AAV capsid enabling gene delivery to the entire CNS through interaction with the transferrin receptor

Abstract

Provided is an engineered AAV capsid. In the engineered AAV capsid, at least one protein on the capsid is modified to include an n - amino - acid - long motif, whereby transduction of the central nervous system (CNS) by the capsid through interaction with the transferrin receptor is promoted. Further embodiments provide a vector system comprising one or more vectors encoding the AAV capsid and a method of delivering a cargo to the CNS. The method includes administering an AAV capsid according to the embodiments described herein, either in vivo or in vitro, wherein the AAV capsid comprises one or more cargo molecules.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 432,336, filed Dec. 13, 2022, and U.S. Provisional Application No. 63 / 368,470, filed Jul. 14, 2022. The entire contents of the applications specified above are hereby incorporated by reference into this specification.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. NS111689 awarded by the National Institutes of Health and Grant No. MH120096 awarded by the National Institute of Mental Health. The government has certain rights in this invention.

[0003] Reference to Electronic Sequence Listing Reference is made to the electronic sequence listing (「BROD - 5515WP_ST26.xml」, size 36,487,898 bytes, created Jul. 14, 2023), the entire contents of which are hereby incorporated by reference into this specification.

[0004] Reference to Electronic Tables Reference is made to Electronic Tables 1 - 20 and 22 filed with the U.S. Patent and Trademark Office together with this application. Also, reference is made to Tables 1 - 13 filed with the U.S. Patent and Trademark Office on Jul. 14, 2022 and assigned Serial No. 63 / 368,470. The tables are hereby incorporated by reference in their entirety into this specification.

[0005] The subject matter disclosed herein generally relates to enhancing transduction of the central nervous system (CNS) by engineered AAV capsids through interaction with the transferrin receptor. In certain examples described herein, at least one protein on the capsid has been modified to include an n-amino acid long motif. Certain examples relate to vector systems having one or more vectors encoding an AAV capsid and to methods of delivering cargo to the CNS, wherein the AAV capsids according to the examples described herein are administered in vivo or in vitro and the AAV capsids contain one or more cargo molecules.

Background Art

[0006] The development of gene therapy for neurodevelopmental disorders and neurological diseases has been constrained by the inability to efficiently deliver genes throughout the CNS. In several studies, engineered AAV9 capsids (particularly the AAV-PHP.B family) that enable highly effective gene transduction throughout the CNS after intravenous administration to adult mice have been reported. However, to date, no engineered AAV capsid that efficiently transduces the mouse brain by crossing the blood-brain barrier (BBB) and exhibits high CNS tropism in primates has been identified. In this study, the applicant adopts a mechanism-oriented approach and designs AAV capsids that interact with the transferrin receptor (TFRC).

[0007] The citation or identification of any document in this application does not admit that such document can be used as prior art for the present invention.

Summary of the Invention

[0008] In one aspect, the present specification provides a composition, the composition comprising a targeting moiety effective to enhance transduction into the central nervous system tissue (CNS) through binding to the transferrin receptor (TFRC), and optionally further comprising a cargo bound or associated with the targeting moiety. Further, one aspect provided herein relates to a vector system, the vector system comprising one or more vectors encoding a targeting moiety effective to enhance transduction into the central nervous system tissue (CNS). Further embodiments provided herein include a polypeptide or particle encoded or produced by the vector system provided herein, or a cell comprising the composition, vector, polynucleotide, or particle provided herein. In one aspect, the present specification provides a method, the method being a method for delivering one or more cargos to the CNS by administering the composition provided herein.

[0009] In one exemplary embodiment, the targeting moiety binds to the extracellular domain of TFRC. In one exemplary embodiment, the targeting moiety binds to one or more of the apical domain of the extracellular domain, the helical domain of the extracellular domain, and / or the protease-like domain of the extracellular domain. In one exemplary embodiment, the targeting moiety binds to the apical domain.

[0010] In one exemplary embodiment, the target-directed moiety comprises an n-amino acid long motif, and the n-amino acid long motif comprises or consists of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 is composed of Y, M, F, and L, X2 is composed of S, H, T, and A, X3 is composed of K and R, X4 is composed of A, G, I, L, M, N, Q, S, T, V, and H, X5 is composed of N, G, A, L, M, Q, S, and T, X6 is composed of A, T, H, N, F, I, P, L, Y, G, S, V, D, E, M, and Q, and X7 is composed of D and N. In one exemplary embodiment, X1 is composed of Y, M, and L, X2 is composed of S, H, T, and A, X3 is composed of K and R, X4 is composed of A, G, I, L, M, N, Q, S, T, and V, X5 includes N, X6 is composed of A, T, H, N, F, I, P, L, and Y, X7 is composed of D and N, or, X1 is composed of Y, M, and L, X2 is composed of S, H, T, and A, X3 is composed of K and R, X4 is composed of A, G, I, L, M, N, Q, S, T, and V, X5 includes G, X6 is composed of A, G, F, H, I, L, N, P, S, T, V, and Y, X7 is composed of D and N, or, X1 is composed of L and Y, X2 is composed of A, H, and S, X3 is composed of K and R, X4 is composed of A, G, H, I, L, M, N, Q, S, T, and V, X5 includes G, X6 includes P, X7 includes D, or, X1 is composed of L and Y, X2 is composed of A, H, and S, X3 is composed of K and R, X4 is composed of A, G, H, I, L, M, N, Q, S, T, and V, X5 includes G, X6 includes P, X7 includes N, or, X1 includes Y, X2 includes S, X3 includes K, X4 is composed of A, I, L, M, N, Q, S, T, and V, X5 includes G, X6 is composed of X, X7 includes Y, P, T, Q, V, F, L, H, S, A, E, D, I, and M, or, X1 is composed of L and Y, X2 includes S, X3 is composed of R and K, X4 is composed of V, I, T, L, and A, X5 is composed of S and A, X6 is composed of P, R, Y, F, H, I, K, and W, X7 includes D, or, X1 is composed of F, L, M, and Y, X2 includes H, X3 is composed of K and R, X4 is composed of A, L,and M, X5 is composed of A, G, L, M, N, Q, S, and T, X6 is composed of A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y, X7 is composed of D and N, or, X1 is composed of L, M, and Y, X2 contains H, X3 is composed of K and R, X4 is composed of A, L, and M, X5 is composed of A, G, L, M, N, Q, S, and T, X6 is composed of A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y, X7 contains N, or, X1 is composed of L, M, and Y, X2 contains H, X3 is composed of K and R, X4 is composed of A, L, and M, X5 is composed of A, G, L, M, N, Q, S, and T, X6 is composed of A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y, X7 contains D, or, X1 is composed of L, M, and Y, X2 contains H, X3 is composed of K and R, X4 contains L, X5 is composed of S, Q, G, T, N, and L, X6 is composed of P, T, V, I, Q, L, and A, X7 contains D, or, X1 contains F, X2 contains S, X3 contains R, X4 contains L, X5 contains G, X6 is composed of A, H, N, L, V, S, P, and T, X7 contains N, or, X1 contains F, X2 contains A, X3 contains R, X4 is composed of T, S, and N, X5 contains G, X6 is composed of Y, F, H, P, and A, X7 contains N, or, X1 contains F, X2 contains H, X3 is composed of K and R, X4 contains L, X5 contains G, X6 is composed of I, P, and S, X7 is composed of N and D. In one exemplary embodiment, the n-amino acid long motif is LHRLGPN (SEQ ID NO: 36834), YSRIGPN (SEQ ID NO: 14632), LHRLGPN (SEQ ID NO: 36834), LHRLGPD (SEQ ID NO: 36413), LHRAGPD (SEQ ID NO: 36894), YSRIGPD (SEQ ID NO: 38223), LSRIGPD (SEQ ID NO: 36274), LARSGPD (SEQ ID NO: 18035), YSRNSDN (SEQ ID NO: 16626), LHKAGPN (SEQ ID NO: 36305), LSRIGPN (SEQ ID NO: 36347), LAKSGPN (SEQ ID NO: 36287), YARNGPN (SEQ ID NO: 14048),It is selected from the group consisting of YSRNSDN (SEQ ID NO: 16626). In one exemplary embodiment, the n-amino acid long motif is YSRIGPN (SEQ ID NO: 14632).

[0011] In one exemplary embodiment, the target-directed moiety comprises an n-amino acid-long motif, and the n-amino acid-long motif comprises or consists of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 is composed of Y or L, X2 contains H, X3 contains A, X4 is composed of K, R, N, and A, X5 is composed of G, Q, L, and S, X6 is composed of P, L, I, N, D, and T, and X7 contains N. In one exemplary embodiment, the target-directed moiety comprises an n-amino acid-long motif, and the n-amino acid-long motif comprises or consists of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 is composed of A, F, H, I, L, N, P, R, S, T, V, and Y, X2 is composed of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, and Y, X3 contains S, X4 is composed of S and T, X5 contains N, X6 contains G, and X7 is composed of I, R, and V. In one exemplary embodiment, X1 is composed of F, L, and Y, X2 is composed of D, E, H, N, Q, S, and T, X3 contains S, X4 is composed of S and T, X5 contains N, X6 contains G, and X7 is composed of I and V, or X1 is composed of V, P, I, S, T, H, and A, X2 is composed of A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, and Y, X3 is composed of S, X4 is composed of S and T, X5 contains N, X6 contains G, and X7 is composed of I and V, or X1 is composed of A, F, I, L, P, S, T, V, and Y, X2 is composed of D, E, N, Q, S, and T, X3 contains S, X4 is composed of T and S, X5 contains N, X6 contains G, and X7 contains R, or X1 contains R, X2 is composed of E, D, Q, and T, X3 contains S, X4 is composed of S and T, X5 contains N, X6 contains G, and X7 is composed of I and V.In one exemplary embodiment, the n-amino acid long motif is selected from the group consisting of FRSTNGV (SEQ ID NO: 16070), VESTNGR (SEQ ID NO: 36431), VDSTNGV (SEQ ID NO: 12206), VQSTNGV (SEQ ID NO: 36423), VSSTNGV (SEQ ID NO: 12333), TESTNGR (SEQ ID NO: 17558), VQSTNGI (SEQ ID NO: 11292), and FVSTNGV (SEQ ID NO: 11162).

