DRG, peripheral nerves, and spinal cord.

[0427] All analytical work was performed by the sponsor using analytical methods developed and certified by its laboratory.

[0428] DNA samples were analyzed using NGS for biodistribution of the vector genome in the CNS, liver, DRG, peripheral nerves, and spinal cord.

[0429] The gene transfer efficacy of each AAV vector was assessed by measuring mRNA transcripts in the CNS, liver, DRG, peripheral nerves, and spinal cord using NGS.

[0430] Appendix A provides a ranked list of 64 targeting peptides selected from 460 peptides based on their CNS targeting ability when incorporated into AAV capsids. Ranking is based on gene transfer efficacy assessed by measuring mRNA transcripts in the CNS. The list provides the targeting peptide's sequence number, peptide sequence, and average LogFC administered by ICM. See Appendix A.

[0431] 7.3. Example 3: AAV in Non-Human Primates to Identify Capsid Mutants with CNS Tropism -Lib1 Analysis of The purpose of this study is to investigate the role of AAVs in non-human primates (NHPs). -mini The modified capsid proteins of the library (i.e., the targeting peptides (and insertion sites) set forth in part in Figures 31-33 and SEQ ID NOS: 3-9, 11-19, or 21-28) were evaluated to identify capsids with targeting peptides (and / or insertion sites) that enhance CNS tropism. Full-length modified capsid proteins containing the targeting peptides described above are as set forth in SEQ ID NOS: 34-38 or 55820-55847.

[0432] NHPs were infected with AAV containing rAAV with modified capsid protein sequences produced as described above. -mini The targeting peptides (SEQ ID NOS: 3-9, 11-19, or 21-28) were inserted into VR VIII of either the Anc80L65 VP1 capsid protein or the AAV9 VP1 capsid protein, as described in part in Figures 31-33. Additionally, Anc80L65 capsid proteins containing one or more additional modifications described in Figures 32A-32D were tested. Specifically, AFT-6, AFT-7, and AFT-8 VP1 capsid proteins were tested. AFT-6 contains an Anc80L65 VP1 capsid protein backbone with the Anc80L65 VR VIII region replaced with the AAV9 VR VIII region (SEQ ID NO: 29). AFT-7 contains an Anc80L65 VP1 capsid protein scaffold with the Anc80L65 VR IV and VIII regions replaced with the AAV9 VR IV region (SEQ ID NO: 30) and AAV9 VIII region (SEQ ID NO: 29), respectively. AFT-8 contains an Anc80L65 VP1 capsid protein scaffold with the Anc80L65 VR IV, VR V, and VIII regions replaced with the AAV9 VR IV region (SEQ ID NO: 30), AAV9 VR V region (SEQ ID NO: 31), and AAV9 VIII region (SEQ ID NO: 29), respectively.

[0433] Controls included pLib-AAV9, pLib-AAV9-D1, pLib-AAV9-Anc80L65 (pLib-AAV9-C1 (AAV9 containing a targeting peptide having the sequence of SEQ ID NO: 55854 inserted between Q558 and A589), pLib-AAV9-C2 (AAV9 containing a targeting peptide having the sequence of SEQ ID NO: 55855 inserted between Q558 and A589), and pLib-AAV9-C3 (AAV9 containing a targeting peptide having the sequence of SEQ ID NO: 55856 inserted between Q558 and A589) in library ATP292).

[0434] Experimental Design Three animals were treated, as summarized in Table 5 below. Immunosuppression of the animals began 7 days before vector administration. The animals received the indicated amounts or concentrations of AAV -mini (containing 37 modified capsid proteins and controls (see Table 5)) were administered IV. [Table 5]

[0435] 7.3.2.Analysis The animals were sacrificed 28 days after AAV vector administration, and their organ samples were collected for analysis. The collected tissue samples included the CNS, liver, DRG, peripheral nerves, and spinal cord.

[0436] All analytical work was performed by the sponsor using analytical methods developed and certified by its laboratory.

[0437] DNA samples will be collected and analyzed using NGS for biodistribution of the vector genome in the CNS, liver, DRG, peripheral nerves, and spinal cord.

[0438] The gene transfer efficacy of each AAV vector was assessed by measuring mRNA transcripts in the CNS, liver, DRG, peripheral nerves, and spinal cord using NGS. In these experiments, each AAV contained a barcoded mRNA representing a single AAV identity. The barcoded mRNA served as a surrogate for the presence of AAV (i.e., AAV containing various capsid proteins) and was the entity sequenced and used to quantify gene transfer efficacy. To aid in quantification, comparison with the test article (TA) was used to normalize for differential AAV abundance in the TA due to manufacturing and was also used as a quantitative measure of expression in the final sample. The sequenced entity of the test article (TA) was identically barcoded DNA from packaged capsids. Gene transfer data are shown in Figure 34 and Table 6.

[0439] Table 6 provides a rank-ordered list of the tested AAVs, with the ranks based on LogFC_mean. "LogMN_FC" refers to the log2 fold change of the sample relative to the injected test article (TA). In particular, LogMN_FC refers to log2 (median normalized counts per million (CPM) of the sample for a single AAV / median normalized CPM of the test article (TA) for the same AAV). "CPM" is the count of the single AAV / total count of all AAVs x 1E6. "Mean normalized CPM" is the CPM of the single AAV / median CPM (CPM of all AAVs in the sample). Table 6 shows the results for AFT-6 and AFT-6. ** (Biological replicate of AFT-6 (SEQ ID NO: 55820)) had the highest RNA expression among the AAVs tested.

