Recombinant AAV with improved tropism and specificity

Abstract

The present disclosure provides a modified AAV capsid protein comprising a targeting peptide in a variable region VIII (VRVIII). The modified AAV capsid protein may form an rAAV having a preferred tropism, specificity, or biological distribution in vivo or in vitro. The rAAVs of the present disclosure can be used in gene therapy targeting specific tissues.

Description

[0001] 1. Cross-references to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 380,170, filed October 19, 2022; U.S. Provisional Application 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.

[0003] 2. Sequence List

[0004] This application contains a sequence list with 55,921 sequences, which has been submitted through the Patent Center and is incorporated herein by reference in its entirety. The XML copy of this application, created on October 16, 2023, is named 53023WO_CRF_sequencelisting.xml and has a size of 48,736,195 bytes.

[0005] This application also incorporates, by reference in its entirety, appendices entitled "Appendix A" and "Appendix B," and the sequence described therein. Appendices A and B are filed together with this application. Background Technology

[0006] Adeno-associated virus (AAV) has become the preferred vector system for in vivo gene therapy. A growing number of recombinant AAVs (rAAVs) designed 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.

[0007] 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, topical application 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 the desired tropism and specificity.

[0008] Generally, the capsid is considered a major determinant of infectivity and host-vector related properties such as adaptive immune response, tropism, specificity, potency, and biodistribution. Indeed, several of these properties are known to differ between natural serotypes and engineered AAV variants. Over the past decade, novel synthetic AAV variants have been developed using various capsid engineering techniques, one of which involves inserting small peptides into an exposed loop in the capsid protein called variable region VIII (VR VIII). In some cases, inserting novel peptides into the wild-type capsid alters the tropism of the variant.

[0009] However, little is known to date about how alterations to the AAV capsid change its biological properties, and AAV vectors with the required tropism and specificity for therapeutic targets such as the central nervous system (CNS) have not yet been obtained. Species-specific differences in AAV tropism, such as between mice and nonhuman primates (NHPs), make it difficult to develop AAV vectors with the required tropism in humans.

[0010] Treatment of CNS diseases remains a challenging problem. Currently, the available therapeutic agents for CNS diseases are limited because many fail to cross the blood-brain barrier when delivered intravenously or do not distribute widely when delivered directly to the brain. Therefore, there is a need for an AAV carrier with preferred and specific tropism for the CNS to treat these CNS diseases. Summary of the Invention

[0011] This disclosure provides a modified AAV capsid protein that can form rAAV with preferred tropism and specificity for a therapeutic target. In some embodiments, the modified adeno-associated virus (AAV) capsid protein having this preferred tropism includes (i) a targeting peptide at a site within the variable region VIII (VR VIII); wherein the targeting peptide has the sequence X1X2X3X4X5X6X7X8X9, wherein 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 containing the targeting peptide having the sequence X1X2X3X4X5X6X7X8X9 at a site within the variable region VIII (VR VIII) includes the deletion of amino acid residues (e.g., A587 and Q588 in the AAV9 capsid).

[0012] The applicant previously demonstrated that a single injection of a rationally designed synthetic vector, Anc80L65 (described in WO2015 / 054653, which is incorporated herein by reference in its entirety), into the CSF of adult cynomolgus monkeys resulted in efficient transduction over a wide range of areas of the CNS and surprisingly outperformed AAV9’s ability to target the cortex and deep nuclei (PCT application PCT / US2022 / 024262, which is incorporated herein by reference in its entirety).

[0013] The applicant now reports a modified AAV capsid protein (e.g., Anc80L65, AAV9, and other AAV capsid proteins) containing a targeting peptide within variable region VIII (VR VIII) that provides a synergistic effect for the specific targeting of rAAV to target tissues (e.g., the CNS). Therefore, the modified AAV capsid proteins of this disclosure (e.g., modified AAV capsid proteins containing the described targeting peptide inserted at the insertion site described herein) can alter the tropism, specificity, and / or biodistribution of AAVs containing such modified AAV capsid proteins.

[0014] Overall, rAAV modified AAV capsid proteins containing the targeting peptide at sites within variable region VIII (VR VIII) exhibit better targeting than AAV capsid proteins containing the targeting peptide at sites not included in variable region VIII, and the transgene is expressed more specifically in target tissues (e.g., the brain).

[0015] On the other hand, this 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, and X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each independently selected from any amino acid residue. In some embodiments,

[0016] (i) X1 is independently selected from proline (P) and glycine (G);

[0017] (ii) X2 is independently selected from lysine (L), threonine (T), serine (S), alanine (A), valine (V) and isoleucine (I);

[0018] (iii) X3 is independently selected from asparagine (N), glutamine (Q) and proline (P);

[0019] (iv)X4 is independently selected from glycine (G) and alanine (A);

[0020] (v)X5 is independently selected from alanine (A), threonine (T), serine (S), valine (V) and glycine (G);

[0021] (vi)X6 is independently selected from valine (V), leucine (L), alanine (A), isoleucine (I), glycine (G), serine (S) and threonine (T);

[0022] (vii)X7 is independently selected from histidine (H), arginine (R), and lysine (K);

[0023] (viii) X8 is independently selected from leucine (L) and valine (V); and

[0024] (ix)X9 is independently selected from tyrosine (Y), arginine (R), histidine (H), lysine (K) and phenylalanine (F).

[0025] In some embodiments, the targeting peptide within VR VIII has a sequence selected from SEQ ID NO: 620-55819.

[0026] In some embodiments, the targeting peptide has the sequence PX2X3GAVX7LY (SEQ ID NO: 2), and X2, X3, and X7 are independently selected from any amino acid residues. In some embodiments,

[0027] (i) X2 is independently selected from lysine (L), isoleucine (I), valine (V) and alanine (A);

[0028] (ii) X3 is asparagine (N) or glutamine (Q); and

[0029] (iii)X7 is independently selected from histidine (H), arginine (R) and lysine (K).

[0030] In some embodiments, the targeting peptide is:

[0031] (i) PLQGAVHLY (SEQ ID NO: 3);

[0032] (ii) PLQGAVRLY (SEQ ID NO: 4);

[0033] (iii) PLQGAVKLY (SEQ ID NO: 5);

[0034] (iv) PINGAVHLY (SEQ ID NO: 6);

[0035] (v) PVNGAVHLY (SEQ ID NO: 7);

[0036] (vi) PANGAVHLY (SEQ ID NO: 8); or

[0037] (vii) PLNGAVHLY (SEQ ID NO: 9).

[0038] 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.

[0039] In some embodiments, the modified AAV capsid protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the AAV9 capsid protein.

[0040] In some embodiments, the targeting peptide is inserted

[0041] (i) Between S586 and T589 of the Anc80L65 capsid protein, thereby replacing A587 and N588 of the Anc80L65 capsid protein.

[0042] (ii) The S586 and A587 of the Anc80L65 capsid protein are replaced between Q585 and N588.

[0043] (iii) Between L584 and A587 of the Anc80L65 capsid protein, thereby replacing Q585 and S586 of the Anc80L65 capsid protein.

[0044] (iv) Between A587 and A590 of the Anc80L65 capsid protein, thereby replacing N588 and T589 of the Anc80L65 capsid protein; or

[0045] (v) Between S586 and A587 of the capsid protein of Anc80L65.

[0046] In some embodiments, the targeting peptide comprises: PLNGAVHLY (SEQ ID NO: 9).

[0047] In some embodiments, the modified AAV capsid protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the Anc80L65 capsid protein.

[0048] In some embodiments, the targeting peptide has the sequence PX2X3GX5X6X7LY (SEQ ID NO: 10), and X2, X3, X5, X6, and X7 are independently selected from any amino acid residues. In some embodiments,

[0049] (i) X2 is independently selected from leucine (L), threonine (T) or serine (S);

[0050] (ii) X3 is independently selected from asparagine (N) and glutamine (Q);

[0051] (iii) X5 is independently selected from alanine (A) and threonine (T);

[0052] (iv) X6 is independently selected from valine (V) and leucine (L); and

[0053] (v)X7 is independently selected from histidine (H), arginine (R), and lysine (K).

[0054] In some embodiments, the targeting peptide is:

[0055] (i)PTNGTVRLY (SEQ ID NO: 11);

[0056] (ii) PTNGTVHLY (SEQ ID NO: 12);

[0057] (iii) PTNGTVKLY (SEQ ID NO: 13);

[0058] (iv)PSNGTLRLY (SEQ ID NO: 14);

[0059] (v) PSNGTLHLY ​​(SEQ ID NO: 15);

[0060] (vi) PSNGTLKLY (SEQ ID NO: 16);

[0061] (vii)PTNGTLRLY (SEQ ID NO: 17);

[0062] (viii)PTNGTLHLY ​​(SEQ ID NO: 18); or

[0063] (ix)PTNGTLKLY (SEQ ID NO: 19).

[0064] In some embodiments, the targeting peptide has the sequence PX2X3GAVX7X8X9 (SEQ ID NO: 20), and X2, X3, X5, X6, and X7 are independently selected from any amino acid residues. In some embodiments,

[0065] (i) X2 is independently selected from leucine (L), threonine (T) or serine (S);

[0066] (ii) X3 is independently selected from asparagine (N) and glutamine (Q);

[0067] (iii) X7 is independently selected from histidine (H) and threonine (T);

[0068] (iv) X8 is independently selected from valine (V) and leucine (L); and

[0069] (v)X9 is independently selected from tyrosine (Y) and arginine (R).

[0070] In some embodiments, the targeting peptide is:

[0071] (i) PTQGAVTVR (SEQ ID NO: 21);

[0072] (ii) PLQGAVTVR (SEQ ID NO: 22);

[0073] (iii) PLQGAVHVR (SEQ ID NO: 23);

[0074] (iv) PLQGAVHVY (SEQ ID NO: 24);

[0075] (v) PSQGAVTLR (SEQ ID NO: 25);

[0076] (vi) PLQGAVTLR (SEQ ID NO: 26);

[0077] (vii)PLQGAVHLR(SEQ ID NO: 27); or

[0078] (viii) PTQGAVTLR (SEQ ID NO: 28).

[0079] In some embodiments, the targeting peptide does not contain PLNGAVHLY (SEQ ID NO: 9).

[0080] 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.

[0081] In another aspect, this disclosure provides 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 NO: 160-619.

[0082] 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 with the sequence of the reference AAV capsid protein.

[0083] In some embodiments, the reference AAV capsid protein is selected from VP1, VP2, and VP3.

[0084] In some embodiments, the reference AAV capsid protein is a capsid protein of AAV 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; r h.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; Anc12 6; Anc127; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc110; and Anc80DI.

[0085] In some embodiments, the reference AAV capsid protein is a capsid protein having a sequence selected from SEQ ID NO: 54-158 or a fragment thereof.

[0086] In some embodiments, the modified AAV capsid protein has one or more modifications compared to the reference AAV capsid protein, including amino acid insertions, deletions, substitutions, or combinations thereof.

[0087] In some embodiments, the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 61 or a fragment thereof.

[0088] In some embodiments, the reference AAV capsid protein is a capsid protein having the sequence of SEQ ID NO: 142 or a fragment thereof.

[0089] In some embodiments, the targeting peptide is located between 576 and 601 within VRVIII of the modified AAV capsid protein.

[0090] In some embodiments, the modified AAV capsid protein is further contained in the N-terminal flanking region of the targeting peptide.

[0091] In some embodiments, the N-terminal flanking region comprises at least four consecutive amino acid residues from amino acid residues 576-585 of the reference AAV capsid protein, wherein these amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0092] In some embodiments, the N-terminal flanking region has the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), and B1, B2, and B3 are each independently selected from any amino acid residue. In some embodiments, B1 is selected from glutamic acid (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H).

[0093] In some implementations, the N-terminal flanking region has the sequence SYGQVATNHQS (SEQ ID NO: 55848).

[0094] In some embodiments, the N-terminal flanking region replaces the N-terminal reference sequence of the 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 at the N-terminus of the targeting peptide insertion site within the reference AAV capsid protein. In some embodiments, the N-terminal flanking region has the sequence SYGQVATNHQS (SEQ ID NO: 55848).

[0095] In some embodiments, the modified AAV capsid protein is further contained in the C-terminal flanking region of the target peptide.

[0096] In some embodiments, the C-terminal flanking region contains at least four consecutive amino acids from amino acid residues 589-602 of the reference AAV capsid protein, wherein these amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0097] In some embodiments, the C-terminal flanking region has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851), and 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).

[0098] In some embodiments, the C-terminal flanking region has the sequence AQAQTGWVQNQGI (SEQ ID NO: 55850).

[0099] In some embodiments, the C-terminal flanking region replaces the C-terminal reference sequence of the 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 at the C-terminus of the targeting peptide insertion site within the reference AAV capsid protein. In some embodiments, the C-terminal reference sequence has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851).

[0100] In some embodiments, the modified AAV capsid protein further comprises:

[0101] The N-terminal flanking region of the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), wherein B1, B2, and B3 are each independently selected from any amino acid residue; and

[0102] The C-terminal flanking region of the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851), where Z l Z2, Z3, and Z4 are each independently selected from any amino acid residue.

[0103] In some embodiments, the modified AAV capsid protein further comprises:

[0104] The sequence B1YGB2VATNB3QS (SEQ ID NO: 55860) has an N-terminal flanking region, wherein B1 is selected from glutamic acid (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H); and

[0105] The sequence has a C-terminal flanking region 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).

[0106] In some embodiments, the modified AAV capsid protein further comprises:

[0107] The N-terminal flanking region of the sequence SYGQVATNHQS (SEQ ID NO: 55848), and

[0108] The C-terminal flanking region of the sequence AQAQTGWVQNQGI (SEQ ID NO: 55850).

[0109] In some embodiments, the modified AAV capsid protein comprises the amino acid sequence 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 glutamic acid (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).

[0110] 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 are different from the sequence of SEQ ID NO: 29, wherein the different amino acids include insertions, deletions or substitutions.

[0111] In some embodiments, the targeting peptide is SEQ ID NO: 29.

[0112] In some embodiments, the one or more modifications include amino acid insertions, deletions, substitutions, or combinations thereof in which the targeting peptide is introduced into VR VIII of the reference AAV capsid protein.

[0113] In some embodiments, the one or more modifications include amino acid modifications other than VR VIII of the reference AAV capsid protein.

[0114] In some embodiments, the one or more modifications other than VR VIII of the reference AAV capsid protein include one or more of VRI, VRII, VRIII, VRIV, VR V, VR VI, or VR VII.

[0115] In some embodiments, the one or more modifications other than VR VIII of the reference AAV capsid protein include one or more modifications of VRIV and VR V.

[0116] In some implementations, the one or more modifications in VRIV result in the introduction of the sequence SEQ ID NO: 30.

[0117] In some implementations, the one or more modifications in VR V result in the introduction of the sequence SEQ ID NO: 31.

[0118] In some modifications, the one or more modifications include the deletion of one or more amino acids within VR VIII.

[0119] In some modifications, the deletion of one or more amino acids includes the deletion of one, two, three, four, or five or more amino acid residues immediately adjacent to the N-terminus of the target peptide within VR VIII.

[0120] In some modifications, the deletion of one or more amino acids includes the 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 reference AAV capsid protein. In some modifications, the deletion of one or more amino acids includes the deletion of an amino acid residue at position 587, relative to a reference sequence numbered according to the reference AAV capsid protein. In some modifications, the deletion of one or more amino acids includes the deletion of an amino acid residue at position 588, relative to a reference sequence numbered according to the reference AAV capsid protein.

[0121] In some modifications, the amino acid deletion includes the deletion of one, two, three, four, or five or more amino acid residues immediately adjacent to the C-terminus of the target peptide within VR VIII. In some modifications, the amino acid deletion includes the deletion of amino acid residues at positions 589, 590, or 591, or combinations thereof, relative to a reference sequence numbered according to the reference AAV capsid protein amino acid sequence number.

[0122] In some modifications, the amino acid insertion includes the insertion of one, two, three, four, or five or more amino acid residues immediately adjacent to the C-terminus of the target peptide within VR VIII.

[0123] In some modifications, the inserted amino acid residues are independently selected from any amino acid residues.

[0124] In some modifications, the inserted amino acid residue(s) are identical to the missing amino acid residues at the N-terminus or C-terminus of the target peptide within VR VIII.

[0125] In some modifications, the inserted amino acid residue is either alanine (A) or asparagine (N). In other modifications, the inserted amino acid is alanine (A) immediately adjacent to the C-terminus of the targeting peptide and asparagine (N) at the next subsequent position, thus having an amino acid sequence of AN immediately adjacent to the C-terminus of the targeting peptide.

[0126] In some modifications, the targeting peptide is:

[0127] (i) PLNGAVHLYN (SEQ ID NO: 32); or

[0128] (ii) PLNGAVHLYAN (SEQ ID NO: 33).

[0129] In some implementations...

[0130] (i) The reference AAV capsid protein is the capsid protein of AAV1 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.

[0131] (ii) The reference AAV capsid protein is the capsid protein of AAV2 or a modification thereof, and the targeting peptide substitutes residues G586 and N587 of the reference AAV capsid protein between R585 and R588.

[0132] (iii) The reference AAV capsid protein is the capsid protein of AAV3 or a variant 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.

[0133] (iv) The reference AAV capsid protein is the capsid protein of AAV4 or a modification thereof, and the targeting peptide is located between D582 and N585 of the reference AAV capsid protein, thereby replacing residues Q583 and S584 of the reference capsid protein.

[0134] (v) The reference AAV capsid protein is the capsid protein of AAV5 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.

[0135] (vi) The reference AAV capsid protein is the capsid protein of AAV6 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.

[0136] (vii) The reference AAV capsid protein is the capsid protein of AAV7 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.

[0137] (viii) The reference AAV capsid protein is the capsid protein of AAV8 or a modification thereof, and the targeting peptide substitutes residues Q589 and N590 of the reference capsid protein between Q588 and A591 of the modified AAV capsid protein.

[0138] (ix) The reference AAV capsid protein is the capsid protein of AAV9 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.

[0139] (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.

[0140] (xi) The reference AAV capsid protein is the capsid protein of AAVpo.1 or a modification thereof, and the targeting peptide is located between N565 and S568 of the reference AAV capsid protein, thereby replacing residues Q566 and N567 of the reference capsid protein.

[0141] (xii) The reference AAV capsid protein is the capsid protein of AAV12 or a modification thereof, and the targeting peptide is located between N590 and A593 of the reference AAV capsid protein, thereby replacing residues Q591 and N592 of the reference capsid protein.

[0142] (xiii) The reference AAV capsid protein is the capsid protein of Anc80 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.

[0143] (xiv) The reference AAV capsid protein is the capsid protein of Anc80-55 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.

[0144] (xv) The reference AAV capsid protein is the capsid protein of Anc80-129 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.

[0145] (xvi) The reference AAV capsid protein is the capsid protein of Anc80-156 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.

[0146] (xvii) The reference AAV capsid protein is the capsid protein of Anc80-751 or a modification thereof, and the targeting peptide is located between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein.

[0147] (xviii) The reference AAV capsid protein is the capsid protein of Anc80-1029 or a modification thereof, and the targeting peptide substitutes residues A587 and N588 of the reference AAV capsid protein between S586 and T589; or

[0148] (xix) The reference AAV capsid protein is the capsid protein of Anc80-1712 or a modification thereof, and the targeting peptide is located between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein.

[0149] In some implementations...

[0150] (i) The reference AAV capsid protein is the capsid protein of AAV1 or a modification thereof, and the targeting peptide is between D590 and P591 or between S588 and T589 of the reference AAV capsid protein.

[0151] (ii) The reference AAV capsid protein is the capsid protein of AAV2 or a modification thereof, and the targeting peptide is between R588 and Q589 or between N587 and R588 of the reference AAV capsid protein.

[0152] (iii) The reference AAV capsid protein is the capsid protein of AAV3 or a modification thereof, and the targeting peptide is between S586 and S587 or between N588 and T589 of the reference AAV capsid protein.

[0153] (iv) The reference AAV capsid protein is the capsid protein of AAV4 or a modification thereof, and the targeting peptide is between S584 and N585 or between S586 and N587 of the reference AAV capsid protein.

[0154] (v) The reference AAV capsid protein is the capsid protein of AAV5 or a modification thereof, and the targeting peptide is between S575 and S576 or between T577 and T578 of the reference AAV capsid protein.

[0155] (vi) The reference AAV capsid protein is the capsid protein of AAV6 or a modification thereof, and the targeting peptide is between D590 and P591 or S588 and T589 of the reference AAV capsid protein.

[0156] (vii) The reference AAV capsid protein is the capsid protein of AAV7 or a modification thereof, and the targeting peptide is between N589 and T590 of the reference AAV capsid protein;

[0157] (viii) The reference AAV capsid protein is the capsid protein of AAV8 or a modification thereof, and the targeting peptide is between N590 and T591 of the modified AAV capsid protein;

[0158] (ix) The reference AAV capsid protein is the capsid protein of AAV9 or a modification thereof, and the targeting peptide is between Q588 and A589 of the reference AAV capsid protein;

[0159] (x) The reference AAV capsid protein is the capsid protein of AAVrh10 or a modification thereof, and the targeting peptide is between N590 and A591 of the reference AAV capsid protein;

[0160] (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.

[0161] (xii) The reference AAV capsid protein is the capsid protein of AAV12 or a modification thereof, and the targeting peptide is between N592 and A593 or between T594 and T595 of the reference AAV capsid protein.

[0162] (xiii) The reference AAV capsid protein is the capsid protein of Anc80 or a modification thereof, and the targeting peptide is between T589 and A590, N588 and T589, or N587 and T588 of the reference AAV capsid protein.

[0163] (xiv) The reference AAV capsid protein is the capsid protein of Anc80-55 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein.

[0164] (xv) The reference AAV capsid protein is the capsid protein of Anc80-129 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein.