[0012] In one exemplary embodiment, the target-directed moiety comprises an n-amino acid-long motif, and the n-amino acid-long motif comprises or consists of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 is composed of R and T, X2 is composed of T, L, M, S, G, D, N, E, R, K, Y, and W, X3 is composed of G, E, D, I, F, H, S, A, M, P, V, Y, W, Q, and T, X4 is composed of D, T, E, H, N, and G, X5 is composed of A, V, S, T, and D, X6 is composed of Y, F, P, and A, and X7 is composed of A and P. In one exemplary embodiment, X1 comprises R, X2 is composed of T, M, L, and S, X3 is composed of Y, S, A, M, I, F, and P, X4 comprises D, X5 is composed of A, V, S, and T, X6 is composed of Y and F, X7 comprises P, or X1 comprises R, X2 is composed of T, M, L, and S, X3 is composed of Y, S, A, M, I, F, and P, X4 comprises D, X5 is composed of A, V, S, and T, X6 is composed of Y and F, X7 comprises A, or X1 comprises R, X2 is composed of G, T, D, S, N, and E, X3 is composed of E, D, P, S, and G, X4 is composed of D, T, E, H, and N, X5 is composed of V, A, and T, X6 is composed of Y and F, X7 comprises P, or X1 comprises R, X2 is composed of G, L, T, D, and S, X3 is composed of D, P, S, and G, X4 is composed of D, E, H, and N, X5 is composed of V and T, X6 is composed of Y and F, X7 comprises P, or X1 comprises T, X2 is composed of R, K, Y, and W, X3 is composed of E, W, Y, Q, S, and T, X4 comprises G, X5 comprises D, X6 is composed of P and A, and X7 is composed of A and P. In one exemplary embodiment, the n-amino acid-long motif is selected from the group consisting of RGEDVYP (SEQ ID NO: 36864), RLEDVFP (SEQ ID NO: 36264), RTYDSYP (SEQ ID NO: 37938), RTYDAYP (SEQ ID NO: 38571), RTYDSFP (SEQ ID NO: 37806), RTETVYP (SEQ ID NO: 36486), RTETVFP (SEQ ID NO: 36389), and RTEHVFP (SEQ ID NO: 36603).

[0013] In one exemplary embodiment, the target-directed moiety comprises an n-amino acid long motif, the n-amino acid long motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 comprises L, X2 comprises C, X3 consists of K and R, X4 comprises P, X5 comprises C, X6 consists of L, S, D, A, N, Q, H, P, and V, and X7 consists of E, T, G, A, D, N, and S. In one exemplary embodiment, the n-amino acid long motif is LCKPCLD (SEQ ID NO: 36437) or LCKPCPT (SEQ ID NO: 36438). In one exemplary embodiment, the target-directed moiety comprises an n-amino acid long motif, the n-amino acid long motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 consists of Y and F, X2 consists of W, F, and Y, X3 consists of S, T, H, A, and Q, X4 comprises G, X5 consists of I, T, V, Q, M, H, K, and R, X6 consists of I, P, H, L, M, A, Q, T, V, K, and R, and X7 consists of A, S, D, E, and N.In an exemplary embodiment, X1 is composed of Y and F, X2 is composed of W, F, and Y, X3 is composed of T and S, X4 includes G, X5 is composed of I, T, V, Q, M, and H, X6 is composed of I, P, H, L, M, A, Q, T, and V, X7 is composed of A, S, D, and E, or X1 is composed of Y and F, X2 is composed of W, F, and Y, X3 is composed of T and S, X4 includes G, X5 is composed of I, T, V, Q, M, and H, X6 is composed of K and R, X7 is composed of A, S, D, and E, or X1 is composed of Y and F, X2 is composed of W, F, and Y, X3 is composed of T and S, X4 includes G, X5 is composed of K and R, X6 is composed of I, P, H, L, M, A, Q, T, and V, X7 is composed of A, S, D, and E, or X1 includes Y, X2 includes F, X3 includes T, X4 includes G, X5 is composed of K, R, Q, M, H, and I, X6 is composed of T, R, H, K, V, and L, X7 includes E, or X1 includes Y, X2 includes F, X3 is composed of T, S, H, and A, X4 includes G, X5 is composed of K, R, and T, X6 is composed of I, P, H, L, M, A, Q, and T, X7 is composed of D and N, or X1 includes Y, X2 includes W, X3 includes T, X4 includes G, X5 is composed of K, M, V, and T, X6 is composed of P, V, I, H, Q, T, M, and L, and X7 is composed of E and D, or X1 is composed of Y and F, X2 includes F, X3 is composed of S, H, A, and Q, X4 includes G, X5 is composed of K, Q, and R, X6 is composed of I, V, L, K, H, R, Q, and M, X7 includes E.

[0014] In one exemplary embodiment, the target-directed moiety comprises an n-amino acid long motif, the n-amino acid long motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 is composed of K, R, S, G, N, T, M, Q, V, D, I, and E, X2 is composed of D, S, N, M, L, G, P, E, and A, X3 is composed of E, D, G, S, A, R, Q, T, P, and N, X4 is composed of F, Y, T, V, S, N, A, G, and H, X5 is composed of T, K, S, R, V, and H, X6 is composed of T, S, G, V, A, K, R, N, D, E, and H, and X7 is composed of F, W, and Y.In one exemplary embodiment, X1 is composed of K and R, X2 is composed of D, S, and N, X3 includes E, X4 includes F, X5 is composed of T, K, S, R, and V, X6 is composed of T, S, G, and V, X7 is composed of F, W, and Y; or X1 is composed of K and R, X2 includes D, X3 includes D, X4 is composed of F and Y, X5 is composed of T, S, V, and H, X6 is composed of T, S, G, V, and A, X7 is composed of F, W, and Y; or X1 is composed of S, R, G, N, T, M, and Q, X2 includes D, X3 includes G, X4 is composed of T, V, S, N, and Y, X5 includes S, X6 is composed of K and R, X7 includes W; or X1 is composed of R, V, D, I, Q, and K, X2 is composed of M, L, and G, X3 is composed of S, E, A, R, and Q, X4 includes D, X5 includes R, X6 is composed of T, A, S, G, K, and N, X7 includes W; or X1 is composed of D, I, E, Q, V, S, and K, X2 is composed of L, M, G, and P, X3 is composed of E, A, S, D, Q, and T, X4 is composed of S and A, X5 includes R, X6 is composed of D, S, E, T, G, and A, X7 includes W; or X1 includes G, X2 is composed of E, S, P, G, and A, X3 is composed of D, E, P, and N, X4 is composed of G, H, T, S, and N, X5 includes V, X6 is composed of R, K, and S, X7 is composed of W and Y; or X1 includes R, X2 includes E, X3 is composed of D, E, P, and N, X4 is composed of G, H, T, S, and N, X5 includes V, X6 is composed of R, K, and S, X7 is composed of W and Y; or X1 includes G, X2 is composed of G and S, X3 is composed of G, E, S, A, P, and D, X4 is composed of T, G, and S, X5 includes S, X6 is composed of S, T, H, K, R, A, and N, X7 includes W. In one exemplary embodiment, the n-amino acid long motif is selected from the group consisting of KDEFTTF (SEQ ID NO: 36308), KDDFTTY (SEQ ID NO: 36336), RDEFTTY (SEQ ID NO: 36615), KDEFSTY (SEQ ID NO: 36390), RDEFTSF (SEQ ID NO: 36701), and REDHVSW (SEQ ID NO: 37067).

[0015] In one exemplary embodiment, the target-directed moiety comprises an n-amino acid long motif, the n-amino acid long motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 is composed of V, I, R, N, and D, X2 is composed of A, G, and S, X3 is composed of L, T, S, H, and G, X4 is composed of K, R, and E, X5 includes G, X6 is composed of W, R, A, and I, and X7 is composed of D and G. In one exemplary embodiment, the n-amino acid long motif is selected from the group consisting of IALKGWD (SEQ ID NO: 36248), NALEGRD (SEQ ID NO: 36407), VALEGRD (SEQ ID NO: 36604), and VALKGWD (SEQ ID NO: 17701). In one exemplary embodiment, the target-directed moiety comprises an n-amino acid long motif, the n-amino acid long motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 is composed of L, M, and W, X2 is composed of F, R, W, K, T, and Y, X3 is composed of D and S, X4 includes G, X5 includes T, X6 is composed of P, G, S, N, A, and R, and X7 is composed of A, P, S, and Y.

[0016] In one exemplary embodiment, the target-directed moiety comprises an n-amino acid long motif, the n-amino acid long motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, where X1 is composed of P, N, and K, X2 is composed of Y and F, X3 is composed of A, X4 is composed of R and K, X5 is composed of S, X6 is composed of P, V, A, R, I, L, S, E, and X7 is composed of E, D, M, and L.