[0440] Figure 34 shows the inverse coefficient of variation (ICV) for the expression levels of single AAVs in brain tissue ("mean LogMN_FC"). In particular, "LogMN_FC" refers to log2 (median normalized counts per million (CPM) of samples for a single AAV / median normalized CPM of test articles (TAs) for the same AAV). "Mean LogMN_FC" refers to the average of the logMN_FC values ​​for a single AAV across all samples from all three animals in a selected tissue set (i.e., brain tissue). ICV is a measure of variability, with higher values ​​indicating lower variability compared to the average, and was calculated as 1 / coefficient of variation for each AAV across all samples. As shown in Figure 34, AFT-6A and AFT-6B (biological replicates of AFT-6 (SEQ ID NO: 55820)) not only had the highest RNA expression among the AAVs tested, but also had the least amount of variability compared to the average of all AAVs tested.

[0441] Overall, the data showed that AAV virions containing the capsid protein AFT-6 (SEQ ID NO: 55820) exhibited the highest RNA expression levels in brain tissue among the capsid proteins tested (see Figure 34 and Table 6). [Table 6-1] [Table 6-2] * shows the AAV9 backbone (SEQ ID NO: 61) with a targeting peptide inserted between Q588 and A589 ** indicates a technical replicate of AFT-6

[0442] Using the sequence data, an enrichment score for each tissue was calculated for each AAV variant containing a unique targeting peptide. Specifically, AAV variants containing each targeting peptide were ranked based on their average log fold-change tissue score (see Table 6). After determining the number of sequences from the sequence read data (FASTQ), AAV variants containing unique targeting peptides were counted and normalized, followed by tissue enrichment analysis for each tissue. The tissue enrichment analysis included sequence-activity relationship (SAR) analysis, network analysis, and structural modeling. SAR analysis identified specific amino acids at specific capsid positions or sequence motifs (combinations of amino acids at several positions) that significantly affect tissue tropism. Network analysis identified modules of variants that share amino acid or peptide sequences with the best-performing variants. Structural modeling provided an understanding of the structural effects of significant amino acids at specific capsid positions for the formulation of mechanistic hypotheses.

[0443] As shown in Figure 35, SAR analysis identified one modified AAV capsid protein (i.e., Anc80L65 capsid protein (the complete capsid sequence is referred to as AFT-6 (SEQ ID NO: 55820)) containing the N2 targeting peptide (SEQ ID NO: 9; C4) located in VR VIII) that increased tissue enrichment in on-target CNS tissues compared to AAV9 (as shown in Figure 35). Notably, the Anc80L65-modified AAV capsid protein containing the targeting peptide N2 had 100-1000 increased expression in various brain regions. The on-target CNS tissues included the frontal lobe, motor cortex, parietal lobe, occipital lobe, temporal lobe, cerebellum, putamen, thalamus, globus pallidus, caudate nucleus, and substantia nigra. This Anc80L65-modified AAV capsid protein containing the N2 targeting peptide also showed decreased tissue enrichment in off-target CNS tissues (as shown in Figure 35) compared to AAV9. Non-targeted CNS tissues included dorsal root ganglia such as cervical, lumbar, thoracic, and liver.

[0444] Further SAR analysis identified three modified AAV capsid proteins that increased tissue enrichment in various CNS tissues compared to controls (e.g., Anc80L65-modified AAV capsid protein containing targeting peptide N2 (SEQ ID NO: 9) inserted in VR VIII (the complete capsid sequence is referred to as AFT-6 (SEQ ID NO: 55820)); AAV9 containing an N3 targeting peptide (PLNGSVHLY (SEQ ID NO: 3603)) located in VR VIII between amino acid residues 586 and 589 and replacing amino acids A587 and Q588; and AAV9 containing an N4 targeting peptide (PLNGTVHLY (SEQ ID NO: 1232)) located in VR VIII between amino acid residues 586 and 589 and replacing amino acids A587 and Q588) (see Figures 36 and 37).

[0445] Overall, this data demonstrated that a subset of the modified AAV capsid proteins described herein have optimal CNS tropism.

[0446] 7.4. Example 4: AAV in Non-Human Primates to Identify Capsid Mutants with CNS Tropism -Lib1 Analysis of The purpose of this study was to investigate the efficacy and safety of AAV in non-human primates (NHPs). -Lib The goal of this study was to evaluate 54,000 modified capsid proteins containing the library (i.e., the targeting peptide in Appendix A inserted between positions Q586 and A589 of AAV, thereby replacing A587 and Q588) to identify capsids bearing targeting peptides that enhance CNS tropism.