[0165] (xvi) The reference AAV capsid protein is the capsid protein of Anc80-156 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein.

[0166] (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.

[0167] (xviii) The reference AAV capsid protein is the capsid protein of Anc80-1029 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein; or

[0168] (xix) The reference AAV capsid protein is the capsid protein of Anc80-1712 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein.

[0169] In another respect, this disclosure provides a polynucleotide that encodes any modified AAV capsid protein described herein.

[0170] In another aspect, this disclosure provides a vector comprising any of the polynucleotides described herein. In some embodiments, the vector comprises a promoter operatively linked to the polynucleotide.

[0171] In another respect, this disclosure provides a host cell comprising any modified AAV capsid protein, any polynucleotide, or any vector described herein.

[0172] In another aspect, this disclosure provides a recombinant AAV virion (rAAV) comprising any modified AAV capsid protein described herein.

[0173] In some embodiments, the rAAV further comprises an exogenous polynucleotide. In some embodiments, the exogenous polynucleotide comprises a template for homology-directed repair. In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic tRNA, miRNA, gene editing guide RNA, or RNA editing guide RNA. In some embodiments, the exogenous polynucleotide comprises an expressible polynucleotide encoding a therapeutic protein. In some embodiments, the therapeutic protein is used to treat and / or prevent diseases of the central nervous system (CNS).

[0174] In some implementations, when administered to a subject at a therapeutically effective amount, the AAV virion exhibits enhanced specificity for central nervous system (CNS) tissues relative to a reference AAV virion that contains the reference AAV capsid protein but lacks the targeting peptide.

[0175] In some implementations, when administered to a subject at a therapeutically effective amount, the AAV virion exhibits enhanced transduction efficiency in the CNS compared to a reference AAV virion containing the reference AAV capsid protein but lacking the targeting peptide.

[0176] In some implementations, when administered to a subject at a therapeutically effective amount, the AAV virion exhibits enhanced blood-brain barrier penetration in the subject relative to a reference AAV virion containing a reference capsid protein but lacking the targeting peptide.

[0177] In another respect, this disclosure provides a pharmaceutical composition comprising any of the AAV virions described herein.

[0178] In another aspect, this disclosure provides a method for treating or improving or preventing a disease or condition in a subject, comprising administering a therapeutically effective amount of any AAV virion or any pharmaceutical composition described herein.

[0179] In some implementations, the disease is a disease of the central nervous system (CNS).

[0180] In some implementations, the CNS disease is lysosomal storage disease (LSD).

[0181] In some implementations, the CNS disease is leukodystrophy.

[0182] In some implementations, the CNS disease is metachromatic leukodystrophy (MLD).

[0183] In some implementations, the CNS disease is Clabberg disease.

[0184] In some implementations, the CNS disease is cancer. In some implementations, the CNS disease is metastatic breast cancer.

[0185] In some embodiments, any modified adeno-associated virus (AAV) capsid protein described herein is used to treat and / or prevent diseases of the central nervous system (CNS).

[0186] On the other hand, this disclosure provides AAV virions comprising any modified AAV capsid protein or any AAV virion described herein, for the treatment and / or prevention of diseases of the central nervous system (CNS).

[0187] On the other hand, this disclosure provides any pharmaceutical composition comprising any modified AAV capsid protein described herein and / or any AAV virion described herein, for the treatment and / or prevention of diseases of the central nervous system (CNS).

[0188] In another aspect, this disclosure provides a method for transferring exogenous polynucleotides to the central nervous system (CNS), comprising the step of administering any of the AAV virions described herein to a subject. In some embodiments, the administration results in the transfer of the exogenous polynucleotide in the CNS at a CNS:liver infection rate greater than 1 when measured by genomic copy number of the AAV virion. In some embodiments, the administration results in the expression of the exogenous polynucleotide in the CNS at a CNS:liver expression rate greater than 10. In some embodiments, the CNS:liver expression rate is greater than 10 when measured by protein expression.

[0189] On the other hand, this disclosure provides the use of any AAV capsid protein described herein and / or any AAV virion described herein for the transfer of exogenous polynucleotides to the central nervous system. In some embodiments, this use is non-therapeutic.

[0190] It is anticipated that the modified rAAV of this disclosure will provide improved therapeutic endpoints compared to control rAAV with unmodified capsid proteins, due to its specific tropism to target tissues and higher expression of therapeutic proteins. Attached Figure Description

[0191] These and other features, aspects, and advantages of the invention will be better understood with reference to the following description and accompanying drawings, in which:

[0192] Figure 1 The NHP study designs described in the examples are summarized.

[0193] Figures 2A-2DImmunohistochemical (IHC) images of brain sections obtained from NHP administration of (i) Anc80L65-CAG-GFP or (ii) AAV9-CAG-GFP via intracranial injection (ICM) or lumbar puncture (LP). Brown staining = GFP expression (arrow). Anc80L65-LP ( Figure 2B The small images in the diagram primarily show neuron staining. Figure 2A The results show GFP expression after administration of Anc80L65 via ICM injection. Figure 2B The results show GFP expression after Anc80L65 was applied via LP. Figure 2C The results show GFP expression after administration of AAV9 via ICM injection. Figure 2D The results show GFP expression after AAV administration via LP.

[0194] Figures 3A-3C From the application carrier ( Figure 3A ), Anc80L65-CAG-GFP ( Figure 3B ) or AAV9-CAG-GFP ( Figure 3C IHC images of brain slices (including the cortex) obtained from NHP. Brown staining = GFP expression.

[0195] Figures 4A-4B These are IHC images of brain slices (including the ependyma and caudate nucleus) obtained from NHPs administered with Anc80L65-CAG-GFP via ICM injection. Figure 4B yes Figure 4A A magnified image of a portion of the image. Brown staining = GFP expression.

[0196] Figures 5A-5B These are IHC images of brain slices (including the caudate nucleus) obtained from NHP administered via ICM injection of Anc80L65-CAG-GFP. Figure 5B yes Figure 5A A magnified image of a portion of the image. Brown staining = GFP expression.

[0197] Figure 6 This is an IHC image of a brain slice (including the substantia nigra) obtained from NHP injected with Anc80L65-CAG-GFP via ICM. Brown staining = GFP expression.

[0198] Figure 7A and Figure 7B This is an IHC image of a brain slice (including perivascular cells) obtained from NHPs injected with Anc80L65-CAG-GFP via ICM injection. Figure 7B yes Figure 7A A magnified image of a portion of the image. Brown staining = GFP expression.

[0199] Figure 8A and Figure 8B These are IHC images of brain slices (including the cortex) obtained from NHP administered via ICM injection of Anc80L65-CAG-GFP. Figure 8B yes Figure 8A A magnified image of a portion of the image. Brown staining = GFP expression.

[0200] Figure 9 This is an IHC image of a brain slice (including the cortex) obtained from NHP administered via lumbar puncture (LP) with Anc80L65-CAG-GFP. Brown staining = GFP expression.

[0201] Figures 10A-10C One-way analysis of transgene expression in different brain regions of animals administered AAV9-CAG-GFP via ICM injection or Anc80L65-CAG-GFP via LP was provided. The expression was determined by measuring the mRNA transcript of eGFP according to the following equation: eGFP expression % = (eGFP cp / uL ÷ RPP30 cp / uL) × 100. Figure 10A Data on the frontal cortex were provided; Figure 10B It provides data on the motor cortex; and Figure 10C Data for the parietal cortex were provided.

[0202] Figures 11A-11B Univariate analysis of transgene expression in various brain regions administered AAV9-CAG-GFP via ICM injection or Anc80L65-CAG-GFP via LP was provided. The expression was determined by measuring the mRNA transcript of eGFP according to the following equation: eGFP expression % = (eGFP cp / uL ÷ RPP30 cp / uL) × 100. Figure 11A Data on the caudate nucleus was provided; and Figure 11B Data for the Pale Sphere was provided.

[0203] Figures 12A-12B Univariate analysis of transgene expression in various brain regions administered AAV9-CAG-GFP via ICM injection or Anc80L65-CAG-GFP via LP was provided. The expression was determined by measuring the mRNA transcript of eGFP according to the following equation: eGFP expression % = (eGFP cp / uL ÷ RPP30 cp / uL) × 100. Figure 12A It provides data on the shell core; and Figure 12B Data on black matter was provided.

[0204] Figures 13A-17Univariate analysis of viral genome (DNA) copy number (VGC / DG) for each diploid genome was provided, determined by measuring genome copy number using ddPCR and calculating the (VGC / DG) value using the following equation: VGC / DG = (eGFP cp / uL ÷ RPP 30 cp / uL) × 2. Each figure provides data for different brain regions or liver areas, including the cerebellar cortex (…). Figure 13A ), cervical dorsal root ganglion ( Figure 13B ), lumbar root ganglia ( Figure 14A ), frontal cortex ( Figure 14B ),liver( Figure 15A ), motor cortex ( Figure 15B ), cervical spinal cord ( Figure 16A ), lumbar spinal cord ( Figure 16B ) and sciatic nerve ( Figure 17 ).

[0205] Figure 18A , Figure 18B , Figure 19A , Figure 19B , Figure 20A , Figure 20B and Figure 21 Univariate analysis of transgene expression was provided, determined by measuring eGFP mRNA transcripts according to the following equation: eGFP expression % = (eGFP cp / uL ÷ RPP30 cp / uL) × 100. Each figure provides data for different brain regions, including the caudate nucleus (…). Figure 18A ), frontal cortex ( Figure 18B ), pale ball ( Figure 19A ), motor cortex ( Figure 19B ), apical cortex ( Figure 20A ), shell and core ( Figure 20B ) and substantia nigra ( Figure 21 ).

[0206] Figures 22A-22D Immunohistochemical (IHC) images of brain sections obtained from NHP administered via intracephalic injection of Anc80L65-CAG-GFP or AAV9-CAG-GFP. Brown staining = GFP expression. Figure 22A The expression of GFP in the cortex after administration of Anc80L65-CAG-GFP was shown. Figure 22B The expression of GFP in the caudate nucleus after administration of Anc80L65-CAG-GFP is shown. Figure 22C The expression of GFP in the cortex after administration of AAV9-CAG-GFP was shown. Figure 22D The expression of GFP in the caudate nucleus after administration of AAV9-CAG-GFP was shown.

[0207] Figure 23 and Figure 24 This study demonstrated the expression of GFP mRNA in the brain and spinal cord of NHP patients, measured by ddPCR, two weeks after delivery of AAV9-CAG-GFP or Anc80L65-CAG-GFP via ICM or LP. Figure 23 GFP expression in the frontal cortex, motor cortex, and parietal cortex was provided. Figure 24 GFP expression in the caudate nucleus, globus pallidus, putamen, and substantia nigra was provided.

[0208] Figure 25 The vector genome copy analysis by qPCR is described. The VGCs (mean vector genome copies / DG) of each cell in NHP cells injected with Anc80L65-CAG-GFP and AAV9-CAG-GFP via LP or ICM are provided.

[0209] Figures 26A-26F Is it the application of Anc80L65-CAG-GFP ( Figure 26A , Figure 26B and Figure 26C ) or AAV9-CAG-GFP ( Figure 26D , Figure 26E and Figure 26F Images of brain slices from AAV using double immunofluorescence (IF) staining. Transgenic expression from AAV was detected by staining against GFP, while cell type was detected by staining against cell type-specific markers, including NeuN for neurons. Figure 26A and Figure 26D GFAP targeting astrocytes ( Figure 26B and Figure 26E ) and Iba1 (targeting microglia) Figure 26C and Figure 26F The example was taken from the motor cortex. In all cases, GFP+ cells appear in red, cell-specific markers appear in green, and the merged image shows double-labeled cells in yellow / orange (arrows indicate double-labeled cells).

[0210] Figures 27A-27F It comes from LP ( Figure 27A , Figure 27B and Figure 27C ) or via ICM ( Figure 27D , Figure 27E and Figure 27F Double immunofluorescence (IF) staining images of brain slices from NHPs treated with Anc80L65-CAG-GFP. Example taken from the motor cortex. Transgenic expression from Anc80L65 was detected by staining against GFP, while oligodendrocytes were detected by staining against the oligodendrocyte-specific marker OLIG2, shown in green. Figure 27A and Figure 27D ). Showing GFP+ cells ( Figure 27B and Figure 27E The merged images are displayed with arrows pointing to the double-labeled cells. Figure 27C and Figure 27F ).

[0211] Figure 28 The structure of the AAV VP1 protein is illustrated, with certain variable regions (VR I, VR IV, VR V, and VR VIII) highlighted. The location of the target peptide insertion site in VR VIII is indicated.

[0212] Figures 29A-29C Sequence alignments of the VP1 sequence for certain AAV variants using AAV2 VP1 as a reference are provided. The location of the insertion site for the target peptide is indicated. Figure 29B ). Figures 29A-29C The SEQ ID NOs disclosed in order of appearance are 55, 54, 58, 56, 64, 59, 60, 89, 111, 61, 63, 62 and 57.

[0213] Figures 30A-30D Sequence alignment of the VP1 sequence of the ancestral AAV using AAV2 as a reference is provided. The location of the insertion site of the target peptide is indicated. Figure 30C One or more representative member sequences of each of the Anc80, Anc81, Anc82, Anc83, Anc84, Anc94, Ac110, Anc113, Anc126, and Anc127 libraries were used for alignment. Figures 30A-30D The SEQ ID NOs disclosed in order of appearance are 55, 55911, 143, 147, 144, 146, 145, 148, 149-150, 142 and 55912-55921.

[0214] Figure 31 The sequence of the VR VIII window of the modified AAV capsid protein (e.g., Anc80L65) is provided, where the targeting peptide is located at different positions within VR VIII. The underlined sequences represent amino acid residues of the targeting peptide. Figure 31 SEQ ID NO: 55875-55881 are disclosed in the order of their appearance.

[0215] Figure 32A The structure of the AAV VP1 capsid protein was explained, focusing on the variable regions (VR)IV, VRV, and VRVIII.

[0216] Figure 32BPartial sequence information is provided for three AAV capsid protein variants with various combinations of VR IV, VR V, and VR VIII regions containing Anc80L65 or AAV9 capsid proteins.

[0217] Figure 32C Sequence alignments of amino acid residues 540-640 for SEQ ID NO: 61 (AAV9 capsid protein), 142 (Anc80L65 capsid protein), and 55853 are provided. Boxes indicate amino acid positions 576-601 corresponding to positions in AAV9 without inserted target peptides. Figure 32C SEQ ID NO: 55853 and 55882-55883 are disclosed in the order of their appearance.

[0218] Figure 32D Sequence alignments are provided for AAV9 (SEQ ID NO: 61), the targeting peptide (SEQ ID NO: 9) inserted between 588 and 589 (removing 586 and 587), and amino acid residues 540-640 of AFT-6. AFT-6 has an Anc80L65 capsid protein backbone containing the N-terminal flanking region from AAV9 (SEQ ID NO: 55848), the targeting peptide from SEQ ID NO: 9, and the C-terminal flanking region from AAV9 (SEQ ID NO: 55850). Boxes indicate amino acid positions 559-635. Figure 32D SEQ ID NOs 55884-55885, 55884 and 55886 are disclosed in the order of their appearance.

[0219] Figure 33 The sequence of a modified AAV capsid protein (e.g., AAV9) is provided in a VR VIII window, wherein the targeting peptide is shown as an underlined amino acid residue and blanks at positions 587 and 588, corresponding to the deletion of A587 and Q588 from the modified AAV9 capsid protein. Each of AFT-9 to AFT-31 is inserted into the AAV9 capsid protein as described. Figure 33 SEQ ID NO: 55875 and 55887-55909 are disclosed in the order of their appearance.

[0220] Figure 34 AAV was displayed -mini A graph showing the average gene transfer efficiency (RNA logMN_fold change) of each rAAV in the library across all brain tissues. ** indicates a technical replicate of AFT-6 (SEQ ID NO: 55820).

[0221] Figure 35A graph showing the tissue enrichment scores (expressed on a log10 scale) of the tissues presented is displayed. The 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)). Targeted 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 the cervical, lumbar, and thoracic dorsal root ganglia; and the liver. Higher tissue enrichment scores represent greater tropism.

[0222] Figure 36 A graph showing the tissue enrichment scores (expressed on a log10 scale) of the tissues presented is displayed. The capsids tested included AAV9, AAV9 containing the N3-targeting peptide (PLNGSVHLY (SEQ ID NO: 3603 in Appendix B) located between amino acid residues 586 and 589 in VR VIII (replacing amino acids A587 and Q588), and AAV9 containing the N4-targeting peptide (PLNGTVHLY (SEQ ID NO: 1232 in Appendix B) located between amino acid residues 586 and 589 in VR VIII (replacing amino acids A587 and Q588)). The CNS tissues analyzed included the frontal lobe, temporal lobe, putamen, thalamus, and globus pallidus. Higher tissue enrichment scores represented greater tropism.

[0223] Figure 37 A graph showing the tissue enrichment fractions (expressed on a log10 scale) of the tissues shown is presented. The capsids tested included AAV9 containing the N1 targeting peptide (SEQ ID NO: 9) located in VR VIII of AAV9; the Anc80L65 capsid (the complete capsid sequence is referred to as AFT-6 (SEQ ID NO: 55820)) containing the N3 targeting peptide (PLNGSVHLY (SEQ ID NO: 3603)) located between amino acid residues 586 and 589 in VR VIII (replacing amino acids A587 and Q588); and the N4 targeting peptide (PLNGTVHLY (SEQ ID NO: 1232)) located between amino acid residues 586 and 589 in VR VIII (replacing amino acids A587 and Q588). The tissues analyzed included the frontal lobe, motor cortex, parietal lobe, occipital lobe, cerebellum, putamen, thalamus, globus pallidus, caudate nucleus, substantia nigra, liver, cervical DRG, lumbar DRG, and thoracic DRG. Higher tissue enrichment scores indicated greater directional tendency.

[0224] Figure 38A sequence of the VR VIII window of an AAV capsid library modified with target peptides (e.g., X1X2X3X4X5X6X7X8X9) is provided, wherein the residues at X1 to X9 are selected from the amino acid residues in the corresponding columns. In this library, the spaces at positions 587 and 588 correspond to the deletion of A587 and Q588 from the modified AAV capsid protein. Figure 38 SEQ ID NO: 55875 and 55910 were disclosed in the order of their appearance. Detailed Implementation

[0225] 6.1. Definition

[0226] "AAV" stands for adeno-associated virus, and can be used to refer to the virus itself or its derivatives. Unless otherwise required, the term covers all subtypes, serotypes, and pseudotypes, as well as naturally occurring and recombinant forms.

[0227] 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 AAV9; AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAVhu.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.2 9-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; r wild-type or modified capsid proteins of h.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.

[0228] The term "modified AAV capsid protein" or simply "modified capsid protein" refers to a capsid protein that has been modified compared to a naturally occurring or synthetic / artificial capsid protein (referred to herein as the "reference AAV capsid protein" or "reference capsid protein"). As used herein, the reference AAV capsid protein may be a naturally occurring AAV variant of the form VPI, VP2, or VP3, or a non-naturally occurring VP1, VP2, or VP3 capsid protein known in the art.

[0229] As used herein, the term "targeting peptide" refers to an amino acid sequence of 5 to 16 amino acids in length within the variable region VIII (VRVIII) of a modified AAV capsid protein introduced through one or more modifications described herein. The localization and distribution of AAVs containing modified capsid proteins and possessing targeting peptides in target cells, tissues, or organs may differ from those of AAVs possessing capsid proteins but lacking the targeting peptide.

[0230] As used herein, the term "amino acid position" within the AAV capsid protein refers to the position of an amino acid residue in the AAV VP1 protein sequence, counting from the first amino acid at the N-terminus. As used herein, the term "amino acid" includes naturally occurring L- and D-amino acids as well as 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.

[0231] To avoid ambiguity, as used herein, the indication of an insertion site at amino acid position X means that the target peptide is inserted between amino acids X and X+1, that is, after the amino acid at position X and before the amino acid at position X+1.

[0232] The term "inverted terminal repeat sequence" (or "ITR") refers to a hairpin-forming polynucleotide sequence located at the end of the AAV genome that contributes to the genome's self-priming capacity (allowing for primase-independent synthesis of the complementary second DNA strand) and enables the genome to be capsidated into AAV particles. ITRs can be wild-type ITRs or variants thereof.

[0233] When the term “modification” is combined with one amino acid residue, multiple amino acid residues or a modified sequence, it refers to (one or more) insertions, deletions, substitutions or combinations thereof.

[0234] The terms "operably linked" and "operatively linked" refer to the functional relationship between a nucleic acid sequence and a regulatory sequence of nucleotides (such as promoters, enhancers, transcription and translation termination sites, and other signaling sequences), indicating that two or more consecutive DNA segments are linked together so that they function together for their intended purpose. For example, an operatively linked nucleic acid sequence (usually DNA) to a regulatory sequence or promoter region refers to the physical and functional relationship between the DNA and that regulatory sequence or promoter, such that transcription of this DNA is initiated from that regulatory sequence or promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.

[0235] The term "parenteral" administration of the composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0236] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably in this document to refer to polymers of amino acid residues.

[0237] The term "pharmaceuticalally acceptable carrier" includes any standard drug carrier, excipient, stabilizer, and excipient. For examples of carriers, excipients, stabilizers, and excipients, see Remington: The Science and Practice of Pharmacy, 22nd revised edition, Pharmaceutical Press, 2012.

[0238] The abbreviation "rAAV" refers to a recombinant adeno-associated virus particle containing at least one AAV capsid protein and capsidated polynucleotides, sometimes referred to as the "genome" in this document. rAAV may include a genome containing heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as heterologous polynucleotides encoding genes delivered to mammalian cells, such as sequences encoding therapeutic proteins).