[0017] In one exemplary embodiment, a composition comprising a target-directed moiety effective to enhance transduction into CNS tissue comprises an n-amino acid long motif, where the n-amino acid long motif comprises or consists of the amino acid sequence of Z1-X1-Z2-X2-X3-X4-X5, where Z1 comprises Y, F, or L, Z2 comprises S, R, or K, and X1 to X5 are independently selected amino acids. In one exemplary embodiment, X1 is optionally composed of A, S, or H, X2 is optionally composed of S, T, L, or I, X3 optionally comprises N or G, X4 optionally comprises G, and X5 is optionally composed of N, D, I, V, or R. In one exemplary embodiment, the n-amino acid long motif is selected from the group consisting of YSRIGPN (SEQ ID NO: 14632), YSRLNMN (SEQ ID NO: 14301), YSRLNKD (SEQ ID NO: 16577), and YHRLSNN (SEQ ID NO: 16636). In one exemplary embodiment, the target-directed moiety comprises an n-amino acid long motif, where the n-amino acid long motif comprises or consists of the amino acid sequence of X1-H-X2-L-X3-X4-X5, where X1 to X5 are independently selected amino acids. In one exemplary embodiment, the n-amino acid long motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608).

[0018] In one exemplary embodiment, the n - amino acid - long motif is PSATNGV (SEQ ID NO: 20486), QVSTNGI (SEQ ID NO: 16021), SYSSNGV (SEQ ID NO: 16234), HQSSNGV (SEQ ID NO: 15978), VGSINGI (SEQ ID NO: 16200), AMSTNGR (SEQ ID NO: 16000), SASTNGV (SEQ ID NO: 16127), YMSTNGV (SEQ ID NO: 16042), YYSSNGV (SEQ ID NO: 16206), VHSTNGI (SEQ ID NO: 16134), PLSTNGV (SEQ ID NO: 16233), VYSTNGI (SEQ ID NO: 16059), IISTNGV (SEQ ID NO: 16054), RSVSSNGV (SEQ ID NO: 20502), YKSSNGV (SEQ ID NO: 16123), FRSTNGV (SEQ ID NO: 16070), and / or FVSTNGV (SEQ ID NO: 11162). In one exemplary embodiment, the n - amino acid length is selected from any one of the amino acid sequences in Tables 1 - 22, or any combination thereof. In one exemplary embodiment, the n - amino acid - long motif is selected from the amino acid sequences of SEQ ID NOs: 10952 - 20481 and 36241 - 42428. In one exemplary embodiment, the target - directed moiety is part of a viral capsid protein comprising an AAV capsid.

[0019] In one exemplary embodiment, the target-directed moiety is inserted or substituted into Loop IV, Loop VIII, or both of the AAV capsid protein. In one exemplary embodiment, the target-directed moiety is IPFSRVNPDT (SEQ ID NO: 20285), LGFARTGAAD (SEQ ID NO: 20274), LGFTKSSGSD (SEQ ID NO: 20270), LRYSKTQGES (SEQ ID NO: 20266), SPYARSSAGV (SEQ ID NO: 20271), VGWSRLDLTT (SEQ ID NO: 20262). In one exemplary embodiment, the n-amino acid length is inserted between two amino acids of one or more capsid proteins such that when the protein containing the n-amino acid length is incorporated into the AAV capsid, the n-amino acid length is positioned outside the AAV capsid. In one exemplary embodiment, the viral capsid protein is an AAV viral capsid protein. In one exemplary embodiment, the n-amino acid length is inserted between amino acids 588 and 589 of the capsid protein of AAV9 or at a similar position of the capsid protein derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAVrh.10. In one exemplary embodiment, the target-directed moiety is inserted between two consecutive amino acids within amino acids 451-460 of the capsid protein of AAV9 or at a similar position of the capsid protein derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAVrh.10. In one exemplary embodiment, the capsid protein is VP1, VP2, VP3, or a combination thereof.

[0020] In one exemplary embodiment, the cargo is a polynucleotide, one or more polypeptides, a ribonucleoprotein complex. In one exemplary embodiment, the polynucleotide encodes one or more polypeptides and / or RNAi oligonucleotides. In one exemplary embodiment, the polynucleotide encodes one or more polypeptides. In one exemplary embodiment, one or more polypeptides include an enzyme or an antibody (or an antigen-binding form thereof) including a therapeutically useful enzyme or antibody. In one exemplary embodiment, the polynucleotide encodes a CRISPR-Cas system. In an exemplary embodiment, the cargo is a recombinant AAV genome incorporated into a capsid containing n amino acids in length, and the recombinant AAV genome encodes a therapeutic protein or nucleic acid and is operably linked to appropriate regulatory sequences that induce expression of the protein or nucleic acid in a target tissue. In one exemplary embodiment, the polynucleotide is operably linked to regulatory sequences that promote expression in the CNS.

[0021] In one aspect, a viral capsid or viral particle includes any composition described herein. In one exemplary embodiment, the viral capsid or viral particle further includes a recombinant viral genome, and the recombinant viral genome encodes a therapeutic protein or nucleic acid, a control polypeptide or nucleic acid, and / or a selectable marker polypeptide or nucleic acid. In one exemplary embodiment, the therapeutic protein or nucleic acid, the control polypeptide or nucleic acid, and / or the selectable marker polypeptide or nucleic acid are operably linked to regulatory sequences that promote expression in the CNS. In one exemplary embodiment, the viral capsid or viral particle is an AAV viral capsid or AAV viral particle. In one exemplary embodiment, the recombinant viral genome is a recombinant AAV viral genome. In one exemplary embodiment, the targeting moiety is inserted between amino acids 588 and 589 of the capsid protein of AAV9 or at a similar position of the capsid protein derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAVrh.10.

[0022] In one aspect, the present specification provides a composition, which comprises one or more vectors encoding a targeting moiety effective to enhance transduction into central nervous system tissue (CNS). In one exemplary embodiment, the vector system comprises one or more vectors, and at least one of the one or more vectors encodes a targeting moiety effective to enhance transduction into central nervous system tissue (CNS) via binding to the transferrin receptor (TFRC), and optionally, at least one of the one or more vectors encodes a recombinant AAV genome containing a transgene encoding a protein or polypeptide. Further provided is a composition, which comprises a polypeptide or viral capsid containing a targeting moiety (e.g., n amino acids in length) effective to enhance transduction into the CNS tissue described herein, and optionally further comprises a cargo (e.g., a recombinant AAV genome encoding a therapeutic protein or nucleic acid incorporated into, bound to, or associated with the targeting moiety containing the polypeptide). In one exemplary embodiment, the targeting moiety binds to the extracellular domain of TFRC. In one exemplary embodiment, the targeting moiety binds to one or more of the apical domain of the extracellular domain, the helical domain of the extracellular domain, and / or the protease-like domain of the extracellular domain. In one exemplary embodiment, the targeting moiety binds to the apical domain. In one exemplary embodiment, the targeting moiety comprises any n amino acid length motif described herein. In one exemplary embodiment, the targeting moiety comprises an n amino acid length motif, and the n amino acid length motif comprises or consists of the amino acid sequence of Z1-X1-Z2-X2-X3-X4-X5, where Z1 is composed of Y, F, and L, Z2 is composed of S, R, and K, and X1 to X5 are independently selected amino acids. In one exemplary embodiment, X1 is optionally composed of A, S, or H, X2 is optionally composed of S, T, L, or I, X3 optionally contains N or G, X4 optionally contains G, and X5 is optionally composed of N, D, I, V, or R.In one exemplary embodiment, the n-amino acid long motif is selected from the group consisting of YSRIGPN (SEQ ID NO: 14632), YSRLNMN (SEQ ID NO: 14301), YSRLNKD (SEQ ID NO: 16577), and YHRLSNN (SEQ ID NO: 16636).

[0023] In one exemplary embodiment, the targeting moiety encoded by a vector system or incorporated into a viral vector comprising an AAV capsid comprises an n-amino acid-long motif, the n-amino acid-long motif comprising or consisting of the amino acid sequence of X1-H-X2-L-X3-X4-X5, where X1 to X5 are independently selected amino acids. In one exemplary embodiment, the n-amino acid-long motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608). In one exemplary embodiment, the n-amino acid-long motif is PSATNGV (SEQ ID NO: 20486), QVSTNGI (SEQ ID NO: 16021), SYSSNGV (SEQ ID NO: 16234), HQSSNGV (SEQ ID NO: 15978), VGSINGI (SEQ ID NO: 16199), AMSTNGR (SEQ ID NO: 16000), SASTNGV (SEQ ID NO: 16127), YMSTNGV (SEQ ID NO: 16042), YYSSNGV (SEQ ID NO: 16206), VHSTNGI (SEQ ID NO: 16134), PLSTNGV (SEQ ID NO: 16233), VYSTNGI (SEQ ID NO: 16059), IISTNGV (SEQ ID NO: 16054), RSVSSNGV (SEQ ID NO: 20502), YKSSNGV (SEQ ID NO: 16123), FRSTNGV (SEQ ID NO: 16070), and / or FVSTNGV (SEQ ID NO: 11162). In one exemplary embodiment, provided herein is a vector or recombinant polypeptide, the vector or recombinant polypeptide comprising an engineered AAV capsid, wherein in the engineered AAV capsid, the n-amino acid length is selected from any one listed in Tables 1 to 22, or any combination thereof. In one exemplary embodiment, a vector is provided, wherein in the vector, the n-amino acid-long motif is selected from SEQ ID NOs: 10952 to 20481 and 36241 to 42428. In one exemplary embodiment, provided herein is a vector, the vector encoding a targeting moiety that is part of a viral capsid protein.