[0447] NHPs are herein referred to as AAV -LibA library containing approximately 54,000 unique sequences of modified capsid proteins, referred to as the library, was administered. The targeting peptide is located in VR VIII of the AAV9 VP1 capsid protein between amino acid residues 586 and 589, replacing amino acids A587 and Q588. The targeting peptides include the sequences set forth in SEQ ID NOs: 620-55819 and 55857-55859. For example, amino acid modifications (insertions, deletions, and substitutions) in the VR VIII region of AAV9 include those shown in Figure 33. Controls include pLib-AAV9, pLib-AAV9-C4, pLib-AAV9-C1, pLib-AAV9-C2, and pLib-AAV9-C3.

[0448] Experimental Design Three animals were treated as summarized in Table 6 below. Animal immunosuppression begins 7 days prior to vector administration. Animals are intravenously administered the indicated amount or concentration of AAV-Lib (containing approximately 55,200 modified capsid proteins and controls) (see Table 8). Animals are sacrificed 28 days after AAV vector administration, and their organ samples are collected for analysis. [Table 8-1]

[0449] 7.4.2.Analysis The animals were sacrificed 28 days after AAV vector administration, and their organ samples were collected for analysis, including nucleus caudate, nucleus pallidus, nucleus nigra, cerebellar cortex, DRG cervical, DRG lumbar, DRG thoracic, frontal lobe, liver, occipital lobe, parietal lobe, putamen, sensorimotor cortex, temporal lobe, and thalamus.

[0450] DNA samples were analyzed using NGS for biodistribution of the vector genome in the CNS, liver, DRG, peripheral nerves, and spinal cord (data not shown).

[0451] The gene transfer efficacy of each AAV vector was assessed by measuring mRNA transcripts in the tissues listed in Appendix B. Tissue enrichment data is also presented in Appendix B, which is incorporated herein by reference in its entirety. The data represent the average LogMN_FC measured and calculated as described above. Appendix B provides 750 targeting peptides selected from among 54,000 modified capsid proteins for their CNS targeting capabilities.

[0452] Overall, this data demonstrated that a subset of modified AAV capsid proteins (e.g., the capsid proteins provided in Appendix B) have optimal CNS tropism.

[0453] Table 7 provides CNS targeting data (mean LogMN_FC) for several AAV vectors, AAV9 or AAV9 with targeting peptides (N3, N4, or N5). [Table 7]

[0454] 7.4.3. Production of rAAV Containing AAV-Lib1 Capsids rAAVs containing various AAV-Lib1 capsids were produced in suspension HEK293 cells and their yield and manufacturability were tested. As shown in Table 8, the study showed that rAAVs containing AAV9-N3 and AAV9-N4 provided good yields at harvest and were stable during formulation. However, rAAVs containing AAV9-N5 capsids had low yields at harvest and precipitated during formulation. [Table 8-2]

[0455] Seroprevalence of rAAV containing AAV-Lib1 capsids Seroprevalence of various AAV-Lib1 capsids was tested in an in vitro neutralizing antibody assay using donor samples (N = 55) approximately representative of the population according to the U.S. Census Bureau (2019). All samples were performed at a 1:5 serum dilution, and titers were determined as the reciprocal of the serum dilution reporting 50% relative light units (RLU) compared to AAV capsid alone. Samples with a titer <5 were defined as seronegative samples, and samples with a titer >5 were defined as seropositive samples.

[0456] Table 9 shows the % of donor samples tested that were seropositive or seronegative. The data show that AAV9-N3, AAV9-N4 have lower seroprevalence than AAV9, but higher seroprevalence than AAV9-C4 and AAV5. [Table 9]

[0457] 8. Arrays Many of the nucleotide sequences provided below are obtained from double-stranded vectors. Therefore, those skilled in the art will understand that, unless the context dictates otherwise, reference to the nucleotide sequences provided herein throughout the specification and claims also includes reference to complementary sequences. Additional sequences can be found in Appendix A and Appendix B, each of which is incorporated herein by reference in its entirety. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5] [Table 16-6]

Table 16-7

Table 16-8

Table 16-9

Table 16-10

Table 16-11

Table 16-12

Table 16-13

Table 16-14

Table 16-15

Table 16-16

Table 16-17

Table 16-18

Table 16-19

Table 16-20

Table 16-21

Table 16-22

Table 16-23

[0458] 9. Equivalents and Incorporation by Reference While the present invention has been particularly shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[0459] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes. [Table 17] [Table 18-1] [Table 18-2] [Table 18-3] [Table 18-4] [Table 18-5] [Table 18-6]

Table 18-7

Table 18-8

Table 18-9

Table 18-10

Table 18-11

Table 18-12

Claims

1. 1. A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide within variable region VIII (VR VIII), The targeting peptide is X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ and X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ , X ８ , and X ９ are each independently selected from any amino acid residue.

2. (i) X １ is independently selected from proline (P) and glycine (G); (ii) X ２ are independently selected from lysine (L), threonine (T), serine (S), alanine (A), valine (V), and isoleucine (I); (iii) X ３ are independently selected from asparagine (N), glutamine (Q), and proline (P); (iv) X ４ is independently selected from glycine (G) and alanine (A); (v) X ５ are independently selected from alanine (A), threonine (T), serine (S), valine (V), and glycine (G); (vi) X ６ are independently selected from valine (V), leucine (L), alanine (A), isoleucine (I), glycine (G), serine (S), and threonine (T); (vii) X ７ are independently selected from histidine (H), arginine (R), and lysine (K); (viii) X ８ is independently selected from leucine (L) and valine (V), and (ix) X ９ is independently selected from tyrosine (Y), arginine (R), histidine (H), lysine (K), and phenylalanine (F).