[0239] The terms “sequence identity percentage” (sequence identity %), “identity percentage” (identity %), etc., refer to the percentage of sequence similarity between two nucleotide sequences or two amino acid sequences. It is calculated by comparing two sequences to determine the number of matching nucleotide or amino acid residues between the two sequences, dividing the number of matching by the length of the aligned region (i.e., the number of aligned nucleotide or amino acid residues) and multiplying by 100 to obtain the sequence identity percentage value. To calculate the sequence identity percentage (Sequence Identity %), the EMBOSS Needle pairwise sequence alignment software tool (available at www.ebi.ac.uk / Tools / psa / emboss_needle) based on the Needleman and Wunsch algorithms was used to align two or more sequences with the following parameters: Matrix: BLOSUM62 (for protein sequences) or DNAfull (for DNA sequences); Gap Open: 10; Gap Extend: 0.5; End Gap Penalty: False; End Gap Open: 10; and End Gap Extend: 0.5.

[0240] Methods for aligning nucleotide and amino acid sequences for comparison are well known in the art. Smith and Waterman (1981) Adv. Appl. Math 2:482's local homology algorithm (BESTFIT) allows for optimal alignment of compared sequences; Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453's homology alignment algorithm (GAP); Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444's similarity search method (Tfasta and Fasta); and computerized implementations of these algorithms, including but not limited to: Intelligenetics, Mountain View, Calif.'s PC / Gene program CLUSTAL, Wisconsin Genetics software package version 8 (available from Genetics Computer Group (GCG)). TMThe procedures (Accelrys, Inc., San Diego, Calif.) include GAP, BESTFIT, BLAST, FASTA, and TFASTA. The CLUSTAL procedures are well described by 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 procedure 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, and is incorporated herein by reference). The family of BLAST procedures that can be used for database similarity searches includes: BLASTN for aligning nucleotide query sequences with sequences in a nucleotide database; BLASTX for aligning nucleotide query sequences with sequences in a protein database; BLASTP for aligning protein query sequences with sequences in a protein database; TBLASTN for aligning protein query sequences with sequences in a nucleotide database; and TBLASTX for aligning nucleotide query sequences with sequences in a nucleotide database. See Current Protocols in Molecular Biology, Chapter 19, edited by Ausubel et al., Greene Publishing and Wiley. - Interscience, New York (1995). Newer versions of the BLAST program family include the BLAST+ suite. (Camacho, C. et al., (December 15, 2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421).

[0241] As used herein, the term “tissue-specific” promoter or expression regulatory element (ERE) refers to a nucleotide sequence that, when operatively linked to a polynucleotide encoded by a gene or specified therein, will result in the production of a gene product in the cell only if the cell is a cell of the tissue type corresponding to that promoter.

[0242] The terms “treatment,” “treating,” etc., are generally used herein to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in the complete or partial prevention of a disease, condition, or its symptoms, and / or therapeutic in the partial or complete cure of a disease or condition and / or attributable to its negative effects. As used herein, “treatment” covers any treatment of a disease or condition in mammals (particularly humans) and includes: (a) preventing the development of the disease or condition in a subject who may be susceptible but has not yet been diagnosed with it; (b) inhibiting the disease or condition (e.g., preventing its development); or (c) alleviating the disease or condition (e.g., causing its deterioration, improving one or more symptoms).

[0243] The terms “vector,” “AAV vector,” and “rAAV vector” refer to rAAVs containing heterologous polynucleotides (e.g., transgenics).

[0244] 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) in the AAV capsid protein previously defined by comparison and alignment of various AAV capsid proteins. See, for example, Govindasamy et al., Structurally mapping the diverse phenotype of adeno-associated virus serotype 4, J. Virol. (2006); Meyer et al., Structure of the genetherapy vector, adeno-associated virus with its cell receptor, AAVR, eLife (2019). VR is known to contain amino acids that result in slight differences in surface topology and different functional phenotypes (e.g., receptor binding, transduction efficiency, and antigen reactivity). The relative positions of VR I, VR IV, VR V, and VIIII are shown in […]. Figure 28 The text states that the specific location of the variable region within the capsid protein can vary depending on the capsid protein and / or sequence alignment method.

[0245] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these methods and compositions of substances pertain. While methods and materials similar to or equivalent to those described herein may be used to practice or test compositions of these methods and substances, suitable methods and materials are described below. Furthermore, these materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety.

[0246] 6.2. Modified AAV capsid protein

[0247] One aspect of this disclosure provides a modified adeno-associated virus (AAV) capsid protein comprising: a targeting peptide within a variable region VIII (VR VIII), wherein the targeting peptide has a sequence of X1X2X3X4X5X6X7X8X9, and X1, X2, X3, X4, X5, X6, X7, X8 and X9 are each independently selected from any amino acid residue.

[0248] 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 NO: 620-55819.

[0249] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a targeting peptide within variable region VIII (VRVIII), wherein the targeting peptide has the sequence PX2X3GAVX7LY (SEQ ID NO: 2), and X2, X3, and X7 are independently selected from any amino acid residues.

[0250] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a targeting peptide within the variable region VIII (VRVIII), wherein the targeting peptide has the sequence Px2x3GX5X6X7LY (SEQ ID NO: 10), and x2, x3, x5, x6, and x7 are independently selected from any amino acid residues.

[0251] In some embodiments, the modified adeno-associated virus (AAV) capsid protein comprises a targeting peptide within the variable region VIII (VRVIII), wherein the targeting peptide has the sequence Px2X3GAVX7X8X9 (SEQ ID NO: 20), and X2, X3, X5, X6, and X7 are independently selected from any amino acid residues.

[0252] 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 these amino acid insertions, deletions, substitutions, or combinations thereof are used to introduce the targeting peptide into the reference AAV capsid protein. In some embodiments, all differences between the modified AAV capsid protein and the reference AAV capsid protein are within the variable region VIII (VR VIII). In some embodiments, all differences between the modified AAV capsid protein and the reference AAV capsid protein are within the targeting peptide.

[0253] In some embodiments, in addition to introducing the targeting peptide into VR VIII of the reference AAV capsid protein, the modified AAV capsid protein further comprises one or more modifications, including amino acid insertions, deletions, substitutions, or combinations thereof.

[0254] In some embodiments, the modified capsid protein further includes one or more modifications in addition to VRVIII of the reference AAV capsid protein.

[0255] In some embodiments, the modified AAV capsid protein contains one or more modifications, including amino acid insertions, deletions, substitutions, or combinations thereof, to introduce the targeting peptide within VR VIII (e.g., where VR VIII corresponds to an amino acid between positions 565 and 595 of the reference AAV capsid protein).

[0256] In some embodiments, the reference AAV capsid protein is the AAV9 capsid protein. In some embodiments, the modified AAV capsid protein has a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the AAV9 capsid protein. In some embodiments, the modified AAV capsid protein has a sequence that differs from the AAV9 capsid protein by (e.g., insertion, deletion, or substitution) 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 some embodiments, the modified AAV capsid protein has a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an AAV9 capsid protein having a targeting peptide in VR VIII (e.g., any targeting peptide described herein). In some embodiments, the modified AAV capsid protein has a sequence differing (e.g., by insertion, deletion, or substitution) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more amino acid residues from an AAV9 capsid protein having a targeting peptide in VR VIII (e.g., any targeting peptide described herein).

[0257] In some embodiments, the reference AAV capsid protein is the Anc80L65 capsid protein. In some embodiments, the modified AAV capsid protein has a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the Anc80L65 capsid protein. In some embodiments, the modified AAV capsid protein has a sequence that differs from the Anc80L65 capsid protein by (e.g., insertion, deletion, or substitution) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more amino acid residues. In some embodiments, the modified AAV capsid protein has a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the Anc80L65 capsid protein having a targeting peptide in VR VIII (e.g., any targeting peptide described herein). In some embodiments, the modified AAV capsid protein has a sequence differing (e.g., by insertion, deletion, or substitution) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more amino acid residues from the Anc80L65 capsid protein having a targeting peptide in VR VIII (e.g., any targeting peptide described herein).

[0258] In some embodiments, the modified AAV capsid protein has a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from SEQ ID NO: 34-38 or 55820-55847. In some embodiments, the modified AAV capsid protein has a sequence selected from SEQ ID NO: 34-38 or 55820-55847. In some embodiments, the modified AAV capsid protein has a sequence selected from SEQ ID NO: 34-39.

[0259] In some embodiments, when administered to a subject at a therapeutically effective amount, AAV virions containing the modified AAV capsid protein have enhanced specificity for central nervous system (CNS) tissues, relative to AAV virions containing a reference capsid protein but without the targeting peptide.

[0260] In some implementations, when administered to a subject at a therapeutically effective amount, AAV virions containing the modified AAV capsid protein have enhanced transduction efficiency in the CNS compared to AAV virions containing a reference capsid protein but without the targeting peptide.

[0261] In some embodiments, when administered to a subject at a therapeutically effective amount, AAV virions containing the modified AAV capsid protein have enhanced blood-brain barrier penetration in the subject, relative to AAV virions containing a reference capsid protein but without the targeting peptide.

[0262] In some implementations, when administered to a subject at a therapeutically effective amount, AAV virions containing the modified AAV capsid protein have reduced specificity for central nervous system (CNS) tissues compared to AAV virions containing a reference capsid protein but without the targeting peptide.

[0263] In some implementations, when administered to a subject at a therapeutically effective amount, AAV virions containing the modified AAV capsid protein have reduced transduction efficiency in the CNS compared to AAV virions containing a reference capsid protein but without the targeting peptide.

[0264] In some embodiments, when administered to a subject at a therapeutically effective amount, AAV virions containing the modified AAV capsid protein have reduced blood-brain barrier penetration in the subject, compared to AAV virions containing a reference capsid protein but without the targeting peptide.

[0265] 6.2.1. Targeted peptides

[0266] In some embodiments, the targeting peptide within the 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 with the sequence X1X2X3X4X5X6X7X8X9 at a site within the variable region VIII (VR VIII), 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. In some embodiments,

[0267] (i) X1 is independently selected from proline (P) and glycine (G);

[0268] (ii) X2 is independently selected from lysine (L), threonine (T), serine (S), alanine (A), valine (V) and isoleucine (I);

[0269] (iii) X3 is independently selected from asparagine (N), glutamine (Q) and proline (P);

[0270] (iv)X4 is independently selected from glycine (G) and alanine (A);

[0271] (v)X5 is independently selected from alanine (A), threonine (T), serine (S), valine (V) and glycine (G);

[0272] (vi)X6 is independently selected from valine (V), leucine (L), alanine (A), isoleucine (I), glycine (G), serine (S) and threonine (T);

[0273] (vii)x7 is independently selected from histidine (H), arginine (R), and lysine (K);

[0274] (viii) X8 is independently selected from leucine (L) and valine (V); and

[0275] (ix)x9 is independently selected from tyrosine (Y), arginine (R), histidine (H), lysine (K) and phenylalanine (F).

[0276] In some embodiments, the modified AAV capsid protein comprises a targeting peptide introduced into VR VIII having at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 620-55819. In some embodiments, the modified AAV capsid protein comprises a targeting peptide introduced into VR VIII, wherein the peptide has a sequence differing from the comparative peptide by substitutions of 0, 1, 2, 3, or 4 residues in the amino acid sequence selected from SEQ ID NO: 620-55819.

[0277] In some embodiments, the targeting peptide has the sequence PX2X3GAVX7LY (SEQ ID NO: 2), and X2, X3, and X7 are independently selected from any amino acid residues. In some embodiments, the modified capsid protein comprises a targeting peptide having the sequence PX2X3GAVX7LY (SEQ ID NO: 2), wherein the targeting peptide has inserted or substituted amino acid residues A587 and Q588. In some embodiments,

[0278] (i) X2 is independently selected from lysine (L), isoleucine (I), valine (V) and alanine (A);

[0279] (ii) X3 is asparagine (N) or glutamine (Q); and

[0280] (iii)X7 is independently selected from histidine (H), arginine (R) and lysine (K).

[0281] In some embodiments, the targeting peptide is selected from:

[0282] (i) PLQGAVHLY (SEQ ID NO: 3);

[0283] (ii) PLQGAVRLY (SEQ ID NO: 4);

[0284] (iii) PLQGAVKLY (SEQ ID NO: 5);

[0285] (iv) PINGAVHLY (SEQ ID NO: 6);

[0286] (v) PVNGAVHLY (SEQ ID NO: 7);

[0287] (vi) PANGAVHLY (SEQ ID NO: 8); or

[0288] (vii) PLNGAVHLY (SEQ ID NO: 9).

[0289] 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).

[0290] In some embodiments, the targeting peptide has a sequence selected from SEQ ID NO: 620-55819.

[0291] In some embodiments, the targeting peptide has the sequence PX2X3GX5X6X7LY (SEQ ID NO: 10), and X2, X3, X5, X6, and X7 are independently selected from any amino acid residues. In some embodiments, the modified capsid protein comprises a targeting peptide having the sequence PX2X3GX5X6X7LY (SEQ ID NO: 10), wherein the targeting peptide has inserted and substituted amino acid residues A587 and Q588. In some embodiments,

[0292] (i) X2 is independently selected from leucine (L), threonine (T) or serine (S);

[0293] (ii) X3 is independently selected from asparagine (N) and glutamine (Q);

[0294] (iii) X5 is independently selected from alanine (A) and threonine (T);

[0295] (iv) X6 is independently selected from valine (V) and leucine (L); and

[0296] (v)X7 is independently selected from histidine (H), arginine (R), and lysine (K).

[0297] In some embodiments, the targeting peptide is selected from:

[0298] (i)PTNGTVRLY (SEQ ID NO: 11);

[0299] (ii) PTNGTVHLY (SEQ ID NO: 12);

[0300] (iii) PTNGTVKLY (SEQ ID NO: 13);

[0301] (iv)PSNGTLRLY (SEQ ID NO: 14);

[0302] (v) PSNGTLHLY ​​(SEQ ID NO: 15);

[0303] (vi) PSNGTLKLY (SEQ ID NO: 16);

[0304] (vii)PTNGTLRLY (SEQ ID NO: 17);

[0305] (Viii)PTNGTLHLY ​​(SEQ ID NO: 18); or

[0306] (ix)PTNGTLKLY (SEQ ID NO: 19).

[0307] In some embodiments, the targeting peptide has the sequence PX2X3GAVX7X8X9 (SEQ ID NO: 20), and X2, X3, X5, X6, and X7 are independently selected from any amino acid residues. In some embodiments, the modified capsid protein comprises a targeting peptide having the sequence PX2X3GAVX7X8X9 (SEQ ID NO: 20), wherein the targeting peptide has inserted substitutions of amino acid residues A587 and Q588. In some embodiments,

[0308] (i) X2 is independently selected from leucine (L), threonine (T) or serine (S);

[0309] (ii) X3 is independently selected from asparagine (N) and glutamine (Q);

[0310] (iii) X7 is independently selected from histidine (H) and threonine (T);

[0311] (iv) X8 is independently selected from valine (V) and leucine (L); and

[0312] (v)X9 is independently selected from tyrosine (Y) and arginine (R).

[0313] In some embodiments, the targeting peptide is selected from:

[0314] (i) PTQGAVTVR (SEQ ID NO: 21);

[0315] (ii) PLQGAVTVR (SEQ ID NO: 22);

[0316] (iii) PLQGAVHVR (SEQ ID NO: 23);

[0317] (iv) PLQGAVHVY (SEQ ID NO: 24);

[0318] (v) PSQGAVTLR (SEQ ID NO: 25);

[0319] (vi) PLQGAVTLR (SEQ ID NO: 26);

[0320] (vii)PLQGAVHLR(SEQ ID NO: 27); or

[0321] (viii) PTQGAVTLR (SEQ ID NO: 28).

[0322] In some embodiments, the targeting peptide is PLNGSVHLY (SEQ ID NO: 3603).

[0323] In some embodiments, the modified AAV capsid protein comprises a modified AAV9 capsid protein containing a targeting peptide having the sequence PLNGSVHLY (SEQ ID NO: 3603) located between amino acid residues 586 and 589 in VR VIII, replacing amino acids A587 and Q588.

[0324] In some embodiments, the targeting peptide is PLNGTVHLY (SEQ ID NO: 1232).

[0325] In some embodiments, the modified AAV capsid protein comprises a modified AAV9 capsid protein containing a targeting peptide having the sequence PLNGTVHLY (SEQ ID NO: 1232) located between amino acid residues 586 and 589 in VR VIII, replacing amino acids A587 and Q588.

[0326] In some embodiments, the targeting peptide is selected from any of those in Appendix B.

[0327] In some embodiments, the targeting peptide does not contain the sequence of SEQ ID NO: 9.

[0328] In some embodiments, the targeting peptide within VR VIII has a sequence selected from SEQ ID NO: 160-619. In some embodiments, the targeting peptide is inserted into the AAV9 or Anc80L65 backbone.

[0329] In some embodiments, the modified AAV capsid protein comprises a targeting peptide introducing VR VIII, having at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% sequence identity with the amino acid sequence selected from SEQ ID NO: 160-619. In some embodiments, the modified AAV capsid protein comprises a targeting peptide introducing VR VIII, wherein the peptide has a sequence differing from the comparative peptide by substitutions of 0, 1, 2, 3, or 4 residues in the amino acid sequence selected from SEQ ID NO: 160-619.

[0330] In some embodiments, the modified AAV capsid protein comprises a targeting peptide having a sequence selected from SEQ ID NO: 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 NO: 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 NO: 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.

[0331] In some embodiments, the targeting peptide is the targeting peptide disclosed in US2017 / 0166926 (which is incorporated herein by reference in its entirety).

[0332] In some embodiments, the targeting peptide is not the targeting peptide disclosed in WO 2020 / 210655; WO 2021 / 222831; WO 2021 / 202651; WO 2021 / 211 753; WO 2021 / 226167; WO 2021 / 230987; or WO 2022 / 040527 (which is incorporated herein by reference in its entirety).

[0333] In some embodiments, the targeting peptide does not have the sequence identified in the variable region VR VIII of the reference AAV capsid protein.

[0334] 6.2.2. Target peptide sites

[0335] The modified AAV capsid protein of this disclosure includes a targeting peptide within VRVIII of the reference AAV capsid protein. Figure 28 ).

[0336] Preferably, the targeting peptide is located at a site exposed outside the capsid, preferably based on structural prediction and / or experimental data. More preferably, the targeting peptide is located at a site exposed outside the AAV capsid in a manner that does not interfere with the activity of the protein during capsid assembly.

[0337] In some embodiments, the one or more modifications include amino acid insertions, deletions, substitutions, or combinations thereof in which the targeting peptide is introduced into VR VIII of the reference AAV capsid protein.

[0338] In some embodiments, the one or more modifications include the deletion of one or more amino acids within VR VIII. In some embodiments, the one or more amino acid deletions include the deletion of one, two, three, four, or five 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 include the 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 include the 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 include the 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 include the deletion of four amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII.

[0339] In some embodiments, the deletion of one or more amino acids includes the 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. In some embodiments, the deletion of one or more amino acids includes the deletion of an amino acid residue at position 587, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the deletion of one or more amino acids includes the deletion of amino acid residues at positions 587 and 588, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the deletion of one or more amino acids includes the deletion of amino acid residues at positions 586, 587, and 588, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the deletion of one or more amino acids includes the deletion of amino acid residues at positions 585, 586, 587, and 588 relative to a reference sequence numbered according to the amino acid sequence number of the reference AAV capsid protein.

[0340] In some embodiments, the deletion of one or more amino acids includes the deletion of one, two, three, four, or five or more amino acid residues immediately adjacent to the C-terminus of the target peptide within VR VIII. In some embodiments, the deletion of one or more amino acids includes the deletion of one amino acid residue immediately adjacent to the C-terminus of the target peptide within VR VIII. In some embodiments, the deletion of one or more amino acids includes the deletion of two amino acid residues immediately adjacent to the C-terminus of the target peptide within VR VIII. In some embodiments, the deletion of one or more amino acids includes the deletion of three amino acid residues immediately adjacent to the C-terminus of the target peptide within VR VIII. In some embodiments, the deletion of one or more amino acids includes the deletion of four amino acid residues immediately adjacent to the C-terminus of the target peptide within VR VIII.

[0341] In some embodiments, the amino acid deletion includes the deletion of an amino acid residue at positions 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. In some embodiments, the amino acid deletion includes the deletion of an amino acid residue at position 589, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the amino acid deletion includes the deletion of an amino acid residue at position 590, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the amino acid deletion includes the deletion of an amino acid residue at position 591, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the amino acid deletion includes the deletion of amino acid residues at positions 589 and 590, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the amino acid deletion includes the deletion of an amino acid residue at positions 590 and 591, relative to a reference sequence numbered according to the amino acid sequence of the reference AAV capsid protein. In some embodiments, the amino acid deletion includes the deletion of amino acid residues at positions 589, 590, and 591 relative to a reference sequence numbered according to the amino acid sequence number of the reference AAV capsid protein.

[0342] In some embodiments, the one or more modifications include an amino acid insertion that introduces the targeting peptide into the VR VIII of the reference AAV capsid protein. In some embodiments, the amino acid insertion includes the insertion of one, two, three, four, or five or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of one amino acid residue immediately adjacent to the C-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of two amino acid residues immediately adjacent to the C-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of three amino acid residues immediately adjacent to the C-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of four amino acid residues immediately adjacent to the C-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of five amino acid residues immediately adjacent to the C-terminus of the targeting peptide within the VR VIII.

[0343] In some embodiments, the one or more modifications include an amino acid insertion that introduces the targeting peptide into the VR VIII of the reference AAV capsid protein. In some embodiments, the amino acid insertion includes the insertion of one, two, three, four, or five or more amino acid residues immediately adjacent to the N-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of one amino acid residue immediately adjacent to the N-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of two amino acid residues immediately adjacent to the N-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of three amino acid residues immediately adjacent to the N-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of four amino acid residues immediately adjacent to the N-terminus of the targeting peptide within the VR VIII. In some embodiments, the one or more amino acid insertions include the insertion of five amino acid residues immediately adjacent to the N-terminus of the targeting peptide within the VR VIII.