[0024] In one exemplary embodiment, the target-directed moiety is inserted or substituted into Loop IV and / or Loop VIII of the AAV capsid protein. In one exemplary embodiment, the target-directed moiety is IPFSRVNPDT (SEQ ID NO: 20285), LGFARTGAAD (SEQ ID NO: 20274), LGFTKSSGSD (SEQ ID NO: 20270), LRYSKTQGES (SEQ ID NO: 20266), SPYARSSAGV (SEQ ID NO: 20271), VGWSRLDLTT (SEQ ID NO: 20262). In one exemplary embodiment, a vector is provided herein, where the n amino acid length is inserted between two amino acids of one or more capsid proteins such that it is outside the AAV capsid. In one exemplary embodiment, a vector is provided herein, where the viral capsid protein is an AAV viral capsid protein. In one exemplary embodiment, a vector is provided herein, where the n amino acid length is inserted between amino acids 588 and 589 of the capsid protein of AAV9 or at a similar position of the capsid protein derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAVrh.10. In one exemplary embodiment, the target-directed moiety is inserted between two amino acids within amino acids 451 - 460 of the capsid protein of AAV9 or at a similar position of the capsid protein derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAVrh.10. In one exemplary embodiment, a vector is provided herein, where the capsid protein is VP1, VP2, VP3, or a combination thereof.

[0025] In one exemplary embodiment, the present specification provides a vector, where the cargo is a polynucleotide, one or more polypeptides, a ribonucleoprotein complex. In one exemplary embodiment, the present specification provides a vector, where the polynucleotide encodes one or more polypeptides and / or RNAi oligonucleotides. In one exemplary embodiment, the present specification provides a vector, where the polynucleotide encodes one or more polypeptides. In one exemplary embodiment, the present specification provides a vector, where one or more polypeptides include an enzyme or an antibody. In one exemplary embodiment, the polynucleotide encodes a CRISPR-Cas system. In one exemplary embodiment, the polynucleotide is operably linked to a regulatory sequence that promotes expression in the CNS.

[0026] In one aspect, the present specification provides a composition, the composition including a polypeptide encoded or produced by the vector system described herein. In one exemplary embodiment, the polypeptide is a capsid protein, optionally an AAV capsid polypeptide. In one aspect, the present specification provides a composition, the composition including particles produced by the vector system described herein. In one exemplary embodiment, the particles are viral particles, optionally AAV particles. In one aspect, the present specification provides a composition, the composition including cells including the composition, vector, polypeptide, or particle described herein.

[0027] In one aspect, the present specification provides a method for delivering one or more cargos to the CNS, the method comprising administering in vivo or in vitro an engineered AAV capsid described herein, or a vector described herein. In one exemplary embodiment, the present specification provides a delivery method, wherein the cargo is a recombinant AAV genome encoding an RNAi oligonucleotide, a recombinant AAV genome encoding a polynucleotide encoding a polypeptide, or a recombinant AAV genome encoding a polypeptide, optionally operably linked to a regulatory sequence that promotes expression in a target tissue (such as the CNS). In one exemplary embodiment, the present specification provides a delivery method, wherein the polypeptide comprises an enzyme or an antibody. In one exemplary embodiment, the present specification provides a delivery method, wherein the cargo encodes a Cas polypeptide, a guide molecule, or both. In one exemplary embodiment, the present specification provides a delivery method, wherein the cargo encodes a nuclease or a nucleic acid component of an RNA-guided nuclease. In one exemplary embodiment, the present specification provides a delivery method, wherein the cargo is one or more polynucleotides encoding a nuclease and a nucleic acid component of an RNA-guided nuclease.

[0028] In one aspect, a method for generating a humanized transgenic non-human animal comprises delivering a vector system or recombinant viral particles comprising a recombinant viral genome to one or more cells of a non-human animal, wherein the vector system or recombinant viral genome encodes a human transferrin polypeptide, and the encoded human transferrin polypeptide is under the control of a tissue-specific promoter or miRNA binding element having selective activity in a desired cell, tissue, or organ.

[0029] In an exemplary embodiment, one or more cells are endothelial cells. In one exemplary embodiment, one or more cells are CNS cells. In an exemplary embodiment, one or more cells are cells of the CNS vasculature, lung, kidney, liver, or any combination thereof. In an exemplary embodiment, the endothelial cells are cells of the CNS vasculature. In one exemplary embodiment, the recombinant virus particles (optionally AAV virus particles) comprise a capsid polypeptide (optionally an AAV capsid polypeptide), and the capsid polypeptide comprises a CNS-specific n-amino acid long motif. In one exemplary embodiment, the CNS-specific n-amino acid long motif comprises X1-N-X3-X4-X5-X6-X7, where X5 is independently selected from K or R, and X1, X3, X4, X6, and X7 are independently selected from any amino acid, and optionally, the overall charge of the n-amino acid long motif at neutral pH is between 0 and +2. In one exemplary embodiment, the CNS-specific n-amino acid long motif comprises or consists of NNSTRGG (SEQ ID NO: 42429), GNSARNI (SEQ ID NO: 42430), and GNSVRDF (SEQ ID NO: 42431). In one exemplary embodiment, the transgenic non-human animal is a rodent, optionally a mouse.

[0030] In one aspect, provided herein is a humanized transgenic non-human animal, the humanized transgenic non-human animal comprising one or more cells that express a human transferrin polypeptide, and optionally, the one or more cells are CNS cells. In one exemplary embodiment, the transgenic non-human animal is a rodent, optionally a mouse. The humanized transgenic non-human animal is produced by any of the methods described herein. In one exemplary embodiment, the humanized non-human animal has a suppressed immune system.

[0031] In one aspect, provided herein is a method for screening an n-amino acid long motif that can effect transduction into central nervous system (CNS) tissue via binding to transferrin receptor (TFRC) in a humanized transgenic non-human animal. The method includes introducing into the humanized non-human transgenic animal described herein one or more compositions comprising a candidate n-amino acid long motif, detecting the binding of the composition that binds to transferrin receptor (TFRC), and / or detecting transduction or uptake by one or more CNS cells of the humanized transgenic non-human animal. In one exemplary embodiment, the candidate n-amino acid long motif comprises or consists of X1-N-X3-X4-X5-X6-X7, where X5 is independently selected from K or R, and X1, X3, X4, X6, and X7 are independently selected from any amino acid, and optionally, the overall charge of the n-amino acid long motif at neutral pH is between 0 and +2. In one exemplary embodiment, the composition is a viral particle comprising one or more capsid proteins each comprising the candidate n-amino acid long motif.

[0032] In one exemplary embodiment, the viral particle is an AAV viral particle, the one or more capsid proteins are AAV capsid proteins, and optionally, the candidate n-amino acid long motif is inserted between amino acids 588 and 589 of the AAV9 capsid polypeptide or at a similar position of a capsid protein derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAVrh.10. In one exemplary embodiment, at least one of the one or more compositions further comprises a cargo. In one exemplary embodiment, the cargo is a therapeutic nucleic acid or polypeptide, a selectable marker, or a control polypeptide or nucleic acid, or encodes a therapeutic nucleic acid or polypeptide, a selectable marker, or a control polypeptide or nucleic acid.

[0033] In one aspect, a method of in vivo modeling includes introducing a second vector system capable of targeting a transgenic polypeptide in the transgenic non-human animals described herein. In one exemplary embodiment, the non-human animal has a suppressed immune system.

[0034] A method of screening for a vector system capable of transducing central nervous system (CNS) tissue via binding to transferrin receptor (TFRC) in a humanized transgenic non-human animal includes: (a) introducing a plurality of vector systems into one or more humanized transgenic non-human animals; and (b) detecting a vector system that binds to transferrin receptor (TFRC).

[0035] These and other aspects, objects, features, and advantages of the exemplary embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the exemplary embodiments.

[0036] The features and advantages of the present invention will be understood by reference to the following detailed description and the accompanying drawings, which illustrate exemplary embodiments in which the principles of the present invention may be utilized. BRIEF DESCRIPTION OF THE DRAWINGS

[0037]

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Mode for Carrying Out the Invention

[0038] The figures of the present application are for illustrative purposes only and are not necessarily drawn to scale.

[0039] General Definitions Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms and techniques in molecular biology can be found in the following: Molecular Cloning: A Laboratory Manual, 2 nd edition (1989) (Sambrook, Fritsch, and Maniatis), Molecular Cloning: A Laboratory Manual, 4 th edition (2012) (Green and Sambrook), Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.), the series Methods in Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.), Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.), Antibodies A Laboratory Manual, 2 ndedition 2013 (E.A. Greenfield ed.), Animal Cell Culture (1987) (R.I. Freshney, ed.), Benjamin Lewin, Genes IX (published by Jones and Bartlet, 2008 (ISBN0763752223)), Kendrew et al. (eds.), The Encyclopedia of Molecular Biology (published by Blackwell Science Ltd., 1994 (ISBN0632021829)), Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (published by VCH Publishers, Inc., 1995 (ISBN9780471185710)), Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2 nd edition (2011).

[0040] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0041] The terms "any" or "optionally" mean that the event, circumstance, or substituent component that follows may or may not be present, and the description is meant to include both the case where the event or circumstance is present and the case where it is not present.

[0042] The description of a numerical range by endpoints includes all numbers and fractions included within each of those ranges, as well as the endpoints described.