3. 3. The modified AAV capsid protein of claim 1 or 2, wherein the targeting peptide within VR VIII has a sequence selected from SEQ ID NOs: 160-55819 and 55857-55859.

4. The targeting peptide is PX ２ X ３ GAVX ７ LY (SEQ ID NO: 2), ２ , X ３ , and X ７ The modified AAV capsid protein of any one of claims 1 to 3, wherein are independently selected from any amino acid residue.

5. (i) X ２ are independently selected from lysine (L), isoleucine (I), valine (V), and alanine (A); (ii) X ３ is asparagine (N) or glutamine (Q), and (iii) X ７ 4. The modified AAV capsid protein of claim 3, wherein is independently selected from histidine (H), arginine (R), and lysine (K).

6. the targeting peptide is (i) PLQGAVHLY (SEQ ID NO: 3); (ii) PLQGAVRLY (SEQ ID NO: 4); (iii) PLQGAVKLY (SEQ ID NO: 5); (iv) PINGAVHLY (SEQ ID NO: 6); (v) PVNGAVHLY (SEQ ID NO: 7); (vi) PANGAVHLY (SEQ ID NO: 8), or (vii) PLNGAVHLY (SEQ ID NO: 9) The modified AAV capsid protein of claim 4 or 5,

7. 7. The modified AAV capsid protein of any one of claims 1 to 6, wherein the targeting peptide is inserted between S586 and A589 of the AAV9 capsid protein, thereby replacing A587 and Q588 of the AAV9 capsid protein.

8. 8. The modified AAV capsid protein of any one of claims 1 to 7, having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AAV9 capsid protein.

9. the targeting peptide is (i) inserted between S586 and T589 of the Anc80L65 capsid protein, thereby replacing A587 and N588 of said Anc80L65 capsid protein; (ii) inserted between Q585 and N588 of the Anc80L65 capsid protein, thereby replacing S586 and A587 of said Anc80L65 capsid protein; (iii) inserted between L584 and A587 of the Anc80L65 capsid protein, thereby replacing Q585 and S586 of said Anc80L65 capsid protein; (iv) inserted between A587 and A590 of the Anc80L65 capsid protein, thereby replacing N588 and T589 of said Anc80L65 capsid protein; or (v) inserted between S586 and A587 of the Anc80L65 capsid protein; A modified AAV capsid protein according to any one of claims 1 to 6.

10. 10. The modified AAV capsid protein of claim 9, wherein the targeting peptide comprises PLNGAVHLY (SEQ ID NO: 9).

11. 11. The modified AAV capsid protein of claim 9 or 10, having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the Anc80L65 capsid protein.

12. The targeting peptide is PX ２ X ３ GX ５ X ６ X ７ LY (SEQ ID NO: 10), ２、 X ３、 X ５、 X ６、 and X ７ The modified AAV capsid protein of claim 1 or 2, wherein: are independently selected from any amino acid residue.

13. (i) X ２ is independently selected from leucine (L), threonine (T) or serine (S); (ii) X ３ is independently selected from asparagine (N) and glutamine (Q); (iii) X ５ is independently selected from alanine (A) and threonine (T); (iv) X ６ is independently selected from valine (V) and leucine (L); and (v) X ７ are independently selected from histidine (H), arginine (R), and lysine (K); The modified AAV capsid protein of claim 12.

14. the targeting peptide is (i) PTNGTVRLY (SEQ ID NO: 11); (ii) PTNGTVHLY (SEQ ID NO: 12); (iii) PTNGTVKLY (SEQ ID NO: 13); (iv) PSNGTLRLY (SEQ ID NO: 14), (v) PSNGTLHLY ​​(SEQ ID NO: 15), (vi) PSNGTLKLY (SEQ ID NO: 16), (vii) PTNGTLRLY (SEQ ID NO: 17); (viii) PTNGTLHLY ​​(SEQ ID NO: 18), or (ix) PTNGTLKLY (SEQ ID NO: 19) 14. The modified AAV capsid protein of claim 12 or 13,

15. The targeting peptide is PX ２ X ３ GAVX ７ X ８ X ９ (SEQ ID NO: 20), ２、 X ３、 X ５、 X ６、 and X ７ The modified AAV capsid protein of claim 1 or 2, wherein: are independently selected from any amino acid residue.

16. (i) X ２ is independently selected from leucine (L), threonine (T) or serine (S); (ii) X ３ is independently selected from asparagine (N) and glutamine (Q); (iii) X ７ is independently selected from histidine (H) and threonine (T); (iv) X ８ is independently selected from valine (V) and leucine (L); and (v) X ９ is independently selected from tyrosine (Y) and arginine (R); 16. The modified AAV capsid protein of claim 15.