[0344] In some embodiments, the inserted amino acid residues are independently selected from any amino acid residues. In some embodiments in which VRVIII includes insertions and deletions of the amino acid sequence relative to the reference AAV capsid protein, the inserted amino acid residue(s) is identical to the deleted amino acid residue(s) adjacent to the N-terminus or C-terminus of the target peptide within 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 is asparagine (N) at the position immediately adjacent to the C-terminus of the target peptide and asparagine (N) at the next subsequent position, thus having an amino acid sequence of AN immediately adjacent to the C-terminus of the target peptide.

[0345] 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).

[0346] In some embodiments, the one or more modifications include amino acid insertions, deletions, and / or substitutions in the VR VIII of the reference AAV capsid protein to introduce the targeting peptide. 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 amino acid residues immediately adjacent to the C-terminus of the targeting peptide and comprises a deletion of one amino acid residue immediately adjacent to the N-terminus of the targeting peptide.

[0347] In some embodiments, the insertion site of the targeting peptide is in Figures 29A-29C and Figures 30A-30D Provided in [the document / reference]. In some embodiments, the insertion site of the targeting peptide is [located in / at a location that is not specified in the document / reference]. Figure 29B Provided in [the document / reference]. In some embodiments, the insertion site of the targeting peptide is [located in / at a location that is not specified in the document / reference]. Figure 30C Provided by China.

[0348] In some embodiments, the insertion sites of the targeting peptide are provided in Table 1. In Table 1, preferred sites are indicated by "-" relative to the wild-type VP1 capsid peptide. In some embodiments, Table 1 includes exemplary insertion sites of targeting peptides selected from SEQ ID NO: 620-55819. In some embodiments, Table 1 includes exemplary insertion sites of targeting peptides selected from SEQ ID NO: 160-619.

[0349]

[0350]

[0351] Three exemplary insertion sites are indicated in Table 1. For insertion sites 1 and 2, the targeting peptide is inserted between two amino acid positions, where the insertion site is indicated by "-". For example, for insertion site 1 of the AAV1 capsid protein, the targeting peptide is inserted between positions D590 and P591. In some embodiments, the AAV capsid protein is modified by inserting the targeting peptide after a mutation or substitution of one or more amino acids. For example, for insertion site 3 in Table 1, two amino acids are deleted before the insertion of the targeting peptide. In such cases, "[]" represents the deleted amino acid residue / position. For example, for insertion site 3 of the AAV9 capsid protein, the targeting peptide is inserted between positions S586 and A589 after the amino acid "AQ" (A587 and Q588) located between A586 and A589 is deleted, indicated by "S[][]-A".

[0352] In some embodiments, the targeting peptide is located between 560 and 610 within VRVIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is located between 565 and 605 within VRVIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is located between 570 and 600 within VRVIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is located between 575 and 595 within VRVIII of the modified AAV capsid protein. In some embodiments, the targeting peptide is located between 580 and 590 within VRVIII of the modified AAV capsid protein.

[0353] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV1 or a modification thereof, and the targeting peptide is between D590 and P591 or between S588 and T589 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV1 or a modification thereof, and the targeting peptide is between positions 587 and 594 or between 585 and 592 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV1 or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, with amino acid residues S587 and S588 deleted.

[0354] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV2 or a modification thereof, and the targeting peptide is located between R585 and Q589 or between N587 and R588 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV2 or a modification thereof, and the targeting peptide is located 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 the capsid protein of AAV2 or a modification thereof, and the targeting peptide is located between R585 and R588, thereby replacing amino acid residues G586 and N587.

[0355] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV3 or a modification thereof, and the targeting peptide is between S586 and S587 or between N588 and T589 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV3 or a modification thereof, and the targeting peptide is between positions 583 and 590 or between 585 and 592 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV3 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.

[0356] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV4, and the targeting peptide is between S584 and N585 or between S586 and N587 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV4 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 the capsid protein of AAV4 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.

[0357] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV5 or a modification thereof, and the targeting peptide is between positions S575 and S576 or between T577 and T578 of the capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV5 or a modification thereof, and the targeting peptide is between positions 572 and 579 or between 574 and 581 of the capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV5, and the targeting peptide is between S575 and T578 of the reference AAV capsid protein, thereby replacing S576 and S577.

[0358] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV6 or a modification thereof, and the targeting peptide is between D590 and P591 or S588 and T589 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV6 or a modification thereof, and the targeting peptide is between positions 587 and 594 or 585 and 592 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV6 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.

[0359] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV7 or a modification thereof, and the targeting peptide is located between N589 and T590 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV7 or a modification thereof, and the targeting peptide is located between positions 586 and 593 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV7 or a modification thereof, and the targeting peptide is located between A587 and T590 of the reference AAV capsid protein, thereby replacing amino acid residues A588 and N589.

[0360] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV8, and the targeting peptide is located between N590 and T591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV8 or a modification thereof, and the targeting peptide is located between positions 587 and 594 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV8 or a modification thereof, and the targeting peptide is located between Q588 and T591 of the modified AAV capsid protein, thereby replacing amino acid residues Q589 and N590.

[0361] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV9, and the targeting peptide is located between Q588 and A589 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV9 or a modification thereof, and the targeting peptide is located between positions 585 and 592 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV9 or a modification thereof, and the targeting peptide is located between S586 and A589 of the reference AAV capsid protein, thereby replacing amino acid residues A587 and Q588.

[0362] In some embodiments, the reference AAV capsid protein is the capsid protein of AAVrh10 or a modification thereof, and the targeting peptide is located between N590 and A591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAVrh10 or a modification thereof, and the targeting peptide is located between positions 587 and 594 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAVrh10 or a modification thereof, and the targeting peptide is located between Q588 and A591 of the reference AAV capsid protein, thereby replacing amino acid residues Q589 and N590.

[0363] In some embodiments, 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 modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAVpo.1 or a modification thereof, and the targeting peptide is between positions 570 and 571 or between positions 566 and 573 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is 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 amino acid residues Q566 and N567.

[0364] In some embodiments, the reference AAV capsid protein is the capsid protein of AAV12 or a modification thereof, and the targeting peptide is between N592 and A593 or between T594 and T595 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV12 or a modification thereof, and the targeting peptide is between positions 589 and 596 or between 591 and 598 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of AAV12 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.

[0365] In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80 or a modification thereof, and the targeting peptide is between T589 and A590, N588 and T589, or S587 and N588 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80 or a modification thereof, and the targeting peptide is between positions 586 and 593 or 584 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80 or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby substituting amino acid residues S587 and N588.

[0366] In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80L65 or a modification thereof, and the targeting peptide is between T589 and A590, N588 and T589, or A587 and N588 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80L65 or a modification thereof, and the targeting peptide is between positions 586 and 593 or 584 and 591 of the modified AAV capsid protein.

[0367] In some embodiments, the reference capsid protein is the capsid protein of Anc80L65 or a modification thereof, and the targeting peptide (i) is located between S586 and T589 of the Anc80L65 capsid protein, thereby replacing A587 and N588 of the Anc80L65 capsid protein; (ii) is located between Q585 and N588 of the Anc80L65 capsid protein, thereby replacing S586 and T589 of the Anc80L65 capsid protein. A587; (iii) between L584 and A587 of the Anc80L65 capsid protein, thereby replacing Q585 and S586 of the Anc80L65 capsid protein; (iv) between A587 and A590 of the Anc80L65 capsid protein, thereby replacing N588 and T589 of the Anc80L65 capsid protein; or (v) between S586 and A587 of the Anc80L65 capsid protein.

[0368] In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-55 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 the capsid protein of Anc80-55 or a modification thereof, and the targeting peptide is between positions 586 and 593 or between 584 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-55 or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby substituting amino acid residues S587 and N588.

[0369] In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-129 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 the capsid protein of Anc80-129 or a modification thereof, and the targeting peptide is between positions 586 and 593 or between 584 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-129 or a modification thereof, and the targeting peptide is between S586 and T589 of the reference AAV capsid protein, thereby substituting amino acid residues A587 and N588.

[0370] In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-156 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 the capsid protein of Anc80-156 or a modification thereof, and the targeting peptide is between positions 586 and 593 or between 584 and 591 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-156 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.

[0371] In some embodiments, 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 A587 and N588 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-751 or a modification thereof, and the targeting peptide is between positions 586 and 593 or between 584 and 591 of the modified AAV capsid protein. In some embodiments, 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 substituting amino acid residues A587 and N588.

[0372] In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-1029 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 the capsid protein of Anc80-1029 or a modification thereof, and the targeting peptide is between positions 586 and 593 or between 584 and 591 of the modified AAV capsid protein. In some embodiments, 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 substituting amino acid residues A587 and N588.

[0373] In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-1712 or a modification thereof, and the targeting peptide is between T589 and A590 or between A587 and T588 of the modified AAV capsid protein. In some embodiments, the reference AAV capsid protein is the capsid protein of Anc80-1712 or a modification thereof, and the targeting peptide is between positions 586 and 593 or between 584 and 591 of the modified AAV capsid protein. In some embodiments, 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 amino acid residues A587 and N588.

[0374] 6.2.3. AAV capsid proteins modified with flanking regions

[0375] In some embodiments, the modified AAV capsid protein is further contained in the N-terminal flanking region of the N-terminus of the targeting peptide, the C-terminal flanking region of the C-terminus of the targeting peptide, or both.

[0376] 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 these amino acids are selected from amino acid residues 578-588, 577-587, or 576-586 of AAV9.

[0377] In some embodiments, the N-terminal flanking region comprises at least four (e.g., at least five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues (e.g., 558-588, 558-587, 558-586, and 558-585) from amino acids 558-589 of the reference AAV capsid protein, wherein these 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 that differs from (e.g., by insertion, deletion, or substitution) amino acids 558-589 of the reference AAV capsid protein by 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.

[0378] In some embodiments, the N-terminal flanking region comprises at least four (e.g., at least five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from amino acids 576-585 of the reference AAV capsid protein, wherein these 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 five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from amino acids 576-586 of the reference AAV capsid protein, wherein these 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 five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from amino acids 576-587 of the reference AAV capsid protein, wherein these 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 five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from the reference AAV capsid protein, wherein these 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 five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from amino acids 576-589 of the reference AAV capsid protein, wherein these amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0379] In some embodiments, the N-terminal flanking region has the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), and 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), wherein B1 is selected from glutamic acid (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H).

[0380] In some embodiments, the N-terminal flanking region has the sequence of SYGQVATNHQS (SEQ ID NO: 55848). In some embodiments, the N-terminal flanking region contains a sequence that differs from the sequence of SYGQVATNHQS (SEQ ID NO: 55848) by 1, 2, 3, 4, 5 or more amino acid residues (e.g., insertion, deletion or substitution).

[0381] In some embodiments, the N-terminal flanking region replaces the N-terminal reference sequence of the 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 at the N-terminus of the targeting peptide insertion site within the reference AAV capsid protein. The modified AAV capsid protein according to claim 34, wherein the N-terminal reference sequence has the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849).

[0382] In some embodiments, the modified AAV capsid protein is further contained in the C-terminal flanking region of the target peptide.

[0383] 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 contains 13 amino acids, wherein these amino acids are selected from amino acid residues 589-602, 588-601, or 587-602 of AAV9.

[0384] In some embodiments, the C-terminal flanking region comprises at least four (e.g., at least five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from amino acids 589-635 of the reference AAV capsid protein, wherein these 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 that differs from amino acid residues 589-635 by (e.g., insertion, deletion, or substitution) by 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.

[0385] In some embodiments, the C-terminal flanking region comprises at least four (e.g., at least five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from amino acids 589-601 of the reference AAV capsid protein, wherein these 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 five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from amino acids 590-601 of the reference AAV capsid protein, wherein these 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 five, at least six, at least seven, at least eight, at least nine, or at least ten) consecutive amino acid residues from amino acids 591-601 of the reference AAV capsid protein, wherein these amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

[0386] 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).

[0387] In some embodiments, the C-terminal flanking region has the sequence AQAQTGWVQNQGI (SEQ ID NO: 55850). In some embodiments, the C-terminal flanking region contains a sequence that differs from AQAQTGWVQNQGI (SEQ ID NO: 55850) by 1, 2, 3, 4, 5 or more amino acid residues (e.g., insertion, deletion or substitution).

[0388] In some embodiments, the C-terminal flanking region replaces the C-terminal reference sequence of the reference AAV capsid protein, wherein 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 at the C-terminus of the targeting peptide insertion site within the reference AAV capsid protein. In some embodiments, the C-terminal reference sequence has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851).

[0389] In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region having the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), wherein B1, B2, and B3 are each independently selected from any amino acid residue; and a C-terminal flanking region having the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851), wherein Z1, Z2, Z3, and Z4 are each independently selected from any amino acid residue.

[0390] In some embodiments, the modified AAV capsid protein further comprises an N-terminal flanking region having the sequence B1YGB2VATNB3QS (SEQ ID NO: 55860), wherein B1 is selected from glutamic acid (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 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).

[0391] 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).

[0392] In some embodiments, the modified AAV capsid protein further comprises the amino acid sequence 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, the modified AAV capsid protein further comprises the amino acid sequence B1YGB2VATNB3QSPLMGAVHLYAQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55862), wherein B1 is selected from glutamic acid (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).

[0393] In one embodiment, the targeting peptide in VR VIII is SEQ ID NO: 29. In some embodiments, the targeting peptide is SEQ ID NO: 29, wherein one or more residues are substituted by the insertion when inserted into VR VIII.

[0394] In some embodiments, the targeting peptide is a peptide having at least 90% (e.g., 92%, 94%, 96%, or 98%) sequence identity with the sequence of SEQ ID NO: 29. In some embodiments, the targeting peptide is a peptide that differs from the sequence of SEQ ID NO: 29 by (e.g., insertion, deletion, or substitution) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues.

[0395] 6.2.4. Additional modifications in modified AAV capsid proteins

[0396] In some embodiments, the modified AAV capsid protein has one or more modifications compared to the reference AAV capsid protein, including amino acid insertions, deletions, substitutions, or combinations thereof.

[0397] In some embodiments, the one or more modifications include amino acid insertions, deletions, substitutions, or combinations thereof in which the targeting peptide is introduced into VR VIII of the reference AAV capsid protein.

[0398] In some embodiments, the one or more modifications include amino acid modifications other than VR VIII of the reference AAV capsid protein.

[0399] In some embodiments, the one or more modifications other than VR VIII of the reference AAV capsid protein include one or more of VR I, VR II, VR III, VR IV, VR V, VR VI, or VR VII.

[0400] In some embodiments, variable region I (VR I) corresponds to the sequence between approximately position 259 and approximately position 275 of the AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR I corresponds to the sequence between approximately position 262 and approximately position 272 of the AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0401] In some embodiments, variable region II (VR II) corresponds to the sequence between approximately position 329 and approximately position 336 of the AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR II corresponds to the sequence between approximately position 330 and approximately position 335 of the AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0402] In some embodiments, variable region III (VR III) corresponds to the sequence between approximately position 378 and approximately position 400 of the AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR III corresponds to the sequence between approximately position 385 and approximately position 394 of the AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0403] In some embodiments, the variable region IV (VR IV) corresponds to the sequence between approximately position 438 and approximately position 480 of the AAV capsid protein (e.g., the reference capsid or a modified capsid). In some embodiments, the VR IV corresponds to the sequence between approximately position 456 and approximately position 476 of the AAV capsid protein (e.g., the reference capsid or a modified capsid). In some embodiments, the VR IV corresponds to the sequence between approximately position 449 and approximately position 468 of the AAV capsid protein (e.g., the reference capsid or a modified capsid).

[0404] In some embodiments, the variable region V (VR V) corresponds to the sequence between approximately position 483 and approximately position 518 of the AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR V corresponds to the sequence between approximately position 494 and approximately position 512 of the AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR V corresponds to the sequence between approximately position 487 and approximately position 504 of the AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0405] In some embodiments, the variable region VI (VR VI) corresponds to the sequence between approximately position 531 and approximately position 549 of the AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR VI corresponds to the sequence between approximately position 533 and approximately position 545 of the AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0406] In some embodiments, variable region VII (VR VII) corresponds to the sequence between approximately position 551 and approximately position 567 in the AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR VII corresponds to the sequence between approximately position 553 and approximately position 563 in the AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0407] In some embodiments, variable region VIII (VR VIII) corresponds to the sequence between approximately positions 570 and 605 of the AAV capsid protein (e.g., the reference capsid or a modified capsid). In some embodiments, variable region VIII (VR VIII) corresponds to the sequence between approximately positions 576 and 601 of the AAV capsid protein (e.g., the reference capsid or a modified capsid). In some embodiments, variable region VIII (VR VIII) corresponds to the sequence between approximately positions 576 and 608 of the AAV capsid protein (e.g., the reference capsid or a modified capsid). In some embodiments, variable region VIII (VR VIII) corresponds to the sequence between approximately positions 579 and 594 of the AAV capsid protein (e.g., the reference capsid or a modified capsid). In some embodiments, VR VIII corresponds to the sequence between approximately positions 585 and 591 of the AAV capsid protein (e.g., the reference capsid or a modified capsid).

[0408] In some embodiments, the variable region IX (VR IX) corresponds to the sequence between approximately position 709 and approximately position 736 of the AAV capsid protein (e.g., a reference capsid or a modified capsid). In some embodiments, VR IX corresponds to the sequence between approximately position 714 and approximately position 721 of the AAV capsid protein (e.g., a reference capsid or a modified capsid).

[0409] In some implementations, the variable regions in the AAV capsid are as follows: 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 As defined in Neurosci., 25(1): 106-115. doi: 10.1038 / s41593-021-00969 (2022), each of which is incorporated herein by reference in its entirety.

[0410] In some embodiments, the variable region in the AAV capsid is defined by amino acids present on a ring exposed to the surface. In some embodiments, the variable region in the AAV capsid is defined by amino acids present on a ring exposed to the surface and the contribution of that ring to tropism (i.e., to the binding of the AAV virion to the receptor). In some embodiments, the presence of a targeting peptide in the modified capsid protein can alter the amino acid residues present on the ring exposed to the surface.

[0411] In some embodiments, the one or more modifications of the reference AAV capsid protein other than VR VIII include one or more modifications of VR IV and VR V.

[0412] 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 with SEQ ID NO: 30. In some embodiments, the one or more modifications in VR VI result in the introduction of a sequence that differs from the sequence of SEQ ID NO: 30 by (e.g., insertion, deletion, or substitution) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues.

[0413] 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 with 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 that differs from the sequence of SEQ ID NO: 31 by (e.g., insertion, deletion, or substitution) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues.

[0414] 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein) and includes VR I derived from a second reference capsid protein (e.g., any reference capsid protein 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein) and includes VR II derived from a second reference capsid protein (e.g., any reference capsid protein 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein) and comprises a VR III derived from a second reference capsid protein (e.g., any reference capsid protein 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein) and comprises a VRIV derived from a second reference capsid protein (e.g., any reference capsid protein 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein) and includes a VR VI derived from a second reference capsid protein (e.g., any reference capsid protein 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein) and includes VR VII derived from a second reference capsid protein (e.g., any reference capsid protein 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein) and includes VR VIII derived from a second reference capsid protein (e.g., any reference capsid protein 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein), and includes VR Ix derived from a second reference capsid protein (e.g., any reference capsid protein described herein).

[0415] 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 with SEQ ID NO: 142 and includes VR I derived 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 with SEQ ID NO: 142 and includes VR IV derived 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 with SEQ ID NO: 142 and includes VR V derived 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 with SEQ ID NO: 142 and contains VR VIII derived from AAV9.

[0416] 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein), and includes two or more variable regions (VR I, II, III, IV, V, VI, VII, VIII, or IX) derived from a second reference capsid protein (e.g., any reference capsid protein 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 with a reference AAV capsid protein (e.g., any reference capsid protein described herein), and comprises VR IV and VR V derived from a second reference capsid protein (e.g., any reference capsid protein 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 with SEQ ID NO: 142, and comprises VR IV and VR V derived from AAV9.

[0417] The modified AAV capsid protein of this disclosure can alter the tropism, specificity, and / or biodistribution of AAVs containing the modified AAV capsid protein. In a preferred embodiment, when administered to a subject, the AAV containing the modified AAV capsid protein exhibits enhanced targeting to target cells, tissues, or organs. In some embodiments, when administered to a subject, the AAV containing the modified AAV capsid protein exhibits reduced distribution outside of target cells, tissues, or organs. In some embodiments, the tropism of the modified AAV capsid protein can be measured using an enrichment score. Non-limiting examples of enrichment scores are based on combinations of amino acid residues present in the modified sequence within VR VIII. Exemplary enrichment formulas are provided below:

[0418] Tissue enrichment score = log2 fold change = log2MN 组织 -log2MN 测试品

[0419] The formula for the change in the scaling factor of log2 is provided below:

[0420] Change in log2 factor after scaling

[0421] = (log2 change in multiple - min(log2 change in multiple))

[0422] / (max(log2 factorial change)-min(log2FC))

[0423] 6.2.5. Reference AAV capsid protein

[0424] The reference AAV capsid protein used in the various embodiments of this disclosure is a VP1, VP2, or VP3 capsid protein of AAV known in the art. It may be a naturally occurring or non-naturally occurring AAV variant's VP1, VP2, or VP3 capsid protein. This reference AAV capsid protein does not contain the targeting peptide disclosed herein in VR VIII.

[0425] Non-naturally occurring VP1, VP2, or VP3 capsid proteins include capsid proteins generated through biological or chemical changes or in-silico design, or variations of naturally occurring AAV capsid proteins. Therefore, this reference AAV capsid protein includes, but is 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 generated through 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 herein by reference in their entirety).