[0043] As used herein when referring to measurable values such as parameters, amounts, lengths of time, etc., the terms "about" or "approximately" mean to include variations from and of the specified value, such variations being, for example, variations of + / - 10% or less, + / - 5% or less, + / - 1% or less, and + / - 0.1% or less of the specified value, as long as such variations are appropriate in the invention in which such variations are disclosed. Of course, the value itself is also specifically, preferably, disclosed for a value with the modifier "about" or "approximately".

[0044] As used herein, a "biological sample" can include whole cells and / or living cells and / or cell debris. A biological sample can include (or be derived from) "body fluids". The present invention includes embodiments in which the body fluid is selected from amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, cerumen (ear wax), chyle, chyme, endolymph, perilymph, exudate, feces, female vaginal fluid, gastric acid, gastric juice, lymph, mucus (including nasal mucus and sputum), pericardial fluid, ascites, pleural effusion, pus, mucosal secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more of them. Biological samples include cell cultures, body fluids, cell cultures derived from body fluids. Body fluids can be obtained from mammals, for example, by puncture or other collection or sampling methods.

[0045] The terms "subject", "individual", and "patient" are used interchangeably herein to refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, monkeys, humans, domestic animals, sport animals, and pet animals. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0046] The following describes various embodiments. Note that a particular embodiment is not intended as an exhaustive description nor as a limitation to the broader aspects described herein. An aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be implemented with any other embodiment(s). References throughout this specification to "one embodiment", "an embodiment", or "an exemplary embodiment" mean that a particular feature, structure, or characteristic described in connection with such an embodiment is included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment", "in an embodiment", or "in an exemplary embodiment" used in various places throughout this specification do not necessarily all refer to the same embodiment, although they may. Further, a particular feature, structure, or characteristic can be combined in any suitable manner in one or more embodiments as will be apparent to those skilled in the art from this disclosure. Additionally, some embodiments described herein may include some features of other embodiments but may not include others, and combinations of features of different embodiments are intended to be within the scope of the present invention. For example, in the appended claims, any combination of the claimed embodiments can be used.

[0047] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference into this specification, and thereby each individual publication, published patent document, or patent application is specifically and individually shown as if incorporated by reference.

[0048] Overview Embodiments disclosed herein provide a targeting moiety that promotes transduction into the CNS through interaction with the transferrin receptor. Such a targeting moiety can be incorporated into a particle, such as a viral capsid delivery particle, to confer directionality to the delivery particle and promote transduction into the CNS. Examples of CNS tissue include brain and spinal cord tissue. Examples of CNS cell types include neurons and glial cells. Further embodiments disclosed herein provide a vector system that includes one or more vectors encoding an AAV capsid according to the embodiments described herein. Accordingly, embodiments disclosed herein provide compositions that can deliver cargo to the CNS vasculature with improved selectivity and efficiency. Embodiments disclosed herein also provide a vector system for generating and loading such delivery particles with cargo. Similarly, embodiments disclosed herein provide methods of using such compositions to target CNS endothelial cells in vitro and in vivo, for both therapeutic and research purposes.

[0049] Further features and advantages of the foregoing embodiments will be described further below.

[0050] Targeting Moiety and Compositions Thereof In an exemplary embodiment, the present specification provides a composition that includes a targeting moiety with enhanced tropism for endothelial cells of the CNS. The targeting moiety with enhanced tropism for endothelial cells of the CNS promotes, enhances, or improves binding to the CNS and, optionally, transduction into the CNS, compared to a native or wild-type targeting moiety. This targeting moiety can be directly conjugated to a delivered cargo, such as an oligonucleotide or a polypeptide. Alternatively, a targeting molecule can be incorporated into delivery particles to confer tropism for endothelial cells of the CNS to the delivery particles. Non-limiting examples of delivery particles include viral capsid particles. In such an embodiment, the targeting moiety can be incorporated into a viral capsid polypeptide such that the targeting moiety is incorporated into the assembled viral capsid. However, other particle delivery systems in which the targeting moiety can be incorporated or attached, for example, into exosomes or liposomes, are also envisioned and encompassed herein as alternative embodiments.

[0051] In one aspect, the present specification provides a composition, which comprises a targeting moiety effective to enhance transduction into the central nervous system tissue (CNS) via binding to the transferrin receptor (TFRC), and optionally further comprises a cargo bound to or associated with the targeting moiety. The targeting moiety that enhances transduction promotes, enhances, or improves binding to the CNS and optionally transduction into the CNS as compared to a native or wild-type targeting moiety. In one exemplary embodiment, the targeting moiety binds to the extracellular domain of TFRC. In one exemplary embodiment, the targeting moiety binds to one or more of the apical domain of the extracellular domain, the helical domain of the extracellular domain, and / or the protease-like domain of the extracellular domain. In one exemplary embodiment, the targeting moiety binds to the apical domain. In one exemplary embodiment, the n amino acid length is an amino acid sequence of length n. The length of the n amino acid length can be any length required for transducing the CNS. In an exemplary embodiment, the n amino acid length is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids in length. In one exemplary embodiment, the length of the n amino acid length motif is at least 7 amino acids. In one exemplary embodiment, a composition comprising a targeting moiety effective to enhance transduction into CNS tissue comprises an n amino acid length motif, the n amino acid length motif comprising or consisting of the amino acid sequence of Z1-X1-Z2-X2-X3-X4-X5, where Z1 is Y, F, or L, Z2 is S, R, or K, and X1 to X5 are independently selected amino acids. In one exemplary embodiment, X1 is optionally composed of A, S, or H, X2 is optionally composed of S, T, L, or I, X3 optionally contains N or G, X4 optionally contains G, and X5 is optionally composed of N, D, I, V, or R. In one exemplary embodiment, the targeting moiety comprises an n amino acid length motif, the n amino acid length motif comprising or consisting of the amino acid sequence of X1-H-X2-L-X3-X4-X5, where X1 to X5 are independently selected amino acids.

[0052] In an exemplary embodiment, an n-amino acid length can be used to enhance transduction in target cells, i.e., CNS cells and tissues. The increase in transduction efficiency (which may correspond to the targeting efficiency) by the n-amino acid length for cells can be compared to a composition that does not contain a target targeting moiety. For example, when one or more target targeting moieties are included in the composition, transduction and / or transduction efficiency can be increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. In an exemplary embodiment, the increase in transduction and / or transduction efficiency is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or more compared to a composition lacking the n-amino acid length. In one exemplary embodiment, transduction and / or transduction efficiency is increased or enhanced in endothelial cells, and in one exemplary embodiment, it is increased in endothelial cells of a vasculature, such as a central nervous system vasculature. In an embodiment, transduction and / or transduction efficiency is increased or enhanced in cells of the central nervous system. In an embodiment, transduction and / or transduction efficiency is increased or enhanced in neurons and glial cells. In one exemplary embodiment, a composition containing an n-amino acid length is selective for target cells compared to other cell types and / or other viral particles. As used herein, "selective" and "cell-selective" mean preferentially targeting a particular cell over other cell types. Preferably, the target targeting moiety is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or more, or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or more selective for the desired target (e.g., a cell, organ, system, e.g., CNS tissue) or set of targets compared to other targets or cells (e.g., CNS).In one exemplary embodiment, a composition comprising a target-directed moiety described herein has an uptake, delivery rate, transduction rate, efficiency, amount, or combination thereof in target cells (e.g., endothelial cells throughout the CNS, e.g., brain endothelium, the arterial-venous axis of the brain, retina, and spinal cord vasculature) that can be increased compared to other cell types (e.g., muscle cells) and / or other viral particles (e.g., AAV without a target-directed moiety) and other compositions that do not include the cell-selective n-amino acid long motif of the present invention.

[0053] In one exemplary embodiment, the n - amino acid - long motif is selected from the group consisting of YSRIGPN (SEQ ID NO: 14632), YSRLNMN (SEQ ID NO: 14301), YSRLNKD (SEQ ID NO: 16577), and YHRLSNN (SEQ ID NO: 16636). In one exemplary embodiment, the n - amino acid - long motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608). In one exemplary embodiment, the n - amino acid - long motif is PSATNGV (SEQ ID NO: 20486), QVSTNGI (SEQ ID NO: 16021), SYSSNGV (SEQ ID NO: 16234), HQSSNGV (SEQ ID NO: 15978), VGSINGI (SEQ ID NO: 16199), AMSTNGR (SEQ ID NO: 16000), SASTNGV (SEQ ID NO: 16127), YMSTNGV (SEQ ID NO: 16042), YYSSNGV (SEQ ID NO: 16206), VHSTNGI (SEQ ID NO: 16134), PLSTNGV (SEQ ID NO: 16233), VYSTNGI (SEQ ID NO: 16059), IISTNGV (SEQ ID NO: 16054), RSVSSNGV (SEQ ID NO: 20502), YKSSNGV (SEQ ID NO: 16123), FRSTNGV (SEQ ID NO: 16070), and / or FVSTNGV (SEQ ID NO: 11162) (including). In one exemplary embodiment, the n - amino acid length is selected from any of the amino acid sequences in Tables 1 - 13, or any combination thereof. In one exemplary embodiment, the n - amino acid - long motif is selected from peptides having any of the amino acid sequences of SEQ ID NOs: 10952 - 20481. In one exemplary embodiment, the target - directed moiety is part of a viral capsid protein comprising an AAV capsid protein (e.g., inserted between consecutive amino acids).

