JP-2025517628-A5 -

Dates

Publication Date

20260511

Application Date

20230504

Description

In another embodiment, the Specified Information provides an AAV VP2 capsid polypeptide, a) an AAV VP2 capsid polypeptide of AAV serotype AAV1, comprising, with respect to the VP1 amino acid sequence of SEQ ID NO: E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N, or any combination thereof, in particular, an AAV VP2 capsid polypeptide comprising the amino acid substitutions: D213A, T162R, and/or P191N, or b) AAV The VP2 capsid polypeptide is of an AAV serotype other than AAV1, and contains at least one amino acid substitution corresponding to at least one of the amino acid substitutions selected from the group consisting of E147S, P185G, P166R, M211V, G199R, D213A, T162R, and P191N relative to the VP1 amino acid sequence of SEQ ID NO: 7, and in particular, bi) AAV The VP2 capsid polypeptide is of an AAV serotype 8, and contains at least one amino acid substitution selected from the group consisting of E147S, P186G, P167R, M212V, G200R, D214A, K163R, and P192N relative to the VP1 amino acid sequence of SEQ ID NO: 4, or any combination thereof, and in particular, AAV The VP2 capsid polypeptide contains the amino acid substitution: D214A, K163R and/or P192N, or bii) AAV VP2 capsid polypeptide is of AAV serotype 9 and contains, with respect to the VP1 amino acid sequence of SEQ ID NO: E147S, P185G, P166R, G199R, D213A, S162R and P191N, or any combination thereof, in particular, the AAV VP2 capsid polypeptide contains the amino acid substitution: D213A, S162R and/or P191N. The present invention includes the following embodiments. <1> An adeno-associated virus (AAV) VP2 fusion polypeptide comprising an AAV VP2 capsid polypeptide and a polypeptide ligand, wherein the polypeptide ligand is fused to the N-terminus of the AAV VP2 capsid polypeptide and selected from the group consisting of GP2 polypeptide, Sso7d polypeptide, and affibody. <2> The AAV VP2 fusion polypeptide according to <1>, wherein the polypeptide ligand is selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1 and an Sso7d polypeptide having at least 80%, 85%, 90%, or 95% sequence identity thereto. <3> The AAV VP2 fusion polypeptide according to <2>, wherein the polypeptide ligand is selected from the group consisting of the Sso7d polypeptide of SEQ ID NO: 1 (wherein amino acid residues X at positions 21, 23, 25, 28, 30, 32, 40, 42, and 44 are independently selected from D, R, H, N, A, I, Y, and W), and Sso7d polypeptides having at least 80%, 85%, 90%, or 95% sequence identity thereto. <4> The AAV VP2 fusion polypeptide according to any one of <1> to <3>, further comprising a peptide linker between the polypeptide ligand and the AAV VP2 capsid polypeptide, wherein the peptide linker is selected from the group consisting of a glycine-serine (GS) linker and an alanine-proline-serine (APS) linker, more specifically, the GS linker is of the formula [GGGGGS]n, and the APS linker is of the formula [APS]n, where n is an integer in the range of 1 to 10, more specifically, n is 1, 2, 3, 4, 5 or 6, more specifically, in the case of the GS linker, n is 1, 2, 3 or 4, and in the case of the APS linker, n is 2, 3, 4 or 5. <5> The AAV VP2 fusion polypeptide according to any one of <1> to <4>, wherein the AAV VP2 capsid polypeptide is an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh. 8, AAVrh. 10, AAVrh. 32, 33, bovine AAV, or avian AAV. 5, particularly from the group consisting of AAV1, AAV6, AAV8, and AAV9. <6> The AAV VP2 capsid polypeptide comprises at least one mutation in at least one binding site to its native receptor, and in particular, here, a) The AAV VP2 fusion polypeptide is of AAV serotype AAV6, and its VP3 region contains at least one amino acid substitution selected from the group consisting of: K531E, V473D, K459S, N500E, G266A, N269Q, and D590A, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 3, and in particular, the AAV The VP2 fusion polypeptide comprises the following amino acid substitutions to the VP1 amino acid sequence of SEQ ID NO: ai) K531E and V473D; aii) K531E, K459S, V473D and N500E; aiii) G266A and N269Q; aiv) G266A, N269Q and D590A; av) K531E, V473D, G266A and N269Q; or avi) K531E, K459S, V473D, N500E, G266A, N269Q and D590A; b) The AAV VP2 fusion polypeptide is of AAV serotype AAV8 and contains within its VP3 region at least one amino acid substitution selected from the group consisting of G268E, N271Q, S387A, A592Q, and A592D, or any combination thereof, relative to the VP1 amino acid sequence of SEQ ID NO: 4, in particular the AAV VP2 fusion polypeptide contains the following amino acid substitutions relative to the VP1 amino acid sequence of SEQ ID NO: 4: bi) G268E and N271Q; bii) S387A; biiii) G268E, N271Q, and S387A; biv) A592Q; or bv) A592D; c) The AAV VP2 fusion polypeptide is of AAV serotype AAV9 and contains within its VP3 region at least one amino acid substitution s