[0426] In some embodiments, the reference AAV capsid protein is 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... The capsid protein of AAV10 (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), or AAVpol (Genbank accession number: FJ688147.1). In some embodiments, the AAV capsid protein is the capsid protein of AAV9 (Genbank accession number: AAS99264.1).

[0427] The reference AAV capsid protein can be a VP1 capsid protein having a sequence selected from the following: 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 (AAVLK03), SEQ ID NO: 65 (AAV hu.68), SEQ ID NO: 64 (AAVLK03), SEQ ID NO: 65 (AAV hu.68), SEQ ID NO: 66 (AAV hu.68 ...6 (AAV hu.68), SEQ ID NO: 66 (AAV hu.68), SEQ ID NO: 66 (AAV hu.68), SEQ ID NO: 66 (AAV hu.6 hu.1 (AAS99260)), SEQ ID NO: 66 (AAVhu.2 (AAS99270)), SEQ ID NO: 67 (AAVhu.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:1 08(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 rh.74); Anc80-55 (SEQ ID NO: 44885); Anc80-129 (SEQ ID NO: 44887); Anc80-156 (SEQ ID NO: 44889); Anc80-751 (SEQ ID NO: 44916); Anc80-1029 (SEQ ID NO: 44917); and Anc80-1712 (SEQ ID NO: 44893). The reference AAV capsid protein can be a VP2 or VP3 protein having a portion of one of these sequences. For example, a VP2 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1, and a VP3 protein can have a sequence corresponding to amino acids 138 to 736 of AAV9 VP1.

[0428] The reference AAV capsid protein may be selected from SEQ ID NO: 132(Anc80), SEQ ID NO: 133(Anc81(AKU89596)), SEQ ID NO: 134(Anc82(AKU89597)), SEQ ID NO: 135(Anc83(AKU89598)), SEQ ID NO: 136(Anc84(AKU89599)), SEQ ID NO: 137(Anc94), SEQ ID NO: 138(Anc110(AKU89600)), SEQ ID NO: 139(Anc113(AKU89601)), SEQ ID NO: 140(Anc126(AKU89602)), SEQ ID NO: 141(Anc127(AKU89603)) and ... SEQ ID NO: 142 (Anc80L65 (AKU89595)) is a VP1 capsid protein of any member sequence of the ancestral AAV library. This reference AAV capsid protein may be a VP2 or VP3 protein having a portion of one of these sequences. For example, a VP2 protein may have a sequence corresponding to amino acids 138-736 of AAV9 VP1, and a VP3 protein may have a sequence corresponding to amino acids 138-736 of AAV9 VP1. When the library sequence is used in this disclosure, it refers to the sequence of any member of that library.

[0429] In some implementations, the reference AAV capsid protein is the liver-toggle mutant described in WO2019 / 217911 (which is incorporated herein by reference in its entirety).

[0430] In some embodiments, the reference AAV capsid protein is a capsid protein (VP1, VP2, or VP3) selected from the following AAV variants: AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; 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; r h.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; hu.42-E; rh.57-E; rh.40-E; rh74; hu.67-E; hu.17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc8 0L36; Anc80L44; Anc80L1; Anc80-55, Anc80-129, Anc80-156, Anc80-751, Anc80-1029, Anc80-1712; Anc110; and Anc80DI. In some embodiments, the reference AAV capsid protein is a capsid protein selected from any member protein of the ancestral AAV library of Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; Anc113; Anc126; and Anc127.

[0431] In some embodiments, the reference AAV capsid protein is a protein having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from SEQ ID NO: 54-131 and 143-158. In some embodiments, the reference AAV capsid protein is a protein differing from (e.g., by insertion, deletion, or substitution) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more amino acid residues from a sequence selected from SEQ ID NO: 54-131 and 143-158. In some embodiments, the reference AAV capsid protein is a protein having a sequence selected from SEQ ID NO: 54-131 and 143-158. In some embodiments, the reference AAV capsid protein is a protein having a VP2 (corresponding to amino acids 138 to 736 of AAV9 VP1) or VP3 (corresponding to amino acids 138 to 736 of AAV9 VP1) portion of a protein having a sequence selected from SEQ ID NO: 54-131 and 143-158.

[0432] 6.2.6. Other modifications

[0433] In some embodiments, the modified AAV capsid protein includes one or more additional modifications compared to the reference AAV capsid protein. These one or more additional modifications may be insertions, deletions, substitutions, or combinations thereof, and may be located within and / or outside the VR VIII.

[0434] In some embodiments, the modified AAV capsid protein differs from the reference AAV capsid protein in that it has one or more amino acid substitutions in the variable region of the reference AAV capsid protein. In some embodiments, the one or more amino acid substitutions are located at the VRI position of the variable region of the reference AAV capsid protein. Figure 28 ).

[0435] 6.3. Polynucleotides encoding modified AAV capsid proteins; vectors; host cells.

[0436] In another aspect, this disclosure provides a polynucleotide encoding the modified AAV capsid protein described herein. In some embodiments, this polynucleotide is codon-optimized for expression in bacterial or mammalian cells.

[0437] In some embodiments, the polynucleotide is inserted into an expression vector. In some embodiments, the polynucleotide is operatively linked to a promoter or induces the expression of a protein sequence from the polynucleotide. This disclosure provides a vector comprising a polynucleotide encoding a modified AAV capsid protein. The vector can be used to generate the modified AAV capsid protein. In some embodiments, the vector is used to generate an AAV virion comprising the modified AAV capsid protein. In some embodiments, the vector further comprises an AAV rep protein or a fragment thereof. In some embodiments, the modified AAV capsid protein and the reference capsid protein of the rep protein originate from AAV of the same evolutionary branch. In some embodiments, the modified AAV capsid protein and the reference capsid protein of the rep protein originate from AAV of different evolutionary branches.

[0438] In some embodiments, the polynucleotide is transfected into a host cell. This disclosure provides a host cell containing a polynucleotide encoding a modified AAV capsid protein. The host cell may be a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a mammalian cell or a yeast cell.

[0439] In some embodiments, the host cell further comprises another polynucleotide encoding the AAV protein. In some embodiments, the host cell comprises a functional rep gene; a recombinant nucleic acid vector containing an AAV inverted terminal repeat (ITR) sequence and an expression polynucleotide; and sufficient auxiliary functions to allow the recombinant nucleic acid vector to be packaged into the modified AAV capsid protein.

[0440] In some embodiments, the host cell provides the desired components, packaged in a modified AAV capsid protein, to the host cell in a trans configuration. In some embodiments, any one or more of the desired components (e.g., the recombinant nucleic acid vector, rep sequence, cap sequence, and / or auxiliary function) are provided by a stable host cell engineered using methods known to those skilled in the art to contain one or more of the desired components. In some embodiments, such a stable host cell contains one or more of the desired components under the control of an inducible promoter. In some embodiments, one or more of the desired components are under the control of a constitutive promoter.

[0441] 6.4. Modified AAV Virus

[0442] This disclosure further provides modified recombinant AAV (rAAV) virions comprising the modified AAV capsid protein described herein. In some embodiments, the modified rAAV comprises the modified AAV capsid protein and a recombinant nucleic acid vector.

[0443] In some embodiments, after administration to mammalian subjects, modified rAAV containing the modified AAV capsid protein achieves higher target infection compared to rAAV containing the corresponding reference AAV capsid protein. In some embodiments, after administration to mammalian subjects, the modified rAAV achieves higher expression of the expressible polynucleotide within the recombinant nucleic acid vector at the target compared to the expression of the expressible polynucleotide administered in rAAV containing the corresponding reference AAV capsid protein.

[0444] In some embodiments, after administration to mammalian subjects, modified rAAV containing the modified AAV capsid protein achieves lower off-target infection compared to rAAV containing the corresponding reference AAV capsid protein. In some embodiments, after administration to mammalian subjects, the modified rAAV achieves lower off-target expression of the expressible polynucleotide in the recombinant nucleic acid vector compared to expression of the expressible polynucleotide administered in rAAV containing the 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 aforementioned targeting peptide.

[0445] In some embodiments, the target is the brain, muscle, spinal cord, eye, or other organ. In some embodiments, the off-target tissue is muscle, liver, or other organ. In one embodiment, the target is the CNS.

[0446] In some embodiments, the modified rAAV exhibits lower hepatotoxicity than rAAV containing the corresponding reference AAV capsid protein, administered via the same route and at the same dose. In some embodiments, this lower hepatotoxicity is due to the liver de-targeting of the modified rAAV.

[0447] 6.5. Methods for generating rAAV

[0448] The rAAV of this disclosure comprises a recombinant nucleic acid vector containing a heteropolynucleotide. In some embodiments, the heteropolynucleotide comprises an expressible polynucleotide operatively linked to an ERE. The expressible polynucleotide and ERE optionally replace the AAV genome coding region (e.g., replace the AAV rep and cap genes). The expressible polynucleotide and ERE typically have flanking AAV inverted terminal repeat (ITR) regions, although a single ITR may be sufficient to achieve the function typically associated with conformations containing two ITRs (see, e.g., WO94 / 13788), and therefore a vector construct containing only one ITR can be used in combination with the rAAV of this disclosure.

[0449] In some embodiments, the rAAV of this disclosure includes a therapeutic protein-coding sequence operatively linked to an ERE. This therapeutic protein-coding sequence and the ERE optionally substitute for the AAV genomic coding region (e.g., substitute the AAV rep and cap genes).

[0450] For replication and packaging of the vector, the missing functions are supplemented by packaging genes or multiple packaging genes that co-encode the essential functions of the various missing red and / or cap gene products. In one embodiment, this packaging gene or gene cassette does not have a flanking AAVITR, and in another embodiment, it does not share any substantial homology with the rAAV genome.

[0451] The rAAV vector construct and the complementary packaging gene construct can be implemented in a variety of different forms. Viral particles, plasmids, and stably transformed host cells can all be used to transiently or stably introduce such constructs into packaging cells.

[0452] In some embodiments of the invention, the AAV vector and (if any) complementary packaging genes are provided in the form of bacterial plasmids, AAV particles, or any combination thereof. In other embodiments, the AAV vector sequence, the (one or more) packaging genes, or both are provided in the form of genetically modified (preferably heritably modified) eukaryotic cells. The development of host cells that are heritably modified to express the AAV vector sequence, the AAV packaging genes, or both provides an established source of material for reliable level expression.

[0453] Therefore, a variety of different genetically modified cells can be used within the context of this invention. For example, mammalian host cells can be used with at least one complete copy of a stably integrated rAAV vector. AAV packaging plasmids containing at least one AAVrep gene operatively linked to a promoter can be used to provide replication functionality (as described in U.S. Patent No. 5,658,776). Alternatively, stable mammalian cell lines having an AAV rep gene operatively linked to a promoter can be used to provide replication functionality (see, for example, WO 95 / 13392; WO 98 / 23018; and U.S. Patent No. 5,656,785). An AAV cap gene providing the capsid protein as described above can be provided together with or separately from the AAV rep gene (see, for example, the patent documents cited above and WO 98 / 27204).

[0454] Therefore, the rAAV of this disclosure can be assembled, for example, by expressing its components in a packaging host cell. 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.

[0455] Once assembled, rAAV particles can be purified using conventional methods (if necessary). As used herein, "purified" viral particles refer to viral particles that have been removed from the components of the mixtures in which they were prepared, such as, but not limited to, viral components (e.g., rep sequences, cap sequences), packaging host cells, and partially or incompletely assembled viral particles.

[0456] 6.6. Pharmaceutical compositions comprising modified rAAV

[0457] In one aspect, this disclosure provides a pharmaceutical composition comprising the modified AAV capsid protein or modified rAAV of this disclosure and a pharmaceutically acceptable carrier. The modified rAAV may comprise the modified AAV capsid protein as described herein and a recombinant nucleic acid carrier containing an expressible polynucleotide.

[0458] In a specific embodiment, this disclosure provides a pharmaceutical composition comprising rAAV whose genome contains a therapeutic protein for treating and / or preventing diseases of the central nervous system, wherein the coding sequence of the therapeutic protein is operatively linked to an expression regulatory element (ERE).

[0459] The pharmaceutical composition may be formulated using one or more carriers, excipients, stabilizers and excipients to, for example: (1) enhance stability; (2) enhance cell transfection or transduction; (3) allow sustained or delayed release; (4) alter biodistribution (e.g., target the rAAV particles to a specific tissue or cell type); (5) enhance the translation of the encoded protein in vivo; and / or (6) alter the release profile of the encoded protein in vivo.

[0460] The formulations of the pharmaceutical compositions provided herein may include, but are not limited to, physiological saline, which may be formulated with various buffer solutions (e.g., phosphate-buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, water, lipids, liposomes, lipid nanoparticles, polymers, cationic lipid complexes, core-shell nanoparticles, peptides, proteins, nanoparticle mimics, and combinations thereof.

[0461] Formulations of the pharmaceutical compositions described herein can be prepared by any method known in or subsequently developed in the field of pharmacology. Generally, such preparation methods involve the steps of associating the active ingredient with a carrier and / or one or more other auxiliary components (e.g., excipients, stabilizers, and excipients).

[0462] The pharmaceutical compositions according to this disclosure can be prepared, packaged, and / or marketed in bulk as a single unit dose and / or multiple single unit doses. As used herein, a unit dose refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or an appropriate proportion of such dose, such as half or one-third of such dose.

[0463] The relative amounts of the active ingredient (e.g., rAAV), pharmaceutically acceptable carrier, and / or any additional ingredients in the pharmaceutical compositions according to this disclosure may vary depending on the identity, size, and / or condition of the subject being treated and further depending on the route of administration of the composition.

[0464] Various carriers, excipients, stabilizers, and excipients used to prepare pharmaceutical compositions, as well as techniques for preparing such compositions, are known in the art (see Remington: The Science and Practice of Pharmacy, 22nd revised edition, Pharmaceutical Press, 2012; incorporated herein by reference in its entirety). The use of suitable conventional carriers, excipients, stabilizers, and excipients is also considered within the scope of this disclosure.

[0465] In some embodiments, the pharmaceutical composition contains about 1×10 1 To approximately 1×10 16 A solution of 1 × 10⁻⁶ genomic copies (GC) / ml rAAV (e.g., containing approximately 1 × 10⁻⁶ GC / ml rAAV). 3 To approximately 1×10 14 In the form of a solution with a concentration of GC / ml.

[0466] 6.6.1. Route of application

[0467] This disclosure provides a method for administering rAAV to transfer polynucleotides to the CNS. In some embodiments, the rAAV is administered locally or systemically.

[0468] In some embodiments, the rAAV is applied locally to the CNS. In some embodiments, the rAAV is applied to the subject's cerebrospinal fluid (CSF). In some embodiments, the rAAV is applied to the subject's cisterna magna, intraventricular space, ventricles, subarachnoid space, intrathecal space, and / or ependyma.

[0469] In some implementations, rAAV is administered via intrathecal, intracranial, intraventricular (ICV) or intraparenchymal administration, or administration to the lateral ventricles of the brain.

[0470] In some implementations, rAAV is administered via lumbar injection (e.g., into the lumbar cistern) and / or into the occipital cistern (ICM).

[0471] In some embodiments, rAAV is applied to the ventricular system. In some embodiments, rAAV is applied to the cerebrospinal lateral ventricle; and / or to the caudal lateral ventricle; and / or to the right ventricle; and / or to the left ventricle; and / or to the right cerebrospinal lateral ventricle; and / or to the left cerebrospinal lateral ventricle; and / or to the right caudal lateral ventricle; and / or to the left caudal lateral ventricle.

[0472] In some embodiments, administration of rAAV results in the rAAV contacting the subject's ependymal cells. These ependymal cells express a polypeptide, and optionally, the polypeptide is expressed by these cells.

[0473] In some embodiments, the polypeptide is expressed and / or distributed in the lateral ventricle, CSF, and / or brain (e.g., striatum, thalamus, medulla oblongata, cerebellum, occipital cortex, and / or prefrontal cortex).

[0474] In some implementations, rAAV is administered intravenously or systemically.

[0475] In some implementations, rAAV is applied inter-digitally.

[0476] To specifically deliver the rAAV to a particular region of the CNS, especially a specific region of the brain, it can be administered via stereotactic microinjection. For example, on the day of surgery, the patient can have a stereotactic frame base secured in place (screwed into the skull). The brain with the stereotactic frame base (MRI-compatible, with fiduciary marking) 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 for the carrier injection. The software directly converts this trajectory into three-dimensional coordinates suitable for the stereotactic frame. A burr hole can be drilled above the entry site and the stereotactic device, and the needle can be inserted to a given depth. The carrier in a pharmaceutically acceptable carrier can then be injected. The AAV carrier can then be administered by direct injection to the primary target site and retrograde transport via axons to the distal target site. Additional administration routes can be used, such as direct visualization of the surface cortex or other non-stereotactic applications.

[0477] In some implementations, rAAV is delivered via a pump. This pump can be implantable. Another convenient way to administer rAAV is using a cannula or catheter.

[0478] In some implementations, rAAV is administered via convection-enhanced delivery (CED) (Nguyen et al., (2003) J. Neurosurg. 98: 584-590), which has been clinically used for gene therapy (AAV2-hAADC) for Parkinson's disease (Fiandaca et al., (2008) Exp. Neurol. 209: 51-57). The basic principle of CED involves pumping the infusion into the brain parenchyma under sufficient pressure to overcome the hydrostatic pressure of the tissue fluid, thereby forcing the infused particles into close contact with the dense perivascular tissue of the brain. The pulsation of these vessels acts as a pump, distributing the particles over a large distance throughout the parenchyma (Hadaczek et al., (2006) Hum. Gene Ther. 17: 291-302). To enhance the safety and efficacy of CED, anti-reflux cannulas (Krauze et al., (2009) Methods Enzymol. 465: 349-362) can be used in conjunction with delivery monitored by real-time MRI. Monitoring delivery allows for the quantification and control of aberrant events such as cannula reflux and infusion leakage into the ventricles (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). US20190111157A1 provides an improved procedure for achieving broad expression of AAV vectors in the cortex and / or striatum.

[0479] In some embodiments, the rAAV is applied to the striatum. In some embodiments, the rAAV is applied to at least the putamen and caudate nucleus of the striatum. In some embodiments, the rAAV is applied to at least the putamen and caudate nucleus of each hemisphere of the striatum. In some embodiments, the rAAV is applied to at least one site in the caudate nucleus and two sites in the putamen.

[0480] In some embodiments, rAAV is delivered to specific regions of the brain via intraparenchymal administration. In some embodiments, rAAV is delivered to the putamen, striatum, basal forebrain, substantia nigra, and / or ventral tegmentum via intraparenchymal administration.

[0481] In some embodiments of the above aspects and implementations, the rAAV is delivered via stereotactic delivery. In some embodiments, the rAAV is delivered via 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 an anti-backflow 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 a permeation pump. In some embodiments, the pump is an infusion pump.

[0482] The modified rAAV of this disclosure can be administered to subjects (e.g., humans or non-human mammals) in suitable carriers. Suitable carriers include physiological saline, which can be formulated with various buffer solutions (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 adequate levels of gene transfer and expression to deliver therapeutic benefit without inappropriate side effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to an organ (e.g., muscle, liver, or lung), oral, intranasal, intratracheal, intrathecal, intravenous, intramuscular, intraocular, subcutaneous, intradermal, or other routes of administration. Routes of administration can be combined if desired.

[0483] 6.6.2. Subjects

[0484] This disclosure provides a method for transferring a polynucleotide into the central nervous system (CNS) of a subject (e.g., a mammal). In some embodiments, the subject is a human. In some embodiments, the subject suffers from a CNS disease. In some embodiments, the subject has a genetic defect associated with a CNS disease or disorder.

[0485] In some embodiments, the CNS disease or disorder is selected from 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), Deafness, Duchenne muscular dystrophy, Epilepsy, Essential tremor, Fragile X syndrome, and Friedreich's ataxia. Ataxia, Gaucher disease, GM1 gangliosidosis, GM2 gangliosidoses, Huntington disease, frontotemporal degeneration (FTD), Lesch-Nyhan syndrome, Maple syrupurine disease, Menkes syndrome, Metachromatic leukodystrophy (MLD), Myotonic dystrophy, Multiple sclerosis, Narcolepsy, Neurofibromatosis, Niemann-Pick diseaseDiseases including 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.

[0486] In some embodiments, the CNS disease or disorder is a demyelinating or white matter disease. In some embodiments, the subject has a single-gene defect. In some embodiments, the subject has a gene defect in a protein expressed in the CNS. In some embodiments, the subject has a single-gene defect in a protein expressed in the CNS.

[0487] In some implementations, the subject suffers from lysosomal storage disease (LDS). In some embodiments, the subject suffers from a disease selected from the following: mucopolysaccharidosis type I, such as Hurler syndrome, 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 disease; Sandhoff disease; GM1 ganglioside storage disease; Fabry disease. (disease); Krabbe's disease; Leukodystrophy; Metachromatic leukodystrophy; Pompe disease; Fucosidosis deficiency; Alpha-mannosidosis deficiency; Beta-mannosidosis deficiency; Gaucher disease; Batten disease; Typical late-stage infant Batten disease; Juvenile Batten disease; Other forms of Batten disease; Niemann-Pick disease; Niemann-Pick disease without sphingomyelinase deficiency; and Wolman disease.

[0488] 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.

[0489] 6.6.3. Dosage

[0490] The dose of the viral vector administered to the subject will depend primarily on factors such as the condition being treated and the subject's age, weight, and health. For example, the therapeutically effective dose of the viral vector administered to a human subject is generally in the range of about 0.1 ml to about 10 ml of a solution containing a concentration of about 1E1 to about 1E16 genomic copies (GC) / 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 the subject is less than 3E14 GC, for example, 1E14 GC or less, 5E13 GC or less, 1E13 GC or less, 5E12 GC or less, or 1E12 GC or less.