[0054] Transferrin receptor (TFRC) binding In a preferred embodiment, the target-directed moiety binds to the transferrin receptor. TFRC (i.e., the TfR1 protein encoded by the TFRC gene, or CD71) is composed of two types of receptors, TFRC1 (or cluster of differentiation 71 (CD71)) and TFRC2. TFRC2 is less common than TFRC1 and is mainly expressed in hepatocytes. TFRC1 binds to transferrin (TF) with high affinity and is generally expressed. TFRC1 is a type II transmembrane glycoprotein of approximately 90 kDa and is composed of approximately 760 amino acids. TFRC1 usually exists as a dimer linked by disulfide bonds on the cell surface (see Figure 4).

[0055] The TFRC1 domain contains an extracellular C-terminal domain (approximately 671 amino acids) and is composed of TF binding sites. The extracellular C-terminal domain is composed of three subdomains: the apical domain, the helical domain, and the protease-like domain (see Figure 4). Furthermore, the extracellular C-terminal domain is composed of three N-linked glycosylation sites at asparagine residues 251, 317, and 727, and one O-linked glycosylation site at threonine 104, which contribute to the proper function of the receptor. TFRC1 is further composed of a transmembrane domain (approximately 29 amino acids) and an intracellular N-terminal domain (approximately 61 amino acids). Instead of iron delivery via TF by TFRC1, iron uptake can also occur by H-ferritin binding via the apical domain. See also Candelaria, P.V.; et al. Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-Cancer Agents. Frontiers in Immunology, 2021, 12 (incorporated herein by reference).

[0056] In one exemplary embodiment, the targeting moiety binds to the extracellular domain of TFRC. In one exemplary embodiment, the targeting moiety binds to one or more of the apical domain, the helical domain, and / or the protease-like domain. In one exemplary embodiment, the targeting moiety binds to the apical domain.

[0057] engineered viral capsid As used herein, various embodiments of engineered viral capsids such as adeno-associated virus (AAV) capsids are described. Such capsids can be engineered to confer cell-selective tropism (e.g., CNS tissue-specific tropism and cell-specific tropism) to engineered viral particles. Engineered viral capsids can be lentiviral, retroviral, adenoviral, or AAV capsids. Engineered capsids can be included in engineered viral particles (e.g., engineered lentiviral particles, engineered retroviral particles, engineered adenoviral particles, or engineered AAV viral particles) to confer cell-selective tropism to the engineered viral particles. Engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein. Engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein, and such engineered viral capsid proteins can include one or more targeting moieties described elsewhere herein.

[0058] The engineered viral capsid can be a variant of the wild-type viral capsid. For example, in some embodiments, the engineered AAV capsid can be a variant of the wild-type AAV capsid. In some embodiments, the wild-type AAV capsid can be composed of VP1, VP2, VP3 capsid proteins, or combinations thereof. That is, the engineered AAV capsid can include one or more variants of the wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins. In some embodiments, the serotype of the reference wild-type AAV capsid can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In some embodiments, the serotype of the wild-type AAV capsid can be AAV-9. The engineered AAV capsid can have a tropism different from that of the reference wild-type AAV capsid.

[0059] In some embodiments, the targeting moiety is incorporated into a viral protein, such as a capsid protein, including but not limited to proteins of lentivirus, adenovirus, AAV, bacteriophage, retrovirus. In some embodiments, the targeting moiety is positioned between two amino acids of the viral protein such that the targeting moiety is outside of the viral capsid (i.e., present on its surface). In one exemplary embodiment, the targeting moiety disclosed herein can be inserted between two consecutive amino acids within a wild-type viral protein (VP) (or capsid protein) that includes a region that is exposed on the surface when incorporated into the viral capsid. In some embodiments, the targeting moiety can be inserted between two consecutive amino acids within a variable amino acid region of the viral capsid protein.

[0060] In some embodiments, the target-directed moiety can be inserted between two consecutive amino acids within a variable amino acid region of the AAV capsid protein. The core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (beta B-beta I) and an alpha helix (alpha A), which are conserved in autonomous parvovirus capsids (see, e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). The structurally variable regions (VRs), also called "loops," are present in surface loops that connect beta strands and cluster to create local changes on the capsid surface. AAV has 12 variable regions (also called hypervariable regions) (see, e.g., Weitzman and Linden. 2011. “Adeno-Associated Virus Biology.” In Snyder, R.O., Moullier, P. (eds.) Totowa, NJ: Humana Press). In one exemplary embodiment, one or more target-directed moieties can be inserted between two amino acids in one or more of the 12 variable regions of the wild-type AVV capsid protein. In one exemplary embodiment, one or more target-directed moieties can be inserted between two amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-III, VR-IX, VR-X, VR-XI, VR-XII, or combinations thereof, respectively. In one exemplary embodiment, the target-directed moiety is inserted or substituted in loop IV and / or loop VIII. In one exemplary embodiment, the target-directed moiety is IPFSRVNPDT (SEQ ID NO: 20285), LGFARTGAAD (SEQ ID NO: 20274), LGFTKSSGSD (SEQ ID NO: 20270), LRYSKTQGES (SEQ ID NO: 20266), SPYARSSAGV (SEQ ID NO: 20271), VGWSRLDLTT (SEQ ID NO: 20262).

[0061] In one exemplary embodiment, the engineered capsid can be a modified AAV1 capsid and can have a target-directed partial motif inserted after or near amino acid 590 (i.e., between amino acids 590 and 591). In one exemplary embodiment, the engineered capsid can be a modified AAV3 capsid and can have a target-directed partial motif inserted after or near amino acid 586. In one exemplary embodiment, the engineered capsid can be a modified AAV4 capsid and can have a target-directed partial motif inserted after or near amino acid 586. In one exemplary embodiment, the engineered capsid can be a modified AAV5 capsid and can have a target-directed partial motif inserted after or near amino acid 575. In one exemplary embodiment, the engineered capsid can be a modified AAV6 capsid and can have a target-directed moiety inserted after amino acids 585 and optionally Y705-731, T492V, K531E, or near them. In one exemplary embodiment, the engineered capsid can be a modified AAV8 capsid and can have a target-directed partial motif inserted after or near amino acids 585 and 590. In one exemplary embodiment, the engineered capsid can be a modified AAV9 capsid and can have a target-directed moiety inserted between amino acids 588 and 589. (Buning, H.; Srivastava, A. Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors. Molecular Therapy-Methods & Clinical Development 2019, 12, 248-265). In one exemplary embodiment, the engineered capsid can have a 7-amino acid-long motif inserted between amino acids 588 and 589 of the AAV9 viral protein. SEQ ID NO: 20506 is a reference AAV9 capsid sequence for at least referring to the above insertion site.In one exemplary embodiment, the engineered capsid can have a 7 - amino - acid - long motif inserted between two consecutive amino acids within amino acids 451 - 460 of the capsid protein of the AAV9 viral protein. SEQ ID NO: 20506 is a reference AAV9 capsid sequence for at least referring to the above - mentioned insertion site. Obviously, the targeting moiety can be inserted at similar positions in AAV viral proteins of other serotypes, such as, without limitation, the capsid polypeptides of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.74, AAVrh.10. In some embodiments as described above, the targeting moiety(ies) can be inserted between any two consecutive amino acids within the AAV viral protein, and in some embodiments, the insertion is made within a variable region.

[0062] In one exemplary embodiment, for the peptide into which the targeting moiety is inserted, 1, 2, 3, or 4 amino acids of the polypeptide preceding the insertion site can be replaced by the first 1, 2, 3, or 4 amino acids of the targeting moiety. Using AAV as another non - limiting example, one or more of the targeting moieties can be inserted, for example, between amino acids 588 and 589 of the AAV9 capsid polypeptide. The insert can replace amino acids 586, 587, and 588, such that the amino acid immediately preceding the targeting moiety after insertion is residue 585. Obviously, this principle can be applied to any other insertion situation and is not necessarily limited to the insertion between residues 588 and 589 of the AAV9 capsid or the insertion at the equivalent position of another AAV capsid. Further obviously, in some embodiments, the amino acids within the polypeptide into which the targeting moiety is inserted are not replaced by the targeting moiety. In one exemplary embodiment, the AAV capsid protein is selected from SEQ ID NO: 20506.

[0063] In some embodiments, in addition to the n-amino acid long motif(s), the target-directed moiety can include a polypeptide, polynucleotide, lipid, polymer, sugar, or a combination thereof.

[0064] The engineered viral capsid and / or capsid protein can be encoded by one or more engineered viral capsid polynucleotides. In some embodiments, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, an engineered lentiviral capsid polynucleotide, an engineered retroviral capsid polynucleotide, or an engineered adenoviral capsid polynucleotide. In some embodiments, the engineered viral capsid polynucleotide (e.g., an engineered AAV capsid polynucleotide, an engineered lentiviral capsid polynucleotide, an engineered retroviral capsid polynucleotide, or an engineered adenoviral capsid polynucleotide) can include a 3' polyadenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal.

[0065] In some embodiments, the engineered polynucleotide can be included in a polynucleotide configured to express an engineered capsid in a host cell system for viral particle production. The host cell system can also include a construct that expresses a recombinant viral genome, the recombinant viral genome including a transgene that encodes a polypeptide or nucleic acid, the transgene being operably linked to one or more regulatory sequences that promote expression of the transgene in a target cell, and such recombinant viral genomes include recombinant AAV genomes in which the transgene and regulatory sequences are flanked by AAV ITR sequences.