17. the targeting peptide is (i) PTQGAVTVR (SEQ ID NO: 21); (ii) PLQGAVTVR (SEQ ID NO: 22); (iii) PLQGAVHVR (SEQ ID NO: 23); (iv) PLQGAVHVY (SEQ ID NO: 24), (v) PSQGAVTLR (SEQ ID NO: 25); (vi) PLQGAVTLR (SEQ ID NO: 26), (vii) PLQGAVHLR (SEQ ID NO: 27), or (viii) PTQGAVTLR (SEQ ID NO: 28) 17. The modified AAV capsid protein of claim 15 or 16,

18. 18. The modified AAV capsid protein of any one of claims 1 to 17, wherein the targeting peptide does not comprise PLNGAVHLY (SEQ ID NO: 9).

19. 18. The modified AAV capsid protein of any one of claims 1 to 17, wherein the targeting peptide comprises PLNGSVHLY (SEQ ID NO: 3603) or PLNGTVHLY (SEQ ID NO: 1232).

20. 20. The modified AAV capsid protein of any one of claims 12 to 19, wherein the targeting peptide is inserted between S586 and A589 of the AAV9 capsid protein, thereby replacing A587 and Q588 of the AAV9 capsid protein.

21. 1. A modified adeno-associated virus (AAV) capsid protein comprising a targeting peptide within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NOs: 160-619.

22. 22. The modified AAV capsid protein of any one of claims 1 to 21, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to the sequence of a reference AAV capsid protein.

23. 23. The modified AAV capsid protein of claim 22, wherein the reference AAV capsid protein is selected from VP1, VP2 and VP3.

24. The reference AAV capsid protein is an AAV capsid protein selected from the group consisting of AAV9, Anc80L65, 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, the modified AAV capsid protein according to claim 22 or 23.

25. The modified AAV capsid protein of any one of claims 22 to 24, wherein the reference AAV capsid protein is a capsid protein having a sequence selected from SEQ ID NOs: 54 to 158, or a fragment thereof.

26. The modified AAV capsid protein of any one of claims 22 to 25, wherein the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 61, or a fragment thereof.

27. The modified AAV capsid protein of any one of claims 22 to 26, wherein the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 142, or a fragment thereof.

28. The modified AAV capsid protein of any one of claims 22 to 27, wherein the targeting peptide is located between positions 576 and 601 within VR VIII of the modified AAV capsid protein.

29. 29. The modified AAV capsid protein of any one of claims 1 to 28, further comprising an N-terminal flanking region at the N-terminus of the targeting peptide.

30. 30. The modified AAV capsid protein of claim 29, wherein the N-terminal flanking region comprises at least four consecutive amino acids from amino acid residues 576 to 585 of the reference AAV capsid protein, the amino acid residues being numbered according to the amino acid sequence of the reference AAV capsid protein.

31. The N-terminal flanking region is B １ YGB ２ VATNB ３ QS (SEQ ID NO: 55849), １ , B ２ , and B ３ The modified AAV capsid protein of claim 29 or 30, wherein each of the amino acid residues is independently selected from any of the amino acid residues.

32. B １ is selected from glutamate (E) or serine (S), and B ２ is selected from threonine (T) or glutamine (Q), and B ３ is selected from leucine (L) or histidine (H).

33. 33. The modified AAV capsid protein of any one of claims 29 to 32, wherein the N-terminal flanking region has the sequence SYGQVATNHQS (SEQ ID NO: 55848).

34. 34. The modified AAV capsid protein of any one of claims 29 to 33, wherein the N-terminal flanking region replaces the N-terminal reference sequence of the reference AAV capsid protein, the N-terminal reference sequence having at least 60% sequence identity with the N-terminal flanking region and located N-terminal to an insertion site for the targeting peptide in the reference AAV capsid protein.

35. The N-terminal reference sequence is B １ YGB ２ VATNB ３ 35. The modified AAV capsid protein of claim 34, having the sequence of QS (sequence number 55849).

36. 36. The modified AAV capsid protein of any one of claims 1 to 35, further comprising a C-terminal flanking region at the C-terminus of the targeting peptide.

37. 37. The modified AAV capsid protein of claim 36, wherein the C-terminal flanking region comprises at least four consecutive amino acids from amino acid residues 589 to 602 of the reference AAV capsid protein, the amino acid residues being numbered according to the amino acid sequence of the reference AAV capsid protein.

38. The C-terminal flanking region is AQAQTGZ １ VZ ２ Z ３ QGZ ４ (SEQ ID NO: 55851), Z １、 Z ２、 Z ３、 and Z ４ 38. The modified AAV capsid protein of claim 36 or 37, wherein each of is independently selected from any amino acid residue.

39. Z １ is selected from threonine (T) or tryptophan (W), and Z ２ is selected from asparagine (N) or glutamine (Q), and Z ３ is selected from serine (S) or asparagine (N), and Z ４ is selected from alanine (A) or isoleucine (I).

40. 40. The modified AAV capsid protein of any one of claims 36 to 39, wherein the C-terminal flanking region has the sequence AQAQTGWVQNQGI (SEQ ID NO: 55850).

41. 41. The modified AAV capsid protein of any one of claims 36 to 40, wherein the C-terminal flanking region replaces a C-terminal reference sequence of the reference AAV capsid protein, the C-terminal reference sequence having at least 60% sequence identity with the C-terminal flanking region and located C-terminal to an insertion site for the targeting peptide in the reference AAV capsid protein.