[0491] In another embodiment, the therapeutically effective dose of the viral vector administered to human subjects is generally in the range of about 0.1 ml to about 10 ml of a solution containing a concentration of about 1E1 to 1E12 genomic copies (GCs) (e.g., about 1E3 to 1E9 GCs). Transduction and / or expression of the transgene can be monitored at different time points after administration by DNA, RNA, or protein assays. In some cases, the expression level of the transgene can be monitored to determine the frequency and / or dosage. Dosing regimens similar to those described for therapeutic purposes can also be used for immunization.

[0492] In some embodiments, the effective dose is rAAV for each subject with 1E10 to 1E16 genomic copy numbers (GC). In some embodiments, the effective dose for human patients corresponds to rAAV for monkeys with 1E12 to 1E15 GC. In some embodiments, the effective dose for human patients corresponds to rAAV for monkeys with 1E13 to 1E14 GC. In some embodiments, the effective dose for human patients corresponds to rAAV for monkeys with approximately 4E13 GC.

[0493] In some embodiments, the effective dose is 1E11 to 1E15 GC of rAAV per gram of brain mass. In some embodiments, the effective dose is 1E11 to 1E13 GC of rAAV per gram of brain mass. In some embodiments, the effective dose is 1E11 to 1E12 GC of rAAV per gram of brain mass. In some embodiments, the effective dose is 1E12 to 1E14 GC of rAAV per gram of brain mass. In some embodiments, the effective dose is approximately 5E11 GC of rAAV per gram of brain mass. In some embodiments, the effective dose is approximately 2.5E11 GC of rAAV per gram of brain mass. In some embodiments, the effective dose is approximately 5E10 GC of rAAV per gram of brain mass. In some embodiments, the effective dose is approximately 2.5E10 GC of rAAV per gram of brain mass.

[0494] In some embodiments, the effective dose is 1E10-1E16 genome copy number (GC) rAAV per kg body weight. In some embodiments, the effective dose is 1E11-1E15 genome copy number (GC) rAAV per kg body weight. In some embodiments, the effective dose is 1E12-5E14 genome copy number (GC) rAAV per kg body weight. In some embodiments, the effective dose is 0.5E13-2E14 genome copy number (GC) rAAV per kg body weight.

[0495] Transduction and / or expression of the transgene can be monitored at different time points after administration by DNA, RNA, or protein assays. In some cases, the expression level of the transgene can be monitored to determine the frequency and / or dosage. Dosing regimens similar to those described for therapeutic purposes can also be used for immunization.

[0496] In one aspect, the present invention provides a unit dose of the rAAV provided herein. This unit dose comprises about 0.1 ml to about 10 ml of a solution containing about 1E9 to 1E17 genomic copies (GC) per ml of the rAAV described herein. In some embodiments, the unit dose contains about 1E10 to 1E16 genomic copies (GC) per ml of the rAAV described herein. In some embodiments, the unit dose contains about 1E11 to 1E15 genomic copies (GC) per ml of the rAAV described herein. In some embodiments, the unit dose contains about 1E12 to 1E14 genomic copies (GC) per ml of the rAAV described herein. In some embodiments, the unit dose contains about 2E13 genomic copies (GC) per ml of the rAAV described herein.

[0497] In some embodiments, the unit dose contains approximately 1E10 to 1E16 genomic copies (GC) of the rAAV described herein. In some embodiments, the unit dose contains approximately 1E11 to 1E15 genomic copies (GC) of the rAAV described herein. In some embodiments, the unit dose contains approximately 1E12 to 1E15 genomic copies (GC) of the rAAV described herein. In some embodiments, the unit dose contains approximately 1E13 to 1E15 genomic copies (GC) of the rAAV described herein.

[0498] This unit dose further includes pharmaceutically acceptable excipients.

[0499] 6.6.4. Targeting

[0500] This pharmaceutical composition can be used to deliver the recombinant nucleic acid vector to a target within a mammalian subject. When administered to a mammalian subject, the modified rAAV achieves higher target cell infection rates compared to rAAV containing the corresponding reference AAV capsid protein administered at the same dose via the same route of administration. In some embodiments, after administration to a mammalian subject, the modified rAAV achieves higher expression of expressible polynucleotides within the recombinant nucleic acid genome in target cells compared to expressible polynucleotides in rAAV containing the corresponding reference AAV capsid protein administered at the same dose via the same route of administration.

[0501] The targeting ability of rAAV can be tested in laboratory animals by measuring rAAV infection or polynucleotide expression. In some implementations, targeting ability is measured in non-human primates (NHPs), mice, rats, birds, rabbits, guinea pigs, hamsters, farm animals (including pigs and sheep), dogs, or cats.

[0502] The targeting of rAAV can be measured after systemic or local administration of rAAV. In some embodiments, the targeting of rAAV is measured after intravenous infusion or local administration of rAAV to the CNS. In some embodiments, the targeting is measured after administration to the CNS via lumbar puncture (LP) via injection into the lumbar cistern (e.g., approximately L3-L4) or intraoccipital cistern (ICM).

[0503] In some embodiments, the targeting of the modified rAAV is measured by measuring the ratio between the copy number of the transgenic transcript and the transcripts of housekeeping genes (e.g., RPP30, actin, GAPDH, or ubiquitin). In specific embodiments, these transcripts are measured by RT-ddPCR. In some embodiments, this ratio is measured after the first administration to a mammal (such as a primate, e.g., a monkey, e.g., a cynomolgus monkey or a rhesus monkey) or a mouse.

[0504] In some embodiments, the rAAV of this disclosure provides an infection rate (i.e., expression) that is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 500-fold, or at least 1000-fold compared to AAV9 in the brain (or target regions of the brain) or other tissues (or non-target regions of the brain).

[0505] In some implementations, brain: comparing tissue infection rates by comparing two individuals or two groups of animals (each administered the test rAAV). 测试The ratio between the copy number of transgenic transcripts and housekeeping gene (e.g., RPP30) transcripts 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) is measured.

[0506]

[0507] In some implementations, compared to AAV9, this rAAV 测试 Infection rates 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% were achieved in the brain. In some implementations, compared to AAV9, this rAAV... 测 Infection rates 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 were achieved at one of the target tissues (caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, and substantia nigra).

[0508] In some embodiments, the targeting of the modified rAAV is measured by measuring the ratio between the copy number of the transgenic DNA genome and the copy number of the host gene or locus (e.g., RPP30). In specific embodiments, these genomes are measured by RT-ddPCR. In some embodiments, this ratio is measured after the first administration to a mammal (e.g., a mouse, or a non-human primate (such as a marmoset or rhesus monkey)).

[0509] In some implementations, brain: comparing tissue infection rates (DNA) by comparing two individuals or two groups of animals (each administered rAAV). 测试 The ratio between the copy number of the transgenic DNA genome and the copy number of 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) is measured.

[0510]

[0511] In some implementations, compared to AAV9, this rAAV 测试 Infection rates 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% were achieved in the brain. In some implementations, compared to AAV9, this rAAV... 测试Infection rates 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 were achieved at one of the target tissues (caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, and substantia nigra).

[0512] In some implementations, when the (transgenic genome / housekeeper genome) in the comparative tissue is zero or below the detection limit, the brain:comparative tissue infection rate is conventionally reported as >10,000.

[0513] In some embodiments, the modified rAAV of this disclosure provides 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 brain:comparative tissue infection rate (DNA). In some embodiments, the muscle is the calf triceps, biceps, heart, or quadriceps.

[0514] In some embodiments, the modified rAAV of this disclosure provides brain:comparative tissue infection rates (DNA) ranging from 0.5 to 1, 0.5 to 5, 0.5 to 10, 1 to 10, 1 to 100, 2 to 8, 5 to 10, 10 to 20, 20 to 80, 10 to 50, 10 to 100, 50 to 80, 100 to 500, 100 to 1000, or 500 to 1000. In some embodiments, the muscle is the calf muscle, biceps, heart muscle, or quadriceps. In some embodiments, the modified rAAV achieves brain:comparative tissue infection rates (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 implementations, the modified rAAV achieves brain:comparative tissue infection rates 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.

[0515] 6.6.4.1 RNA Data - Brain: Liver Infection Rate

[0516] In some embodiments, the targeting of the modified rAAV is measured by measuring the ratio between the copy numbers of the transgenic transcript and the housekeeping gene (e.g., RPP30) transcript. In specific embodiments, these transcripts are measured by RT-ddPCR. In some embodiments, this ratio is measured after the first administration to a mammal (e.g., a mouse, or a non-human primate (such as a marmoset or rhesus monkey)).

[0517] In some implementations, the brain:liver infection rate (RNA) is measured by comparing the copy number of transgenic transcripts and housekeeping gene (e.g., RPP30) transcripts in these two different organs (e.g., brain to liver).

[0518]

[0519] In some embodiments, the modified rAAV of this disclosure provides a ratio 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 (transgenic transcript / housekeeper transcript) in the liver.

[0520] In some implementations, when the number of (transgenic transcripts / housekeeper transcripts) in the liver is zero or below the detection limit, the brain:liver infection rate is conventionally reported as >10,000.

[0521] In some embodiments, the modified rAAV of this disclosure provides brain:liver infection rates (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, and at least 1000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra.

[0522] In some embodiments, the modified rAAV of this disclosure provides brain:liver infection rates (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.

[0523] 6.6.4.2 DNA Data - Brain: Liver Infection Rate

[0524] In some embodiments, the targeting of the modified rAAV is measured by measuring the ratio between the copy number of the transgenic DNA genome and the copy number of the host gene or locus (e.g., RPP30). In specific embodiments, these genomes are measured by RT-ddPCR. In some embodiments, this ratio is measured after the first administration to a mammal (e.g., a mouse, or a non-human primate (such as a marmoset or rhesus monkey)).

[0525] In some implementations, the brain:liver infection rate (DNA) is measured by comparing the copy number of the transgenic DNA genome and the housekeeping gene (e.g., RPP30) genome in these two different organs (e.g., brain to liver).

[0526]

[0527] In some embodiments, the modified rAAV of this disclosure provides a (transgenic 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.

[0528] In some implementations, when the (transgenic genome / housekeeping genome) in the liver is zero or below the detection limit, the brain:liver infection rate is conventionally reported as >10,000.

[0529] In some embodiments, the modified rAAV of this disclosure provides a brain:liver infection rate (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.

[0530] In some embodiments, the modified rAAV of this disclosure provides brain:liver infection rates (DNA) ranging from 0.5 to 1, 0.5 to 5, 0.5 to 10, 1 to 10, 1 to 100, 2 to 8, 5 to 10, 10 to 20, 20 to 80, 10 to 50, 10 to 100, 50 to 80, 100 to 500, 100 to 1000, or 500 to 1000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra. In some embodiments, the modified rAAV achieves brain:liver infection rates (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 implementations, the modified rAAV achieves brain:liver infection rates 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.

[0531] 6.6.4.3 IHC Data - Brain:Hepatic Infection Rate

[0532] In some embodiments, the targeting of the modified rAAV is calculated using the percentage of cells in the tissue that have been successfully transduced and expressed with the transgene (e.g., eGFP). In specific embodiments, this transgene expression is measured by immunohistochemistry. In some embodiments, the ratio is measured after the first administration to a mammal (e.g., a mouse, or a non-human primate (such as a marmoset or rhesus monkey)).

[0533] In some implementations, the brain:liver infection rate (IHC) is measured by comparing the percentage of transgenic GFP+ cells and the percentage of cells with housekeeping genes (e.g., RPP30) GFP+ in these two different organs (e.g., brain to liver).

[0534]

[0535] In some embodiments, the modified rAAV of this disclosure provides a ratio of less than 1, less than 5, less than 10, 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 (transgenic GFP% / housekeeper GFP%) in the liver.

[0536] In some implementations, when the (transgenic GFP% / housekeeper GFP%) in the liver is zero or below the detection limit, the brain:liver infection rate is conventionally reported as >10,000.

[0537] In some embodiments, the modified rAAV of this disclosure provides brain:liver infection rates (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, and at least 1000. In some embodiments, the muscle is the caudate nucleus, frontal cortex, globus pallidus, motor cortex, parietal cortex, putamen, or substantia nigra.

[0538] In some embodiments, the modified rAAV of this disclosure provides brain:liver infection rates (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.

[0539] 6.7. Instructions for Use

[0540] The modified rAAVs described herein can be used for research and / or therapeutic applications. In some embodiments, the modified rAAVs are used for gene modification of cells in vitro or in vivo. In some embodiments, the modified rAAVs are used for gene therapy or vaccination in humans or animals. More specifically, the modified rAAVs can be used for gene addition, gene enhancement, gene delivery of peptide therapeutics, gene vaccines, gene silencing, genome editing, gene therapy, RNAi delivery, cDNA delivery, mRNA delivery, miRNA delivery, miRNA sponging, gene immunization, photogene therapy, transgenics, DNA vaccination, or DNA immunization of hepatocytes or non-hepatocytes.

[0541] In some embodiments, the modified rAAV of this disclosure is used to treat or improve or prevent a disease or condition in a subject. In some embodiments, the disease is a disease of the central nervous system (CNS).

[0542] In some embodiments, the modified rAAV of this disclosure is used to transfer exogenous polynucleotides into the central nervous system (CNS). In some embodiments, when measured by genome copy number of the AAV virion, transfer of the exogenous polynucleotide into the CNS results in a CNS:liver infection rate greater than 1. 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 rate greater than 10. In some embodiments, when measured by protein expression, transfer of the exogenous polynucleotide into the CNS results in expression of the exogenous polynucleotide in the CNS at a CNS:liver expression rate greater than 10.

[0543] The modified rAAV disclosed herein can be administered to subjects in a suitable drug carrier.

[0544] The rAAV disclosed herein is typically administered in an amount sufficient to transduce or infect desired cells and provide adequate levels of gene transfer and expression to provide therapeutic benefit to patients with a disease. In a specific embodiment, the rAAV is administered in an amount sufficient to provide therapeutic benefit to a subject with a disease of the central nervous system (CNS).

[0545] Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to an organ (e.g., the brain), intracerebrospinal fluid (ICM), interdigital, intravenous, oral, intranasal, intratracheal, intrathecal, intramuscular, intraocular, subcutaneous, intradermal, or other routes of administration. Routes of administration may be combined if necessary.

[0546] The transduction and / or expression of this transgene can be monitored at different time points after application by DNA, RNA, or protein assays.

[0547] Therefore, this disclosure provides a method for treating and / or preventing muscle diseases and / or muscle degeneration by applying rAAV modified as described herein.

[0548] 7. Example

[0549] 7.1. Example 1: Evaluation of CNS tropism of Anc80L65 compared to AAV9

[0550] The applicant assessed the distribution of the AAV9 and Anc80L65 vectors encoding the EGFP reporter gene 14 days after lumbar puncture (LP) into the lumbar cisternae (approximately L3-L4) or intracranial cistern (ICM) of adult cynomolgus monkeys (4E13 gc / animal; 2E13 vg / ml). The applicant demonstrated that a single injection of Anc80L65 into the CSF of adult cynomolgus monkeys resulted in efficient transduction of a wide region of the CNS.

[0551] Following ICM injection, Anc80L65 showed a more extensive distribution throughout the cortex and deep nuclei compared to AAV9. Following LP injection, the distribution of Anc80L65 throughout the cortex was comparable to that of ICM delivery and superior to the observed distribution of AAV9 delivered via ICM. Following LP delivery, AAV9 showed limited transduction in the cortex. Both AAV9 and Anc80L65 effectively transduced spinal ventral horn motor neurons via these two administration routes.

[0552] Specifically, Anc80L65 transduces both neurons and astrocytes. Rare oligodendrocyte transduction was also observed in the cortical region with Anc80L65; however, microglia transduction was not detected using the microglia marker Ibal. AAV9 showed similar tropism to Anc80L65 in the non-human primate CNS, primarily transducing neurons and astrocytes. Similar to Anc80L65, dual microglia labeling was not observed. For AAV9, oligodendrocyte transduction was not observed; however, overall transduction in the CNS was less than with Anc80L65, making comparison difficult.

[0553] This work demonstrates the ability of Anc80L65 to target a wide range of regions of the CNS via the CSF delivery pathway, and its superior distribution compared to 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 brain and spinal cord in the NHP supports the potential use of the Anc80L65 vector for the treatment of a broad range of neurological disorders.

[0554] 7.1.1. Experimental Procedure

[0555] 7.1.1.1 Lumbar puncture (LP) injection

[0556] Inject the anesthetic into the animal and place it in a lateral decubitus position. Percutaneously insert a 22-gauge Gerti Marx spinal needle into the lumbar cistern (approximately L3-L4). Use fluoroscopy for guidance if necessary. Once the needle is in place, remove the stylet and confirm positive cerebrospinal fluid (CSF) flow, collecting CSF before administration. Then attach the test syringe to the needle and slowly infuse the test sample manually as a slow bolus over approximately 120 ± 5 seconds. After injection, remove the needle and apply brief pressure manually at the injection site. Place the animal in the Trendelenburg position (30°, head down) for at least approximately 10 minutes. Then allow the animal to recover naturally from anesthesia. Lumbar puncture is an intrathecal injection.

[0557] 7.1.1.2 Intracaver magnum (ICM) injection

[0558] Inject the anesthetic into the animal and place it in a lateral recumbent position. Percutaneously advance a 22-gauge spinal puncture needle into the cisterna magna, verifying proper needle placement by the presence of positive cerebrospinal fluid (CSF) flow, and collect CSF before administration. Then connect a suitable test syringe to the spinal puncture needle and administer the test sample manually via a slow bolus injection (120 ± 5 seconds). After injection, remove the syringe and apply pressure briefly manually. Then place the animal in the Trend-Lombard recumbent position (30°, head down) for at least approximately 10 minutes. The animal is then allowed to recover naturally from anesthesia.

[0559] 7.1.1.3 Immunohistochemistry (IHC)

[0560] 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. The brain was placed in pre-chilled brain matrix and sliced ​​into 4 mm sections, followed by hemisection. Even-numbered hemisections (slabs) were stored in 10% NBF and used for immunohistochemistry (IHC). Odd-numbered hemisections were frozen on dry ice and stored at -60°C to -90°C until used for ddPCR analysis.

[0561] To detect GFP expression, slides were incubated with an antibody against GFP diluted 1:1,000 in Monet Blue dilution buffer (Biocare Medical, PD901) (GeneTex, GTX20290). Slides were washed with Valent wash buffer (Biocare Medical, VLT8013MX) and incubated for 30 minutes with an anti-rabbit antibody conjugated with Farma HRP (Biocare Medical, BRR4009). Slides were washed and then reacted with Betazoid DAB for 5 minutes (Biocare Medical, BDB2004) and counterstained with Mayer's hematoxylin for 5 minutes (StatLab, HXMMHPT). After reacting with either Betazoid DAB or Mayer's hematoxylin, slides were washed with Aqua Rinse (Biocare Medical, VLT8012MX).

[0562] GFP staining was performed using 3,3'-diaminobenzidine (DAB): sections (three sections per 6 mm block, spaced 2 mm apart) were washed three times in PBST and then treated with 1% H2O2. As previously described, sections were stained with anti-GFP primary antibody diluted 1:1000 in Da Vinci Green buffer (Lluis Samaranch, Ernesto A. Salegio, Waldy SanSebastian, 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).

[0563] To detect trastuzumab expression, slides were incubated with an antibody against IgG (Fc). IgG (Fc) can be used as a surrogate marker for trastuzumab expression.

[0564] 7.1.1.4 Dual immunofluorescence:

[0565] As previously described, GFP was used to perform fluorescent immunostaining on different cellular markers (NeuN, GFAP, Ibal, Olig2+) (San Sebastian et al., 2013).

[0566] Sample collection:

[0567] Tissue samples were collected and stored in 10% neutral buffered formalin (NBF) for 48–72 hours, then transferred to 70% ethanol. The brain was placed in pre-chilled brain matrix and sectioned into 4 mm sections, followed by hemisection. Even-numbered hemisections were stored in 10% NBF for immunohistochemistry (IHC). Odd-numbered hemisections were frozen on dry ice and stored at -60°C to -90°C until used for ddPCR analysis.

[0568] Immunohistochemical protocol for GFP expression:

[0569] ○ Bake the slices at 55-65℃ for 15 minutes to remove the paraffin wax.

[0570] ○ Load the slides onto the Valent staining platform (Biocare Medical)

[0571] ○ Val DePar 8 minutes (Biocare Medical, VLT8001MM)

[0572] ○ At 98°C and low pH AR for 60 minutes (Biocare Medical, VLT8004MM)

[0573] ○ Peroxidazed 1 lasts for 5 minutes (Biocare Medical, PX968)

[0574] ○ Background Punisher lasts 5 minutes (Biocare Medical, BP974)

[0575] ○ GFP (GeneTex, GTX20290) at a 1:1,000 ratio in Monet Blue dilution (Biocare Medical, PD901)

[0576] ○ Rabbit on Farma HRP lasts for 30 minutes (Biocare Medical, BRR4009)

[0577] ○ Betazoid DAB lasts for 5 minutes (Biocare Medical, BDB2004)

[0578] Counterstain with Mayer's hematoxylin for 5 minutes (StatLab, HXMMHPT).

[0579] Valent wash buffer (Biocare Medical, VLT8013MX) was used after all steps except for Betazoid DAB and Mayer's hematoxylin. Aqua Rinse (Biocare Medical, VLT8012MX) was used after these reagents.

[0580] Double staining method using GFP, IBA1, NeuN, and GFAP:

[0581] Reagents:

[0582] ● Monet Blue dilution buffer (Biocare Medical, PD901) contains GFP (GeneTex, GTX20290) at a ratio of 1:1,000 and GFAP (Cell Signaling, 3670) at a ratio of 1:500.

[0583] ● Monet Blue dilution buffer (Biocare Medical, PD901) contains GFP (GeneTex, GTX20290) at a ratio of 1:1,000 and IBA1 (Millipore, MABN92) at a ratio of 1:250.