[0066] In some embodiments, the polynucleotide encoding the engineered AAV capsid can be included in a polynucleotide configured to express the engineered capsid in a host cell system for AAV viral particle production. The host cell system can also include a construct that expresses a recombinant AAV viral genome, the recombinant AAV viral genome including a transgene encoding a polypeptide or nucleic acid, the transgene being operably linked to one or more regulatory sequences that promote expression of the transgene in a target cell, and such recombinant AAV viral genomes including a recombinant AAV genome in which the transgene and regulatory sequences are flanked by AAV ITR sequences. In some embodiments, the polynucleotide encoding the engineered AAV capsid can be operably linked to a polyadenylation tail. In some embodiments, the polyadenylation tail can be an SV40 polyadenylation tail. In some embodiments, the polynucleotide encoding the AAV capsid can be operably linked to a promoter. In some embodiments, the regulatory sequence that controls expression of the transgene is a promoter, which can be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific for muscle (e.g., heart, skeletal, and / or smooth muscle), neurons and supporting cells (e.g., astrocytes, glial cells, Schwann cells, etc.), adipose, spleen, liver, kidney, immune cells, cerebrospinal fluid cells, synovial fluid cells, skin cells, cartilage, tendon, connective tissue, bone, pancreas, adrenal gland, blood cells, bone marrow cells, placenta, endothelial cells, and combinations thereof. In some embodiments, the promoter can be a constitutive promoter. Suitable tissue-specific promoters and constitutive promoters are described elsewhere in this specification, are generally known in the art, and are available commercially. Suitable neuron tissue / cell-specific promoters include, but are not limited to, the GFAP promoter (astrocytes), SYN1 promoter (neurons), and NSE / RU5’ (mature neurons).

[0067] Further Capsid Modifications In one exemplary embodiment, the viral capsid protein can include one or more mutations relative to the wild type. In one exemplary embodiment, the one or more mutations include the K449R substitution in the capsid polypeptide of AAV9 20507, or substitutions at similar positions in capsid polypeptides derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAVrh.10. In one exemplary embodiment, the K449R substitution AAV capsid is selected from SEQ ID NO: 20507.

[0068] In an exemplary embodiment, the viral capsid protein can include additional target-directed motifs in addition to the n-amino acid long motif of the present disclosure. Without being bound by theory, the additional target-directed moiety can be an antibody or a fragment thereof. In some embodiments, the additional target-directed moiety can be any molecule or composition that can recognize, bind to, adhere to, or interact with a binding partner that may be present on the surface of the target cell. Binding partners include, but are not limited to, nucleic acids, proteins, peptides, sugars, fats, or combinations thereof, or any other single or plural molecules present on the surface of the target cell. In some embodiments, the binding partner is specific to a cell type or cell state, or is specific to a group of related cell types or cell states. In some embodiments, the binding partner is a receptor, channel, or other complex present on the surface of the target cell. These additional target-directed moieties can be used, for example, to target specific cell types or cell states within the set of target cells targeted by the n-amino acid long motif. As used herein, "cell state" is used to describe a transient aspect of a cell's identity. A cell state can be considered a transient profile of a cell's properties or phenotype. A cell state can occur transiently as a one-way progression over time (e.g., during differentiation or after environmental stimulation) or as a fluctuating state that is not necessarily one-way (where the cell can return to its original state). The fluctuating process can be periodic (e.g., the cell cycle or circadian rhythm) or can transition between states without a defined order (e.g., by probabilistic or environmentally controlled molecular events). These time-dependent processes can occur transiently within a stable cell type (such as in the case of a transient environmental response) or can lead to new different types (such as in the case of differentiation). See, for example, Wagner et al., 2016. Nat Biotechnol. 34(11):1145-1160.

[0069] In some embodiments, the additional targeting moiety is or comprises a peptide or polypeptide. In some embodiments, the additional targeting moiety is or comprises an antibody or a fragment thereof. Exemplary antibodies and fragments thereof are described in more detail elsewhere in this specification; see, for example, the description regarding exemplary cargo. In some embodiments, the additional targeting moiety is or comprises an aptamer. In some embodiments, the additional targeting moiety is or comprises a small molecule. In some embodiments, the additional targeting moiety is or comprises a nucleic acid (e.g., DNA or RNA). In some embodiments, the additional targeting moiety is or comprises a receptor. In some embodiments, the additional targeting moiety is or comprises a carbohydrate (e.g., sugar). In some embodiments, the additional targeting moiety is or comprises a lipid. In some embodiments, the additional targeting moiety is an engineered protein scaffold. In some embodiments, the additional targeting moiety is an affibody. In some embodiments, the additional targeting moiety is an antibody mimetic. In some embodiments, the additional targeting moiety is an engineered binding protein, such as a designed ankyrin repeat protein (DARPin) (see, e.g., Pluckthun et al., Annu. Rev. Pharmacol. Toxicol. (2015) 55(1):489-511), an avimer (see, e.g., Silverman et al., Nat. Biotechnol. (2005) 23(12):1556-1561 and Jeong et al., Nat. Biotechnol. (2005) 23(12):1493-1494), or an affibody (see, e.g., Nord et al., Nat. Biotechnol. (1997) 15(8):772-777). In an exemplary embodiment, the additional targeting moiety is a receptor ligand or a binding protein.In some embodiments, the additional targeting moiety is attached or bound to the capsid surface. In some embodiments, the additional targeting moiety is encoded by a vector that produces the capsids of the invention described herein.

[0070] Engineered vectors and engineered vector systems Also provided herein are vectors and vector systems that can include one or more of the engineered polynucleotides described herein that encode one or more of the targeted moieties of the invention, including but not limited to engineered viral polynucleotides (e.g., polynucleotides encoding engineered AAV capsid proteins). In a preferred embodiment, provided herein is a vector system that includes one or more vectors encoding a targeting moiety effective to enhance transduction into central nervous system tissue (CNS), optionally further including a cargo that binds or associates with the targeting moiety, or further including a construct encoding a cargo (including a recombinant viral genome containing a transgene). In a preferred embodiment, the targeting moiety encoded by the vector system binds to the transferrin receptor (TFRC). As used in this context, an engineered viral capsid polynucleotide refers to one or more of the polynucleotides described herein that can encode an engineered viral capsid as described elsewhere herein, and / or polynucleotides (s) that can encode one or more of the engineered viral capsid proteins described elsewhere herein. Further, when a vector includes an engineered viral capsid polynucleotide as described herein, the vector can also be referred to and considered, even in the absence of such specific recitation, as an engineered vector or an engineered vector system. In embodiments, the vector can include one or more polynucleotides encoding one or more elements of the engineered viral capsids described herein. Vectors and their systems can be useful for the production of bacteria, fungi, yeast, plant cells, animal cells, and transgenic animals that can express one or more components of the engineered viral capsids, particles, or other compositions described herein. Within the scope of the present disclosure are vectors that include one or more of the polynucleotide sequences described herein. One or more of the polynucleotides that are part of the engineered viral capsids and their systems described herein can be included in a vector or vector system.

[0071] In an exemplary embodiment, the vector used for the production of rAAV disclosed herein contains the rep gene and the cap gene. The rep gene typically encodes the Rep78, Rep68, Rep52, and Rep40 of a single ORF. These replication factors assist in the replication of the AAV genome and the assembly of viral particles. The cap gene typically also encodes three capsid proteins of a single ORF (i.e., virion protein 1 (VP1), VP2, and VP3). Furthermore, the three capsid proteins are controlled by transcription from the start codon (ACG) and alternative splicing. The cap gene also encodes the assembly activation protein (AAP) of an in-frame shifted ORF. AAP is essential for the assembly of the capsid.

[0072] In some embodiments, the vector can comprise an engineered viral (e.g., AAV) capsid polynucleotide having a 3' polyadenylation signal. In some embodiments, the 3' polyadenylation signal is the SV40 polyadenylation signal. In some embodiments, the vector does not have a splice regulatory element. In some embodiments, the vector comprises one or more minimal splice regulatory elements. In some embodiments, the vector can further comprise a modified splice regulatory element, which is modified such that the splice regulatory element is inactivated. In some embodiments, the modified splice regulatory element is a polynucleotide sequence sufficient to induce splicing between the rep protein polynucleotide and the engineered viral (e.g., AAV) capsid protein variant polynucleotide. In some embodiments, the polynucleotide sequence can be sufficient to induce splicing as a splice acceptor or a splice donor. In some embodiments, the viral (e.g., AAV) capsid polynucleotide is an engineered viral (e.g., AAV) capsid polynucleotide as described elsewhere herein. In some embodiments, the vector does not comprise one or more minimal splice regulatory elements, modified splice regulatory factors, splice acceptors, and / or splice donors.

[0073] Using a vector and / or vector system, for example, one or more engineered virus (e.g., AAV) capsids and / or other polynucleotides can be expressed intracellularly in cells such as producer cells, and other compositions can be generated including engineered virus (e.g., AAV) particles, and / or engineered virus (e.g., AAV) capsids or other compositions including the n - amino acid - long motif of the invention described elsewhere herein (e.g., polypeptides, particles, etc.). Other uses of the vectors and vector systems described herein are also within the scope of the disclosure. Generally, throughout this specification, this term refers to a tool that enables or facilitates the transfer of an entity from one environment to another. In the context that will be apparent to those skilled in the art, the technical term "vector" can refer to a nucleic acid molecule that can transport another nucleic acid to which it is linked. A vector is a replicon such as a plasmid, phage, cosmid, etc., into which another DNA segment can be inserted to effect the replication of the inserted segment. Generally, a vector becomes replicable when associated with appropriate regulatory elements.