42. The C-terminal reference sequence is AQAQTGZ １ VZ ２ Z ３ QGZ ４ 42. The modified AAV capsid protein of claim 41 having the sequence of (sequence number 55851).

43. (i) B １ YGB ２ VATNB ３ an N-terminal flanking region having the sequence of QS (SEQ ID NO: 55849); １ , B ２ , and B ３ are each independently selected from any amino acid residue), an N-terminal flanking region, and (ii) AQAQTGZ １ VZ ２ Z ３ QGZ ４ A C-terminal flanking region having the sequence of (SEQ ID NO: 55851), １ , Z ２ , Z ３ , and Z ４ are each independently selected from any amino acid residue, 29. The modified AAV capsid protein of any one of claims 1 to 28, further comprising:

44. (i) B １ YGB ２ VATNB ３ an N-terminal flanking region having the sequence of QS (SEQ ID NO: 55860); １ is selected from glutamate (E) or serine (S), and B ２ is selected from threonine (T) or glutamine (Q), and B ３ is selected from leucine (L) or histidine (H), and (ii) AQAQTGZ １ VZ ２ Z ３ QGZ ４ A C-terminal flanking region having the sequence of (SEQ ID NO: 55861), １ is selected from threonine (T) or tryptophan (W), and Z ２ is selected from asparagine (N) or glutamine (Q), and Z ３ is selected from serine (S) or asparagine (N), and Z ４ is selected from alanine (A) or isoleucine (I), 29. The modified AAV capsid protein of any one of claims 1 to 28, further comprising:

45. (i) an N-terminal flanking region having the sequence SYGQVATNHQS (SEQ ID NO: 55848); and (ii) a C-terminal flanking region having the sequence AQAQTGWVQNQGI (SEQ ID NO: 55850) 29. The modified AAV capsid protein of any one of claims 1 to 28, further comprising:

46. B １ YGB ２ VATNB ３ QSPLMGAVHLYAQAQTGZ １ VZ ２ Z ３ QGZ ４ (SEQ ID NO: 55852), １ , B ２ , B ３ , Z １ , Z ２ , Z ３ , and Z ４ The modified AAV capsid protein of any one of claims 1 to 28, wherein each of the following is independently selected from any amino acid residue:

47. B １ is selected from glutamate (E) or serine (S), and B ２ is selected from threonine (T) or glutamine (Q), and B ３ is selected from leucine (L) or histidine (H), and Z １ is selected from threonine (T) or tryptophan (W), and Z ２ is selected from asparagine (N) or glutamine (Q), and Z ３ is selected from serine (S) or asparagine (N), and Z ４ is selected from alanine (A) or isoleucine (I).

48. 29. The modified capsid protein of any one of claims 1 to 28, wherein the targeting peptide is a peptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues that differ from the sequence of SEQ ID NO:29, and the different amino acids comprise an insertion, deletion, or substitution compared to the sequence of SEQ ID NO:

29.

49. 49. The modified AAV capsid protein of claim 48, wherein the targeting peptide has the sequence of SEQ ID NO:

29.

50. 50. The modified AAV capsid protein of any one of claims 22 to 49, wherein the modified AAV capsid protein has one or more modifications comprising amino acid insertions, deletions, substitutions, or a combination thereof, compared to a reference AAV capsid protein.

51. 51. The modified AAV capsid protein of any one of claims 22-50, wherein the one or more modifications comprise an amino acid insertion, deletion, substitution, or a combination thereof, to introduce the targeting peptide into VR VIII of the reference AAV capsid protein.

52. 51. The modified AAV capsid protein of any one of claims 22-50, wherein the one or more modifications comprise an amino acid modification outside of VR VIII of the reference AAV capsid protein.

53. 53. The modified AAV capsid protein of Claim 52, wherein the one or more modifications outside VR VIII of the reference AAV capsid protein comprise one or more modifications of VR I, VR II, VR III, VR IV, VR V, VR VI, or VR VII.

54. 54. The modified AAV capsid protein of Claim 53, wherein the one or more modifications outside VR VIII of the reference AAV capsid protein comprise one or more modifications in VR IV and VR V.

55. 55. The modified AAV capsid protein of any one of claims 52 to 54, wherein the one or more modifications of VR IV result in the introduction of the sequence of SEQ ID NO:

30.

56. 56. The modified AAV capsid protein of any one of claims 52 to 55, wherein the one or more modifications in VR V result in the introduction of the sequence of SEQ ID NO:

31.

57. 53. The modified AAV capsid protein of any one of claims 1 to 52, wherein the one or more modifications comprise a deletion of one or more amino acids within VR VIII.

58. 58. The modified AAV capsid protein of Claim 57, wherein the one or more amino acid deletion comprises a deletion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII.

59. 59. The modified AAV capsid protein of claim 57 or 58, wherein the one or more amino acid deletions comprise deletion of at least one amino acid residue at a position selected from 584, 585, 586, 587, or 588, or a combination thereof, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein.

60. 60. The modified AAV capsid protein of claim 59, wherein the deletion of one or more amino acids comprises a deletion of the amino acid residue at position 587 relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein.