[0584] ● Monet Blue dilution buffer (Biocare Medical, PD901) contains GFP (GeneTex, GTX20290) at a ratio of 1:1,000 and NeuN (Abcam, ab104224) at a ratio of 1:250.

[0585] plan:

[0586] ○ Bake the slices at 55-65℃ for 15 minutes to help remove the paraffin wax.

[0587] ○ Load the slides onto the Valent staining platform (Biocare Medical)

[0588] ○ Val DePar 8 minutes (Biocare Medical, VLT8001MM)

[0589] ○ At 98°C and low pH AR for 60 minutes (Biocare Medical, VLT8004MM)

[0590] ○ Peroxidazed 1 lasts for 5 minutes (Biocare Medical, PX968)

[0591] ○ Background Punisher lasts 10 minutes (Biocare Medical, BP974)

[0592] ○ Primary antibody mixture (cocktail): Rabbit 594nm (Invitrogen, A32740) 1:500, mouse 488nm (Invitrogen, A-21202) 1:500, mixed together in Da Vinci Green (cocktail) for 60 minutes (Biocare Medical, PD900).

[0593] ○ Mount the slide with Prolong Diamond Antifade reagent containing DAPI.

[0594] ○ Use Valent wash buffer (Biocare Medical, VLT8013MX) after all steps.

[0595] 7.1.1.5 ddPCR

[0596] Following euthanasia and bloodletting, the brain was placed in pre-cooled brain matrix and sectioned into 4 mm sections, followed by hemisection. Odd-numbered hemisections were frozen on dry ice and then stored at -60°C to -90°C until analysis. Brain regions were isolated using tissue drills with a diameter of 2 mm or 3 mm (Miltex, catalog numbers: 95039-098 and 98PUN6-4), followed by nucleic acid isolation.

[0597] Tissues were homogenized in Qiagen Tissuelyser II (20 rpm for 2 minutes) according to standard Qiagen protocols in lysis buffer from either the Qiagen Dneasy Blood and Tissue Kit or the Qiagen RNeasy Lipotissue Microkit. Samples were eluted in 50 μL buffer. Prior to analysis, the concentration and quality of DNA and RNA were determined using a NanoDrop One nucleic acid (DNA or RNA) program. Vector genomic biodistribution of DNA samples was analyzed using a duplex ddPCR method with a targeted transgene (eGFP) and a reference gene (RPP30). eGFP transgene expression in RNA samples was analyzed using a duplex one-step RT-ddPCR method with the reference gene (RPP30).

[0598] Double staining method for Olie2 and GFP (performed at StaeeBio):

[0599] Reagents:

[0600] ● Monet Blue dilution (Biocare Medicaj, PD901) GFP (GeneTex, GTX20290) 1:1,000, Olig2 (Millipore, MABN50) 1:250

[0601] plan:

[0602] ○ Bake the slices at 55-65℃ for 15 minutes to help remove the paraffin wax.

[0603] ○ Load the slides onto the Valent staining platform (Biocare Medical)

[0604] ○ Val DePar 8 minutes (Biocare Medical, VLT8001MM)

[0605] ○ At 98°C and low pH AR for 60 minutes (Biocare Medical, VLT8004MM)

[0606] ○ Peroxidazed 1 lasts for 5 minutes (Biocare Medical, PX968)

[0607] ○ Background Punisher lasts 10 minutes (Biocare Medical, BP974)

[0608] ○ Primary antibody mixture: Biotinylated mice (Vector Laboratories, BA-9200) in Da Vinci Green dilution 1:500

[0609] ○ Rabbit 594nm (Invitrogen, A32740) 1:500, streptavidin 488nm (Invitrogen, S11223) 1:500, mixed together in Da Vinci Green for 60 minutes (Biocare Medical, PD900).

[0610] Mount the slides with Prolong Diamond Antifade reagent containing DAPI. After all steps, wash with Valent wash buffer (Biocare Medical, VLT8013MX).

[0611] DNA analysis:

[0612] To isolate DNA, tissues were homogenized in Qiagen Tissuelyser II (20 rpm for 2 minutes) using standard Qiagen protocols in lysis buffer from the Qiagen DNeasy Blood and Tissue Kit (product model 69506). Samples were eluted in 50 μL of AE buffer. Prior to analysis, DNA concentration and quality were determined using the nucleic acid (DNA) program with NanoDrop One.

[0613] The vector genomic biodistribution of DNA samples was analyzed using a dual ddPCR method targeting the transgene (eGFP or trastuzumab) and a reference gene (RPP30). Specific primer and probe sequences are listed in the table below.

[0614]

[0615] Samples were analyzed according to the standard Bio-Rad ddPCR protocol for probe-based DNA biodistribution analysis. Briefly, a reaction mixture containing two primer probe sets, DNA sample, and Bio-Rad ddPCR Supermix for Probes (dUTP-free) (product model 186-3024) was prepared according to the formulation in the table below.

[0616] reagents Volume / Reaction 2X ddPCR Supermix 10 20X RPP30 PnP 1 20X eGFP PnP or 20X trastuzumab PnP 1 water 3 sample* 3

[0617] *Pre-dilute DNA samples to 2 ng / μL (liver), 10 ng / μL (DRG, samples with concentrations <10 ng / μL do not need to be diluted), and 20 ng / μL (other samples) using nuclease-free water.

[0618] After droplet formation, the reaction is scaled up using the following thermal cycling procedure.

[0619]

[0620]

[0621] Data are reported as vector genomes (VGC / DG) for each diploid genome replication. The formula for calculating the output is VGC / DG = (eGFP cp / μL ÷ RPP30 cp / μL) × 2 for eGFP, or VGC / DG = (trastuzumab cp / μL ÷ RPP30 cp / μL) × 2 for trastuzumab.

[0622] RNA analysis:

[0623] To isolate mRNA, tissues were homogenized in 1 ml of Qiazol from the Qiagen RNeasy Lipotissue Microkit (product model 74804) using Qiagen Tissuelyser II (20 rps for 1 min) according to the standard Qiagen protocol. Samples were eluted in 50 μL of nuclease-free water. RNA concentration and quality were determined using the nucleic acid (RNA) program with NanoDrop One prior to analysis.

[0624] The expression of eGFP or trastuzumab transgenes in DNA samples was analyzed using a two-step RT-ddPCR method targeting the transgene (eGFP or trastuzumab) and a reference gene (RPP30). Specific primer and probe sequences are listed in the table below.

[0625]

[0626] Samples were analyzed according to the standard Bio-Rad RT-ddPCR protocol for probe-based RNA expression analysis. Briefly, a reaction mixture containing a 2-primer probe set, RNA sample, and Bio-Rad One-Step RT-ddPCR Advanced Kit for Probes (product model 186-4021) was prepared according to the formulation in the table below.

[0627] reagents Volume (uL) / reaction Supermix 5 300mM DTT 1 reverse transcriptase 2 20X RPP30 PnP 1 20X eGFP PnP or 20x trastuzumab PnP 1 Nuclease-free water 5 RNA sample* 5

[0628] Pre-dilute the RNA sample to 20 ng / μL using nuclease-free water.

[0629] After droplet formation, the reaction is scaled up using the following thermal cycling procedure.

[0630]

[0631] 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.

[0632] 7.1.2. Anc80L65's extensive CNS penetration and wide distribution compared to AAV9

[0633] The aim of this study was to determine the biodistribution and preliminary feasibility of the Anc80L65 vector compared to the AAV9 vector when administered via a single lumbar puncture or intracephalic administration. Results confirmed the extensive penetration and distribution of Anc80L65 compared to AAV9.

[0634] Two AAV constructs were used in the experiment: (i) Anc80L65-CAG-GFP and (ii) AAV9-CAG-GFP, each containing an AAV genome construct with a GFP coding sequence. GFP was used to detect AAV distribution and transgene expression. Cynomolgus monkeys were used as subject animals.

[0635] A total of 14 animals were divided into 6 groups, such as Figure 1 As outlined in Table 2. Animals in Groups 1 and 4 were control animals administered the vector. Animals in Groups 2 and 5 were administered Anc80L65 containing 4E13vg (viral genome or Gc), while animals in Groups 3 and 6 were administered AAV9 containing 4E13vg. Two administration routes were tested—ICM administration for animals in Groups 1–3, and LP administration for animals in Groups 4–6. Animals were sacrificed on day 14 or 15 following vector or AAV administration, and their organ samples were collected for analysis.

[0636]

[0637] The collected samples were processed for IHC and stained with an antibody against GFP. Images of the IHC staining are shown in [images]. Figures 2A-9 and Figures 22A-22D Provided by China. Figures 2A-2D Immunohistochemical (IHC) images of cortical tissue from brain slices obtained by intracephalic injection or lumbar puncture with NHP of Anc80L65 or AAV9 are provided. Figures 22A-22DIHC images of the cortex and caudate nucleus from brain slices obtained by intracerebrospinal injection of Anc80L65 or AAV9 via NHP are provided.

[0638] These results show that, through both ICM and LP administration, Anc80L65 exhibits superior transgenic (GFP) expression capacity compared to AAV9. Compared to AAV9, Anc80L65 administration resulted in more cells in the cortex and caudate nucleus being stained due to GFP expression. Figures 2A-2D Furthermore, it was shown that ICM administration of both vectors (i.e., Anc80L65 and AAV9) provided better results than LP administration in terms of widespread distribution within the brain.

[0639] It also provides information from other parts of the brain—specifically, the cortex ( Figures 3A-3C , Figure 8A -Figure 8C and Figure 9 ), ependyma and caudate nucleus ( Figures 4A-4B ), caudate nucleus ( Figures 5A-5B ), melanin ( Figure 6 ) and perivascular cells ( Figures 7A-7B The IHC results from [reference needed] show that Anc80L65 has a wider penetration and distribution compared to AAV9.

[0640] To characterize cell types expressing GFP after administration of Anc80L65 or AAV9, NHP brain slices were double-stained with GFP and cell type-specific markers. Figures 26A-26F and Figures 27A-27F Images of double staining are provided—targeting GFP and neuronal markers (NeuN) in the motor cortex transfected with Anc80L65 or AAV9. Figure 26A and Figure 26D ), targeting GFP and astrocyte markers ( Figure 26B and Figure 26E ), targeting GFP and microglia markers (ibal), targeting GFP and oligodendrocyte markers ( Figure 27A , Figure 27B and Figure 27C In all cases, GFP+ cells appeared red, cell-specific markers appeared green, and merged images showed double-labeled cells as yellow / orange (arrows). Staining results showed that, following a single LP or ICM injection, Anc80L65 could mediate efficient transgene expression in neurons, astrocytes, and oligodendrocytes across a large region of the NHP brain. This suggests that Anc80L65 could be used clinically to treat a wide range of neurological disorders, particularly using relatively non-invasive routes of administration such as LP.

[0641] The transgene transfer and expression capabilities of Anc80L65 and AAV9 administered to NHPs via ICM or LP were also tested using ddPCR, measuring the amount of transgene (eGFP) DNA and mRNA in the brain and spinal cord of NHPs two weeks after ICM or LP delivery. The DNA genomic copy and mRNA transcript copy of the transgene (eGFP) were quantified by comparing them to the DNA genomic copy or mRNA transcript copy of the housekeeping gene (RPP30), respectively. Specifically, the DNA genomic copy report was the vector genomic copy (VGC / DG) for each diploid genome. The output was calculated using the formula VGC / DG = (eGFP cp / μL ÷ RPP30 cp / μL) × 2. The RNA transcript copy report was the % of eGFP expression, calculated using the formula eGFP expression % = (eGFP cp / μL ÷ RPP30 cp / μL) × 100.

[0642] The viral DNA genome copy (VGC) of each diploid genome measured in the experiment (i.e., the VGC of each cell) Figures 13A-17 Provided in [the text]. Each figure provides data corresponding to a different brain region or liver, including the cerebellar cortex ([the text is incomplete and likely refers to a different part of the original text]). Figure 13A ), cervical dorsal root ganglion ( Figure 13B ), lumbar root ganglia ( Figure 14A ), frontal cortex ( Figure 14B ),liver( Figure 15A ), motor cortex ( Figure 15B ), cervical spinal cord ( Figure 16A ), lumbar spinal cord ( Figure 16B ) and sciatic nerve ( Figure 17 VGC data in Figure 25 Further analysis and overview are provided below.

[0643] Data shows that, regardless of the injection route, Anc80L65 resulted in more vector genome copies per cell in the frontal cortex, motor cortex, and spinal cord (cervical and lumbar) compared to AAV9. Figure 25 As shown.

[0644] RNA transcripts measured experimentally Figure 18A , Figure 18B , Figure 19A , Figure 19B , Figure 20A , Figure 20B and Figure 21 Provided in [the text]. Each figure provides data corresponding to a different brain region, including the caudate nucleus (…). Figure 18A ), frontal cortex ( Figure 18B ), pale ball ( Figure 19A ), motor cortex ( Figure 19B ), apical cortex ( Figure 20A ), shell and core (Figure 20B ) and substantia nigra ( Figure 21 Anc80L65 administration 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 ICM and LP administration, the parietal cortex after ICM and LP administration, and the putamen after LP administration.

[0645] Single-factor statistical analysis of expressed data Figures 10A-12B Provided in [the document / platform]. The analysis results are also available in [the document / platform]. Figure 23 and Figure 24 The list is displayed in the middle. Figures 10A-10C and Figure 23 Provided information from the frontal cortex ( Figure 10A , Figure 23 ), motor cortex ( Figure 10B , Figure 23 ) and the cortical apical leaf ( Figure 10C , Figure 23 Analysis of the data showed that, compared with AAV9 injection via ICM or LP, animals injected with Anc80L65 via ICM or LP exhibited significantly higher GFP expression in their cortex. Figures 11A-11B , Figures 12A-12B and Figure 24 Showing the caudate nucleus ( Figure 11A , Figure 24 ), pale ball ( Figure 11B , Figure 24 ), shell and core ( Figure 12A , Figure 24 ) and substantia nigra ( Figure 12B , Figure 24 Similar analyses were performed in [the study]. These figures also showed that GFP expression was significantly higher in most brain regions of animals injected with Anc80L65 via ICM or LP compared to AAV9 injection via ICM or LP. These results suggest that both ICM and LP injection of Anc80L65 can be efficient methods for delivering and expressing transgenes, superior to ICM administration of AAV9.

[0646] Statistical analysis of the ddPCR data is also provided in Table 3 below. This table provides data from Tukey. - The Kramer HSD test, including fold-over differences and p-values, compared GFP transcript (RNA) expression between Anc80L65(ICM) and AAV9(ICM), Anc80L65(LP) and AAV9(LP) in different tissues. Positive differences indicate the degree of expression dominance attributable to Anc80L65. Statistically significant p-values ​​are indicated in red (asterisk). The analysis showed that the dominance of Anc80L65 compared to AAV9 was statistically significant in all brain regions.

[0647]

[0648] 7.2. Example 2: Analysis of AAV in non-human primates -Lib460 To identify capsid variants with enhanced CNS tropism

[0649] The aim of this study was to evaluate the efficacy of AAV-derived proteins in non-human primates (NHPs). -Lib460 460 modified capsid proteins were identified to determine the capsid of the VR VIII variant with enhanced CNS tropism. The 460 targeting peptides include the sequence SEQ ID NO: 160-619. The targeting peptides are inserted between Q588 and A589 of the Anc80L65 VP1 capsid protein.

[0650] The NHP was administered a library containing the unique sequences of the Anc80L65 VP1 capsid protein with 460 modifications as described above (referred to herein as AAV). -Lib460 For each of the 460 modified Anc80L65 VP1 capsid variants, the targeting peptide is located between Q588 and A589 in VR VIII. Controls include 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).

[0651] 7.2.1. Experimental Design

[0652] Three cynomolgus monkeys were treated as outlined in Table 4 below. All animals received a specified amount or concentration of AAV via ICM, IG, or IV. -Lib460 (All three treatments included 460 modified capsid proteins and controls (Anc80L65 capsid protein, AAV9 capsid protein, and AAV9-Retro capsid protein)) (see Table 4).

[0653]

[0654] 7.2.2. Intracaver magnum (ICM) injection

[0655] ICM was performed as described in Example 1.

[0656] 7.2.3. Interdigital injection

[0657] Inject the animal with an anesthetic and place it in a lateral recumbent position. Insert a 25-gauge needle containing the appropriate test sample into the finger pad of the animal's upper limb. Specifically, inject an equal volume of 0.2 mL of test sample (total 0.4 mL) into each of the middle and index fingers of the right hand (both connected to the median nerve).

[0658] 7.2.4. Intravenous injection

[0659] Inject the animal with anesthesia and place it in a lateral recumbent position. Restrict the animal to a position that allows access to the vein. Prepare the injection site surgically (shaving and sterile cleaning). Dilate the vein by compressing a vein closer to the heart than the catheter entry site. Apply pressure manually. Visualize the vein and insert and advance the catheter into it, keeping the stylet stationary as the catheter is slowly advanced into the vessel until the catheter hub reaches the skin puncture site. Administer the dose using a syringe with an injection cap. After administering the test sample, flush the catheter with sterile saline.

[0660] 7.2.5. Analysis

[0661] Animals were sacrificed on day 28 following AAV vector administration, and tissue samples were collected for analysis. Collected tissue samples included the CNS, liver, DRG, peripheral nerves, and spinal cord.

[0662] All analyses were performed by the sponsor using the analytical methods developed and validated by the laboratory.

[0663] NGS analysis was used to determine the biodistribution of vector genomes in DNA samples in the CNS, liver, DRG, peripheral nerves, and spinal cord.

[0664] Gene transfer efficiency of each AAV vector was assessed by measuring mRNA transcripts in the CNS, liver, DRG, peripheral nerves, and spinal cord using NGS.

[0665] Appendix A provides a ranked list of 64 target peptides selected from 460 peptides based on their CNS targeting ability when incorporated into the AAV capsid. The ranking is based on gene transfer efficiency assessed by measuring mRNA transcripts in the CNS. The list includes the SEQ ID NO; peptide sequence; and the average LogFC value obtained by ICM administration. See Appendix A.

[0666] 7.3. Example 3: Analysis of AAV- in non-human primates Lib1 To identify capsid variants with CNS orientation

[0667] The aim of this study was to evaluate AAV in non-human primates (NHP). -mini Modified capsid proteins in the library (i.e., such as Figures 31-33The target peptides (and insertion sites) described in SEQ ID NO: 3-9, 11-19 or 21-28 are used to identify capsids with target peptides (and / or insertion sites) that enhance CNS tropism. Full-length modified capsid proteins containing the aforementioned target peptides are as described in SEQ ID NO: 34-38 or 55820-55847.

[0668] Administering AAV containing rAAV with the modified capsid protein sequence as described above to NHP -mini Library. Target peptides (SEQ ID NO: 3-9, 11-19, or 21-28) are inserted into VR VIII of Anc80L65 VP1 capsid protein or AAV9 VP1 capsid protein, partially as follows: Figures 31-33 As described above. Additionally, tests were conducted containing, for example... Figures 32A-32D One or more additionally modified Anc80L65 capsid proteins were described. Specifically, AFT-6, AFT-7, and AFT-8VP1 capsid proteins were tested. AFT-6 comprises an Anc80L65 VP1 capsid protein backbone in which the Anc80L65 VR VIII region is replaced by the AAV9 VR VIII region (SEQ ID NO: 29). AFT-7 comprises an Anc80L65 VP1 capsid protein backbone in which the Anc80L65 VRIV and VIII regions are replaced by the AAV9 VRIV region (SEQ ID NO: 30) and the AAV9 VIII region (SEQ ID NO: 29), respectively. AFT-8 comprises an Anc80L65 VP1 capsid protein backbone in which the Anc80L65 VR IV, VR V, and VIII regions are replaced by the AAV9 VRIV region (SEQ ID NO: 30), the AAV9 VR V region (SEQ ID NO: 31), and the AAV9 VIII region (SEQ ID NO: 29), respectively.

[0669] The controls include pLib-AAV9, pLib-AAV9-D1, pLib-Anc80L65 (pLib-AAV9-C1 (AAV9 containing a target peptide with the sequence SEQ ID NO: 55854 inserted between Q558 and A589), pLib-AAV9-C2 (AAV9 containing a target peptide with the sequence SEQ ID NO: 55855 inserted between Q558 and A589), and pLib-AAV9-C3 (AAV9 containing a target peptide with the sequence SEQ ID NO: 55856 inserted between Q558 and A589) in library ATP292).

[0670] 7.3.1. Experimental Design

[0671] Three animals were treated as outlined in Table 5 below. Immunosuppression of the animals began 7 days prior to vector administration. The specified dose or concentration of AAV was administered to the animals via IV. -mini (Including 37 modified capsid proteins and controls (see Table 5)).

[0672]

[0673] 7.3.2. Analysis

[0674] Animals were sacrificed on day 28 after AAV vector administration, and organ samples were collected for analysis. Collected tissue samples included the CNS, liver, DRG, peripheral nerves, and spinal cord.

[0675] All analyses were performed by the sponsor using the analytical methods developed and validated by the laboratory.

[0676] DNA samples will be collected and NGS analysis will be used to analyze the biodistribution of the vector genome in the CNS, liver, DRG, peripheral nerves, and spinal cord.

[0677] Gene transfer efficiency of each AAV vector was assessed by measuring mRNA transcripts in the CNS, liver, DRG, peripheral nerves, and spinal cord using NGS. For these experiments, each AAV contained a barcoded mRNA representing the ID of a single AAV. The barcoded mRNA served as a surrogate indicator of the presence of AAVs (i.e., AAVs containing various capsid proteins) and was the entity sequenced and used to quantify gene transfer efficiency. To aid quantification, comparisons with test samples (TAs) were used to normalize the varying AAV abundances in the manufactured TAs and also as a quantitative measure of expression in the final sample. The entity sequenced in the test sample (TA) was the DNA from the same barcoded capsid within the package. Gene transfer data are shown in […]. Figure 34 And in Table 6.