61. 60. The modified AAV capsid protein of claim 59, wherein the deletion of one or more amino acids comprises a deletion of the amino acid residue at position 588 relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein.

62. 62. The modified AAV capsid protein of any one of claims 57-61, wherein the amino acid deletion comprises deletion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII.

63. 63. The modified AAV capsid protein of claim 62, wherein the amino acid deletion comprises a deletion of an amino acid residue at position 589, 590, or 591, or a combination thereof, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein.

64. 64. The modified AAV capsid protein of any one of claims 50-63, wherein the amino acid insertion comprises insertion of 1, 2, 3, 4, or 5 or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII.

65. 65. The modified AAV capsid protein of claim 64, wherein the inserted amino acid residues are independently selected from any amino acid residue.

66. 66. The modified AAV capsid protein of claim 64 or 65, wherein the inserted amino acid residue is identical to the amino acid residue deleted adjacent to the N-terminus or C-terminus of the targeting peptide within VR VIII.

67. 67. The modified AAV capsid protein of any one of claims 64 to 66, wherein the inserted amino acid residue is alanine (A) or asparagine (N).

68. 66. The modified AAV capsid protein of Claim 65, wherein the inserted amino acids are an alanine (A) at the position immediately adjacent to the C-terminus of the targeting peptide and an asparagine (N) at the next position, thereby having an amino acid sequence of AN immediately adjacent to the C-terminus of the targeting peptide.

69. the targeting peptide is (i) PLNGAVHLYN (SEQ ID NO: 32), or (ii) PLNGAVHLYAN (SEQ ID NO: 33) The modified AAV capsid protein of any one of claims 22 to 61,

70. (i) the reference AAV capsid protein is an AAV1 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and S588 of the reference capsid protein; (ii) the reference AAV capsid protein is an AAV2 capsid protein or a modification thereof, and the targeting peptide is between R585 and R588 of the reference AAV capsid protein, thereby replacing residues G586 and N587 of the reference capsid protein; (iii) the reference AAV capsid protein is an AAV3 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and N588 of the reference capsid protein; (iv) the reference AAV capsid protein is an AAV4 capsid protein or a modification thereof, and the targeting peptide is between D582 and N585 of the reference AAV capsid protein, thereby replacing residues Q583 and S584 of the reference capsid protein; (v) the reference AAV capsid protein is an AAV5 capsid protein or a modification thereof, and the targeting peptide is between S575 and T578 of the reference AAV capsid protein, thereby replacing residues S576 and S577 of the reference capsid protein; (vi) the reference AAV capsid protein is an AAV6 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and S588 of the reference capsid protein; (vii) the reference AAV capsid protein is an AAV7 capsid protein or a modification thereof, and the targeting peptide is between A587 and T590 of the reference AAV capsid protein, thereby replacing residues A588 and N589 of the reference capsid protein; (viii) the reference AAV capsid protein is an AAV8 capsid protein or a modification thereof, and the targeting peptide is between Q588 and A591 of the modified AAV capsid protein, thereby replacing residues Q589 and N590 of the reference capsid protein; (ix) the reference AAV capsid protein is an AAV9 capsid protein or a modification thereof, and the targeting peptide is between S586 and A589 of the reference AAV capsid protein, thereby replacing residues A587 and Q588 of the reference capsid protein; (x) the reference AAV capsid protein is an AAVrhlO capsid protein or a modification thereof, and the targeting peptide is between Q588 and A591 of the reference AAV capsid protein, thereby replacing residues Q589 and N590 of the reference capsid protein; (xi) the reference AAV capsid protein is an AAVpo.1 capsid protein or a modification thereof, and the targeting peptide is between N565 and S568 of the reference AAV capsid protein, thereby replacing residues Q566 and N567 of the reference capsid protein; (xii) the reference AAV capsid protein is an AAV12 capsid protein or a modification thereof, and the targeting peptide is between N590 and A593 of the reference AAV capsid protein, thereby replacing residues Q591 and N592 of the reference capsid protein; (xiii) the reference AAV capsid protein is an Anc80 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and N588 of the reference capsid protein; (xiv) the reference AAV capsid protein is an Anc80-55 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues S587 and N588 of the reference capsid protein; (xv) the reference AAV capsid protein is an Anc80-129 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein; (xvi) the reference AAV capsid protein is an Anc80-156 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein; (xvii) the reference AAV capsid protein is the capsid protein of Anc80-751 or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein; (xviii) the reference AAV capsid protein is the capsid protein of Anc80-1029 or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein; or (xix) The modified AAV capsid protein of any one of claims 1 to 69, wherein the reference AAV capsid protein is an Anc80-1712 capsid protein or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein.

71. (i) 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; (ii) the reference AAV capsid protein is an AAV2 capsid protein or a modification thereof, and the targeting peptide is between R588 and Q589 or between N587 and R588 of the reference AAV capsid protein; (iii) 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; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) 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 modified AAV capsid protein; (ix) the reference AAV capsid protein is an AAV9 capsid protein or a modification thereof, and the targeting peptide is between Q588 and A589 of the reference AAV capsid protein; (x) 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 reference AAV capsid protein; (xi) 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 reference AAV capsid protein; (xii) 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; (xiii) the reference AAV capsid protein is an Anc80 capsid protein or a modification thereof, and the targeting peptide is between T589 and A590, between N588 and T589, or between N587 and T588 of the reference AAV capsid protein; (xiv) 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 reference AAV capsid protein; (xv) 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 reference AAV capsid protein; (xvi) 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 reference AAV capsid protein; (xvii) the reference AAV capsid protein is the capsid protein of Anc80-751 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; (xviii) 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 reference AAV capsid protein; or (xix) The modified AAV capsid protein of any one of claims 1 to 69, wherein 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 reference AAV capsid protein.