[0678] Table 6 provides a ranking list of the tested AAVs, where the ranking is based on the LogMN_mean. "LogMN_FC" refers to the log2 fold change in the sample relative to the injected test article (TA). Specifically, LogMN_FC is log2(median normalized counts per million reads (CPM) of a single AAV in the sample / median normalized counts per million reads of the same AAV in the test article (TA)). "CPM" is the count of a single AAV / the total count of all AAVs x 1E6. "Mean normalized CPM" is the CPM of a single AAV / median (CPM of all AAVs in the sample). Table 6 shows that AFT-6 and AFT-6** (a biological repeat of AFT-6 (SEQ ID NO: 55820)) had the highest RNA expression among the tested AAVs.

[0679] Figure 34 The inverse coefficient of variation (ICV) is shown to represent the expression level of a single AAV in brain tissue. Specifically, "LogMN_FC" refers to log2 (median normalized counts per million readings (CPM) of a single AAV in the sample / median normalized counts per million readings of the same AAV in the test item (TA). "Mean LogMN_FC" refers to the average logMN_FC value of a single AAV in the selected tissue set (i.e., brain tissue) from all samples from all 3 animals. ICV is a measure of variability, where a larger value indicates less variability compared to the mean, and is calculated as 1 / coefficient of variation for each AAV across all samples. Figure 34 As shown, AFT-6A and AFT-6B (biological duplicates of AFT-6 (SEQ ID NO: 55820)) not only had the highest RNA expression in the tested AAVs, but also had the smallest amount of variability compared to the average of all tested AAVs.

[0680] Overall, the data shows that, among the tested capsid proteins, AAV virions containing the capsid protein AFT-6 (SEQ ID NO: 55820) exhibited the highest RNA expression levels in brain tissue (see [link to relevant documentation]). Figure 34 (and Table 6).

[0681]

[0682]

[0683] * indicates that the targeting peptide is inserted into the AAV9 backbone between Q588 and A589 (SEQ ID NO: 61).

[0684] **Indicates technical repetition in AFT-6

[0685] Using sequence data, an enrichment score was calculated for each AAV variant containing a unique target peptide in each tissue. Specifically, AAV variants containing each target peptide were ranked based on the tissue score of mean log fold change (see, for example, Table 6). After determining sequence counts from sequence read data (FASTQ) following sequencing, AAV variants containing unique target peptides were counted and normalized, followed by tissue enrichment analysis for each tissue. Tissue enrichment analysis included sequence activity relation (SAR) analysis, network analysis, and structural modeling. SAR analysis identified specific amino acids or sequence motifs (combinations of amino acids at several positions) at specific capsid sites that significantly influenced tissue orientation. Network analysis identified variant modules that shared amino acid or peptide sequences of the best-performing variant. Structural modeling provided an understanding of the structural effects of important amino acids at specific capsid sites, contributing to the formation of mechanistic hypotheses.

[0686] like Figure 35 As shown, SAR analysis identified a modified AAV capsid protein (i.e., the Anc80L65 capsid containing the N2 targeting peptide (SEQ ID NO: 9; C4) located in VR VIII, the complete capsid sequence being designated AFT-6 (SEQ ID NO: 55820))) which, compared to AAV9, exhibited enhanced tissue enrichment in target CNS tissues (e.g., Figure 35 (As shown). Specifically, the expression of the AAV capsid protein modified with Anc80L65 containing the targeting peptide N2 increased by 100-1000 in different brain regions. 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. Compared to AAV9, the tissue enrichment of this AAV capsid protein modified with Anc80L65 containing the N2 targeting peptide was also reduced in off-target CNS tissues (e.g., ...). Figure 35 (As shown). Off-target CNS tissues include dorsal root ganglia; such as the cervical, lumbar, and thoracic dorsal root ganglia; and the liver.

[0687] Additional SAR analysis identified three modified AAV capsid proteins that, compared to controls, enhanced tissue enrichment in various CNS tissues (e.g., the Anc80L65-modified AAV capsid protein containing the targeting peptide N2 (SEQ ID NO: 9) inserted into VR VIII (the complete capsid sequence is referred to as AFT-6 (SEQ ID NO: 55820)); AAV9 containing the N3 targeting peptide (PLNGSVHLY (SEQ ID NO: 3603)) located between amino acid residues 586 and 589 in VR VIII, replacing amino acids A587 and Q588; and AAV9 containing the N4 targeting peptide (PLNGTVHLY (SEQ ID NO: 1232)) located between amino acid residues 586 and 589 in VR VIII, replacing amino acids A587 and Q588) (see [link to SAR analysis]). Figure 36 and Figure 37 ).

[0688] Overall, this data shows that the subset of modified AAV capsid proteins described in this paper exhibits the best CNS tropism.

[0689] 7.4. Example 4: Analysis of AAV- in non-human primates Lib1 To identify capsid variants with CNS orientation

[0690] The aim of this study was to evaluate the efficacy of AAV-containing organisms in non-human primates (NHPs). -Lib A library of 54,000 modified capsid proteins (i.e., the targeting peptides in Appendix A, and inserted between positions Q586 and A589 in AAV, thereby replacing A587 and Q588) was used to identify capsids of targeting peptides that enhance CNS tropism.

[0691] The NHP was administered a library of approximately 54,000 unique sequences containing modified capsid proteins, referred to herein as AAV. -Lib The targeting peptide is located between amino acid residues 586 and 589 in the VRVIII region of the AAV9 VP1 capsid protein, with amino acids A587 and Q588 substituted. The targeting peptide comprises sequences as described in SEQ ID NO: 620-55819 and 55857-55859. For example, amino acid modifications (insertions, deletions, substitutions) in the VR VIII region of AAV9 include... Figure 33 The modifications shown are as follows. References include pLib-AAV9, pLib-AAV9-C4, pLib-AAV9-C1, pLib-AAV9-C2, and pLib-AAV9-C3.

[0692] 7.4.1. Experimental Design

[0693] Three animals were treated as outlined in Table 6 below. Immunosuppression in the animals began 7 days prior to vector administration. The specified dose or concentration of AAV was administered to the animals via IV. -Lib (Contains approximately 55,200 modified capsid proteins and controls) (see Table 8). Animals were sacrificed on day 28 after AAV vector administration, and their organ samples were collected for analysis.

[0694]

[0695] 7.4.2 Analysis

[0696] Animals were sacrificed on day 28 after AAV vector administration, and organ samples were collected for analysis. Tissue samples included: basal ganglia-caudate nucleus; basal ganglia-globus pallidus; basal ganglia-substantia nigra; cerebellar cortex; DRG-cervical; DRG-lumbar; DRG-thoracic; frontal lobe; liver; occipital lobe; parietal lobe; putamen; sensorimotor cortex; temporal lobe; and thalamus.

[0697] NGS analysis was used to analyze the biodistribution of vector genomes in the CNS, liver, DRG, peripheral nerves, and spinal cord (data not shown).

[0698] The gene transfer efficacy of each AAV vector was assessed by measuring mRNA transcripts in the tissues described in Appendix B. Tissue enrichment data are also presented in Appendix B (which is incorporated herein by reference in its entirety). These data represent the mean LogMN_FC measured and calculated as described above. Appendix B provides 750 targeting peptides selected from 54,000 modified capsid proteins based on CNS targeting capabilities.

[0699] Overall, the data show that a subset of modified AAV capsid proteins (e.g., the capsid proteins provided in Appendix B) exhibit the best CNS tropism.

[0700] Table 7 provides CNS targeting data (mean LogMN_FC) for some AAV vectors, AAV9, or AAV9 with targeting peptides (N3, N4, or N5).

[0701]

[0702]

[0703] 7.4.3. Generating rAAV containing the AAV-Lib1 capsid

[0704] rAAVs containing various AAV-Lib1 capsids were generated in suspended HEK293 cells to test their yield and manufacturability. As shown in Table 8, the study revealed that rAAVs containing AAV9-N3 and AAV9-N4 provided good yields at harvest and remained stable during formulation. However, rAAVs containing AAV9-N5 capsids provided lower yields at harvest and precipitated during formulation.

[0705]

[0706] 7.4.4. Serum prevalence of rAAV containing the AAV-Lib1 capsid

[0707] Serological prevalence of various AAV-Lib1 capsids was tested in an in vitro neutralizing antibody assay using donor samples (N=55) that were approximately representative of the 2019 U.S. Census Bureau census data. All samples were run at a 1:5 serodilution, and titer was determined as the reciprocal of the serodilution reporting 50% relative optical units (RLU) compared to the AAV capsid alone. Samples with a titer <5 were defined as seronegative, while samples with a titer >5 were defined as seropositive.

[0708] Table 9 shows the percentage of donor samples that were seropositive or serone in the tests. This data indicates that the seroprevalence of AAV9-N3 and AAV9-N4 is lower than that of AAV9 but higher than that of AAV9-C4 and AAV5.

[0709]

[0710] 8. Sequence

[0711] Many of the nucleotide sequences provided below were obtained from double-stranded vectors. Therefore, those skilled in the art will understand that, unless the context otherwise requires, reference to the nucleotide sequences provided herein also includes reference to complementary sequences. Additional sequences are found in Appendices A and B, each of which is incorporated herein by reference in its entirety.

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[0744] 9. Equivalents and incorporation by reference

[0745] Although the invention has been specifically shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0746] All references, authorized patents and patent applications cited in the text of this specification are incorporated herein by reference in their entirety for all purposes.

[0747] Appendix A

[0748]

[0749] Appendix B

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[0761]

Claims

1. A modified adeno-associated virus (AAV) capsid protein comprising... Targeting peptides within variable region VIII (VR VIII), The targeted peptide has the sequence X1X2X3X4X5X6X7X8X9, and X1, X2, X3, X4, X5, X6, X7, X8 and X9 are each independently selected from any amino acid residue.

2. The modified AAV capsid protein according to claim 1, wherein... (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).

3. The modified AAV capsid protein according to claim 1 or 2, wherein the targeting peptide within VR VIII has a sequence selected from SEQ ID NO: 160-55819 and 55857-55859.

4. The modified AAV capsid protein according to any one of claims 1-3, wherein the targeting peptide has the sequence PX2X3GAVX7LY (SEQ ID NO: 2), and X2, X3 and X7 are independently selected from any amino acid residues.

5. The modified AAV capsid protein according to claim 3, wherein... (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).

6. The modified AAV capsid protein according to claim 4 or 5, wherein 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).

7. The modified AAV capsid protein according to any one of claims 1-6, 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. The modified AAV capsid protein according to any one of claims 1-7, having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the AAV9 capsid protein.

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

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

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

12. The modified AAV capsid protein according to claim 1 or 2, wherein the targeting peptide has the sequence PX2X3GX5X6X7LY (SEQ ID NO: 10), and x2, x3, x5, x6 and x7 are independently selected from any amino acid residues.

13. The modified AAV capsid protein according to claim 12, wherein... (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).

14. The modified AAV capsid protein according to claim 12 or 13, wherein 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).

15. The modified AAV capsid protein according to claim 1 or 2, wherein the targeting peptide has the sequence PX2X3GAVX7X8X9 (SEQ ID NO: 20), and X2, X3, X5, X6 and X7 are independently selected from any amino acid residues.

16. The modified AAV capsid protein according to claim 15, wherein... (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).

17. The modified AAV capsid protein according to claim 15 or 16, wherein 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).

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

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

20. The modified AAV capsid protein according to any one of claims 12-19, 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. A modified adeno-associated virus (AAV) capsid protein comprising: The targeting peptide within VR VIII, wherein the targeting peptide has a sequence selected from SEQ ID NO: 160-619.

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

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

24. The modified AAV capsid protein according to claim 22 or 23, wherein the reference AAV capsid protein is a capsid protein of AAV 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; AAVhu.37; AAVrh.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; r h.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; Anc12 6; Anc127; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc110; and Anc80DI.

25. The modified AAV capsid protein according to any one of claims 22-24, wherein the reference AAV capsid protein is a capsid protein or a fragment thereof having a sequence selected from SEQ ID NO: 54-158.

26. The modified AAV capsid protein according to any one of claims 22-25, 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 according to any one of claims 22-26, 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 according to any one of claims 22-27, wherein the targeting peptide is located between 576 and 601 in VR VIII of the modified AAV capsid protein.

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

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 576-585 of the reference AAV capsid protein, wherein the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

31. The modified AAV capsid protein according to claim 29 or 30, wherein the N-terminal flanking region has the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), and B1, B2 and B3 are each independently selected from any amino acid residues.

32. The modified AAV capsid protein according to claim 31, wherein B1 is selected from glutamic acid (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H).

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

34. The modified AAV capsid protein according to any one of claims 29-33, wherein the N-terminal flanking region replaces the N-terminal reference sequence of the 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 at the N-terminus of the insertion site of the targeting peptide within the reference AAV capsid protein.

35. The modified AAV capsid protein according to claim 34, wherein the N-terminal reference sequence has the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849).

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

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 589-602 of the reference AAV capsid protein, wherein the amino acid residues are numbered according to the amino acid sequence of the reference AAV capsid protein.

38. The modified AAV capsid protein according to claim 36 or 37, wherein the C-terminal flanking region has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851), and Z1, Z2, Z3 and Z4 are each independently selected from any amino acid residues.

39. The modified AAV capsid protein according to claim 38, 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).

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

41. The modified AAV capsid protein according to any one of claims 36-40, wherein the C-terminal flanking region replaces the C-terminal reference sequence of the 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 at the C-terminus of the insertion site of the target peptide within the reference AAV capsid protein.

42. The modified AAV capsid protein according to claim 41, wherein the C-terminal reference sequence has the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851).

43. The modified AAV capsid protein according to any one of claims 1-28, further comprising: (i) An N-terminal flanking region having the sequence B1YGB2VATNB3QS (SEQ ID NO: 55849), wherein B1, B2, and B3 are each independently selected from any amino acid residue; and (ii) A C-terminal flanking region having the sequence AQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55851), wherein Z1, Z2, Z3 and Z4 are each independently selected from any amino acid residue.

44. The modified AAV capsid protein according to any one of claims 1-28, further comprising: (i) Having an N-terminal flanking region of the sequence B1YGB2VATNB3QS (SEQ ID NO: 55860), wherein B1 is selected from glutamic acid (E) or serine (S), B2 is selected from threonine (T) or glutamine (Q), and B3 is selected from leucine (L) or histidine (H); and (ii) A C-terminal flanking region having 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).

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

46. ​​The modified AAV capsid protein according to any one of claims 1-28, comprising the amino acid sequence B1YGB2VATNB3QSPLMGAVHLYAQAQTGZ1VZ2Z3QGZ4 (SEQ ID NO: 55852), wherein B1, B2, B3, Z1, Z2, Z3 and Z4 are each independently selected from any amino acid residue.

47. The modified AAV capsid protein according to claim 46, wherein B1 is selected from glutamic acid (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).

48. The modified capsid protein according to any one of claims 1-28, wherein the targeting peptide is a peptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues different from the sequence of SEQ ID NO: 29, wherein the different amino acids include insertions, deletions or substitutions compared to the sequence of SEQ ID NO:

29.

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

29.

50. The modified AAV capsid protein according to any one of claims 22-49, wherein the modified AAV capsid protein has one or more modifications, including amino acid insertion, deletion, substitution or combinations thereof, compared to the reference AAV capsid protein.

51. The modified AAV capsid protein according to any one of claims 22-50, wherein the one or more modifications comprise amino acid insertions, deletions, substitutions, or combinations thereof incorporating the targeting peptide into VR VIII of the reference AAV capsid protein.

52. The modified AAV capsid protein according to any one of claims 22-50, wherein the one or more modifications comprise amino acid modifications other than VR VIII of the reference AAV capsid protein.

53. The modified AAV capsid protein of claim 52, wherein the one or more modifications other than VR VIII of the reference AAV capsid protein include one or more modifications of VRI, VRII, VRIII, VRIV, VR V, VR VI, or VR VII.

54. The modified AAV capsid protein of claim 53, wherein the one or more modifications other than VR VIII of the reference AAV capsid protein include one or more modifications of VR IV and VR V.

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

30.

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

31.

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

58. The modified AAV capsid protein of claim 57, wherein the deletion of one or more amino acids comprises the deletion of one, two, three, four, or five or more amino acid residues immediately adjacent to the N-terminus of the targeting peptide within VR VIII.

59. The modified AAV capsid protein according to claim 57 or 58, wherein the deletion of one or more amino acids comprises the 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 number of the reference AAV capsid protein.

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

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

62. The modified AAV capsid protein according to any one of claims 57-61, wherein the amino acid deletion comprises the deletion of one, two, three, four, or five or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII.

63. The modified AAV capsid protein according to claim 62, wherein the amino acid deletion comprises the 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 number of the reference AAV capsid protein.

64. The modified AAV capsid protein according to any one of claims 50-63, wherein the amino acid insertion comprises the insertion of one, two, three, four, or five or more amino acid residues immediately adjacent to the C-terminus of the targeting peptide within VR VIII.

65. The modified AAV capsid protein according to claim 64, wherein the inserted amino acid residue is independently selected from any amino acid residue.

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

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

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

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

70. The modified AAV capsid protein according to any one of claims 1-69, wherein (i) The reference AAV capsid protein is the capsid protein of AAV1 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 the capsid protein of AAV2 or a modification thereof, and the targeting peptide is located 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 the capsid protein of AAV3 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 the capsid protein of AAV4 or a modification thereof, and the targeting peptide is located 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 the capsid protein of AAV5 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 the capsid protein of AAV6 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 the capsid protein of AAV7 or a modification thereof, and the targeting peptide is located 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 the capsid protein of AAV8 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 the capsid protein of AAV9 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 located 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 the capsid protein of AAV12 or a modification thereof, and the targeting peptide is located 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 the capsid protein of Anc80 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 the capsid protein of Anc80-55 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 the capsid protein of Anc80-129 or a modification thereof, and the targeting peptide is located 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 capsid protein of Anc80-156 or a modification thereof, and the targeting peptide is located 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 located 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 substitutes for residues A587 and N588 of the reference AAV capsid protein between S586 and T589; or (xix) The reference AAV capsid protein is the capsid protein of Anc80-1712 or a modification thereof, and the targeting peptide is located between S586 and T589 of the reference AAV capsid protein, thereby replacing residues A587 and N588 of the reference capsid protein.

71. The modified AAV capsid protein according to any one of claims 1-69, wherein: (i) The reference AAV capsid protein is the capsid protein of AAV1 or a modification thereof, and the targeting peptide is between D590 and P591 or between S588 and T589 of the reference AAV capsid protein. (ii) The reference AAV capsid protein is the capsid protein of AAV2 or a modification thereof, and the targeting peptide is between R588 and Q589 or between N587 and R588 of the reference AAV capsid protein. (iii) The reference AAV capsid protein is the capsid protein of AAV3 or a modification thereof, and the targeting peptide is between S586 and S587 or between N588 and T589 of the reference AAV capsid protein. (iv) The reference AAV capsid protein is the capsid protein of AAV4 or a modification thereof, and the targeting peptide is between S584 and N585 or between S586 and N587 of the reference AAV capsid protein. (v) The reference AAV capsid protein is the capsid protein of AAV5 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 the capsid protein of AAV6 or a modification thereof, and the targeting peptide is between D590 and P591 or S588 and T589 of the reference AAV capsid protein. (vii) The reference AAV capsid protein is the capsid protein of AAV7 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 the capsid protein of AAV8 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 the capsid protein of AAV9 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 the capsid protein of AAVrh10 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 the capsid protein of AAV12 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 the capsid protein of Anc80 or a modification thereof, and the targeting peptide is between T589 and A590, N588 and T589, or N587 and T588 of the reference AAV capsid protein. (xiv) The reference AAV capsid protein is the capsid protein of Anc80-55 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein. (xv) The reference AAV capsid protein is the capsid protein of Anc80-129 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV capsid protein. (xvi) The reference AAV capsid protein is the capsid protein of Anc80-156 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the 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 the capsid protein of Anc80-1029 or a modification thereof, and the targeting peptide is between T589 and A590 or between N587 and T588 of the reference AAV 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 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 NO: 34-39.

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

74. A vector comprising the polynucleotide according to claim 72.

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

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

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

78. The AAV virion according to claim 77, further comprising exogenous polynucleotides.

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

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

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

83. The AAV virion according to any one of claims 77-82, wherein when administered to a subject in a therapeutically effective amount, the AAV virion has enhanced specificity for central nervous system (CNS) tissues relative to a reference AAV virion containing a reference AAV capsid protein but without the target peptide.

84. The AAV virion according to any one of claims 77-83, wherein when administered to a subject in a therapeutically effective amount, the AAV virion has enhanced transduction efficiency in the CNS relative to a reference AAV virion containing a reference AAV capsid protein but without the target peptide.

85. The AAV virion according to any one of claims 77-84, wherein when administered to a subject in a therapeutically effective amount, the AAV virion has enhanced blood-brain barrier penetration in the subject relative to a reference AAV virion containing a reference capsid protein but without the target peptide.

86. A pharmaceutical composition comprising AAV virus according to any one of claims 77-85.

87. A method for treating or improving or preventing a disease or condition of a subject, comprising administering a therapeutically effective amount of AAV virus according to any one of claims 77-85 or a pharmaceutical composition according to claim 86.

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

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

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

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

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

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

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

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

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

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

98. A method for transferring exogenous polynucleotides to the central nervous system (CNS), comprising the step of administering an AAV virion as specified in any one of claims 77-85 to a subject.

99. The method of claim 98, wherein, when measured by genomic copy number of the AAV virion, the administration results in the transfer of the exogenous polynucleotide in the CNS at a CNS:liver infection rate greater than 1.

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

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

102. Use of the modified AAV capsid protein according to any one of claims 1-71 and / or the AAV virion according to any one of claims 77-85 for the transfer of exogenous polynucleotides to the central nervous system.

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

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