72. A modified AAV capsid protein having a sequence selected from SEQ ID NOs: 34-39.

73. A polynucleotide encoding the modified AAV capsid protein of any one of claims 1 to 72.

74. 73. A vector comprising the polynucleotide of claim 72.

75. 75. The vector of claim 74, further comprising a promoter operably linked to the polynucleotide.

76. 76. A host cell comprising a modified AAV capsid protein according to any one of claims 1 to 71, a polynucleotide according to claim 72, or a vector according to claim 74 or 75.

77. A recombinant AAV virion (rAAV) comprising a modified AAV capsid protein according to any one of claims 1 to 71.

78. 78. The AAV virion of claim 77, further comprising an exogenous polynucleotide.

79. 79. The AAV virion of claim 78, wherein the exogenous polynucleotide comprises a template for homology-directed repair.

80. 79. The AAV virion of claim 78, wherein the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA editing guide RNA.

81. 81. The AAV virion of claim 80, wherein the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein.

82. 82. The AAV virion of claim 81, wherein the therapeutic protein is used to treat and / or prevent a disease of the central nervous system (CNS).

83. 83. The AAV virion of any one of claims 77 to 82, which, when administered to a subject in a therapeutically effective amount, has increased specificity for central nervous system (CNS) tissue relative to a reference AAV virion comprising a reference AAV capsid protein that does not contain the targeting peptide.

84. 84. The AAV virion of any one of claims 77 to 83, which, when administered to a subject in a therapeutically effective amount, has increased transduction efficiency in the CNS relative to a reference AAV virion comprising a reference AAV capsid protein that does not comprise the targeting peptide.

85. 85. The AAV virion of any one of claims 77 to 84, which, when administered to a subject in a therapeutically effective amount, has increased blood-brain barrier permeability in the subject relative to a reference AAV virion comprising a reference capsid protein that does not comprise the targeting peptide.

86. A pharmaceutical composition comprising an AAV virion according to any one of claims 77 to 85.

87. 86. A method for treating or ameliorating or preventing a disease or symptom in a subject, the method comprising administering a therapeutically effective amount of an AAV virion of any one of claims 77 to 85 or a pharmaceutical composition of claim 86.

88. 88. The method of treating, ameliorating or preventing a disease according to claim 87, wherein the disease is a disease of the central nervous system (CNS).

89. 89. The method of treating, ameliorating, or preventing a disease according to claim 88, wherein the CNS disease is a lysosomal storage disease (LSD).

90. 89. The method of treating or ameliorating or preventing a disease according to claim 88, wherein the CNS disease is a leukodystrophy.

91. 89. The method of treating, ameliorating, or preventing a disease according to claim 88, wherein the CNS disease is metachromatic leukodystrophy (MLD).

92. 89. The method of treating, ameliorating, or preventing a disease according to claim 88, wherein the CNS disease is Krabbe's disease.

93. 89. The method of treating or ameliorating or preventing a disease according to claim 88, wherein the CNS disease is cancer.

94. 89. The method of treating or ameliorating or preventing a disease according to claim 88, wherein the CNS disease is metastatic breast cancer.

95. A modified adeno-associated virus (AAV) capsid protein according to any one of claims 1 to 71 for use in the treatment and / or prevention of diseases of the central nervous system (CNS).

96. 86. An AAV virion comprising a modified AAV capsid protein according to any one of claims 1 to 71 or an AAV virion according to any one of claims 77 to 85, for use in the treatment and / or prevention of a disease of the central nervous system (CNS).

97. 86. A pharmaceutical composition comprising a modified AAV capsid protein according to any one of claims 1 to 71 and / or an AAV virion according to any one of claims 77 to 85 for use in the treatment and / or prevention of diseases of the central nervous system (CNS).

98. 86. A method of introducing an exogenous polynucleotide into the central nervous system (CNS), comprising the step of administering to a subject an AAV virion of any one of claims 77-85.

99. 99. The method of claim 98, wherein said administration results in introduction of said exogenous polynucleotide in the CNS at a CNS:liver infection ratio of greater than 1 as measured by genome copies of said AAV virion.

100. 100. The method of claim 98 or 99, wherein said administration results in expression of the exogenous polynucleotide in the CNS at a CNS:liver expression ratio of greater than 10.

101. 101. The method of any one of claims 98-100, wherein the CNS:liver expression ratio is greater than 10 as measured by protein expression.

102. 86. Use of a modified AAV capsid protein according to any one of claims 1 to 71 and / or an AAV virion according to any one of claims 77 to 85 for introducing an exogenous polynucleotide into the central nervous system.

103. 103. The use of claim 102, wherein the use is a non-therapeutic use.

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