[0074] Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded, nucleic acid molecules containing one or more free ends, nucleic acid molecules without free ends (e.g., circular), nucleic acid molecules containing DNA, RNA, or both, and various other polynucleotides known in the art. One type of vector is a "plasmid". This refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning methods. Another type of vector is a viral vector, where a DNA sequence or RNA sequence derived from a virus is present within the vector for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by the virus for transfection of host cells. Certain vectors can replicate themselves in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and are thereby replicated along with the host genome. Furthermore, certain vectors can induce the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". General expression vectors useful in recombinant DNA technology are often in the form of plasmids.

[0075] A recombinant expression vector can contain the nucleic acid (e.g., polynucleotide) of the present invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vector contains one or more regulatory elements that are operably linked to the nucleic acid sequence to be expressed and can be selected based on the host cell for expression. "Operably linked" and "operably bound" within the recombinant expression vector are used synonymously herein and are further defined elsewhere in this specification. With respect to a vector, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) such that expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or, when the vector is introduced into a host cell, in the host cell) is enabled.

[0076] In some embodiments, the vector is a bicistronic vector. In some embodiments, a bicistronic vector can be used for one or more elements of the engineered viral (e.g., AAV) capsid system described herein. In some embodiments, expression of the elements of the engineered viral (e.g., AAV) capsid system described herein can be driven by an appropriate constitutive promoter or tissue-specific promoter. When the element of the engineered viral (e.g., AAV) capsid system is RNA, its expression can be driven by a Pol III promoter such as the U6 promoter. In some embodiments, these two are combined.

[0077] Cell-based vector amplification and expression The vector can be designed to express one or more elements of an engineered viral (e.g., AAV) capsid system or other compositions (e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof) containing the n-amino acid long motif of the present invention within a suitable host cell. In one exemplary embodiment, a composition containing a targeting moiety effective to enhance transduction into CNS tissue contains an n-amino acid long motif, where the n-amino acid long motif comprises or consists of the amino acid sequence of Z1-X1-Z2-X2-X3-X4-X5, where Z1 is Y, F, or L, Z2 is S, R, or K, and X1 to X5 are independently selected amino acids. In one exemplary embodiment, X1 is optionally composed of A, S, or H, X2 is optionally composed of S, T, L, or I, X3 optionally contains N or G, X4 optionally contains G, and X5 is optionally composed of N, D, I, V, or R. The n-amino acid long motif is selected from the group consisting of YSRIGPN (SEQ ID NO: 14632), YSRLNMN (SEQ ID NO: 14301), YSRLNKD (SEQ ID NO: 16577), and YHRLSNN (SEQ ID NO: 1663). In one exemplary embodiment, the targeting moiety contains an n-amino acid long motif, where the n-amino acid long motif comprises or consists of the amino acid sequence of X1-H-X2-L-X3-X4-X5, where X1 to X5 are independently selected amino acids. In one exemplary embodiment, the n-amino acid long motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608).In one exemplary embodiment, the n-amino acid long motif is PSATNGV (SEQ ID NO: 20486), QVSTNGI (SEQ ID NO: 16021), SYSSNGV (SEQ ID NO: 16234), HQSSNGV (SEQ ID NO: 15978), VGSINGI (SEQ ID NO: 16200), AMSTNGR (SEQ ID NO: 16000), SASTNGV (SEQ ID NO: 16127), YMSTNGV (SEQ ID NO: 16042), YYSSNGV (SEQ ID NO: 16206), VHSTNGI (SEQ ID NO: 16134), PLSTNGV (SEQ ID NO: 16233), VYSTNGI (SEQ ID NO: 16059), IISTNGV (SEQ ID NO: 16054), RSVSSNGV (SEQ ID NO: 20502), YKSSNGV (SEQ ID NO: 16123), FRSTNGV (SEQ ID NO: 16070), and / or FVSTNGV (SEQ ID NO: 11162) (including).

[0078] In some embodiments, a suitable host cell is a prokaryotic cell. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. The vector may be virus-based or non-virus-based. In some embodiments, a suitable host cell is a eukaryotic cell. In some embodiments, a suitable host cell is a suitable bacterial cell. Suitable bacterial cells include, but are not limited to, bacterial cells derived from bacteria of the Escherichia coli species. Many strains of E. coli suitable for vector expression are well known in the art. These include, but are not limited to, Pir1, Stbl2, Stbl3, Stbl4, TOP10, XL1Blue, XL10Gold. In some embodiments, the host cell is a suitable insect cell. Suitable insect cells include cells derived from Spodoptera frugiperda. Suitable strains of S. frugiperda cells include, but are not limited to, Sf9 and Sf21. In some embodiments, the host cell is a suitable yeast cell. In some embodiments, the yeast cell may be derived from Saccharomyces cerevisiae. In some embodiments, the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed for expressing vectors. Suitable mammalian cells include, but are not limited to, HEK293, Chinese hamster ovary cells (CHO), mouse myeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH3T3, L929, N2a, MCF-7, Y79, SO-Rb50, HepG G2, DIKX-X11, J558L, baby hamster kidney cells (BHK), and chicken embryo fibroblast cells (CEF). Suitable host cells are further described in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

[0079] In some embodiments, the vector can be a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6:229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30:933-943), pJRY88 (Schultz et al., 1987. Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, California), and picZ (Invitrogen Corp, San Diego, California). As used herein, a "yeast expression vector" refers to a nucleic acid that contains one or more sequences encoding RNA and / or polypeptides, and further may contain any desired elements that control the expression of the nucleic acid(s), as well as any elements that enable the replication and maintenance of the expression vector within yeast cells. Many suitable yeast expression vectors and their characteristics are known in the art, for example, various vectors and techniques are shown in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press, New York, 2007) and Buckholz, R.G. and Gleeson, M.A. (1991) Biotechnology (NY) 9(11):1067-72. Yeast vectors can include, without limitation, centromere (CEN) sequences, autonomously replicating sequences (ARS), a promoter such as an RNA polymerase III promoter operably linked to the sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., a nutritional marker, an antibiotic marker, or other selectable marker). Examples of expression vectors for use in yeast include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids, yeast replicating plasmids, shuttle vectors, episomal plasmids, and the like.

[0080] In some embodiments, the vector is a baculovirus vector or a baculovirus expression vector and may be suitable for the expression of polynucleotides and / or proteins in insect cells. Baculovirus vectors that can be used for protein expression in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39). rAAV (recombinant adeno-associated virus) vectors are preferably produced in insect cells grown in serum-free suspension culture, such as Spodoptera frugiperda Sf9 insect cells. Serum-free insect cells can be purchased from vendors such as Sigma Aldrich (EX-CELL405).

[0081] In some embodiments, the vector is a mammalian expression vector. In some embodiments, the mammalian expression vector can express one or more polynucleotides and / or polypeptides in mammalian cells. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. Nature 329:840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6:187-195). Mammalian expression vectors can include one or more appropriate regulatory elements that can control the expression of one or more polynucleotides and / or proteins in mammalian cells. For example, commonly used promoters are obtained from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and other promoters known in the art disclosed herein. Details of appropriate regulatory elements are described elsewhere in this specification.

[0082] For other expression vectors and vector systems suitable for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0083] In some embodiments, the recombinant mammalian expression vector can preferentially induce the expression of nucleic acids in specific cell types (e.g., tissue-specific regulatory elements are used for the expression of nucleic acids). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific, Pinkert, et al., 1987. Genes Dev. 1:268-277), the lymphoid-specific promoter (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), particularly the promoter of the T cell receptor (Winoto and Baltimore, 1989. EMBO J. 8:729-733), and immunoglobulins (Baneiji, et al., 1983. Cell 33:729-740, Queen and Baltimore, 1983. Cell 33:741-748), the neuron-specific promoter (e.g., the neurofilament promoter, Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), the pancreatic-specific promoter (Edlund, et al., 1985. Science 230:912-916), and the mammary gland-specific promoter (e.g., the milk whey promoter, U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters are also included, such as the mouse hox promoter (Kessel and Gruss, 1990. Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546). U.S. Patent No. 6,750,059 is referred to with respect to these prokaryotic and eukaryotic vectors, and the entire content thereof is incorporated herein by reference. In other embodiments, viral vectors can be utilized, and U.S. Patent Application No. 13 / 092,085 is referred to with respect to this, and the entire content thereof is incorporated herein by reference.Tissue-specific regulatory elements are known in the art, and U.S. Patent No. 7,776,321 is hereby incorporated by reference in its entirety. In some embodiments, the regulatory element can be operably linked to a transgene within a recombinant genome packaged by an engineered AAV capsid system, thereby enabling tissue-specific driving of the expression of one or more elements of the transgene delivered by the viral vectors described herein.

[0084] The vector can be introduced into and propagated in a prokaryote or prokaryotic cell. In some embodiments, a prokaryote is used to amplify copies of the vector for introduction into eukaryotic cells or as an intermediate vector in the production of vectors for introduction into eukaryotic cells (e.g., amplifying a plasmid as part of a viral vector packaging system). In some embodiments, for example, to obtain a source of one or more proteins for delivery to a host cell or host organism, a prokaryote is used to amplify copies of the vector and express one or more nucleic acids.

[0085] In some embodiments, the vector is a fusion vector or a fusion expression vector. In some embodiments, the fusion vector adds several amino acids to the protein encoded therein (e.g., the amino terminus, carboxy terminus, or both of the recombinant protein). Such fusion vectors can serve one or more purposes, for example, (i) increasing the expression of the recombinant protein, (ii) enhancing the solubility of the recombinant protein, and (iii) assisting in the purification of the recombinant protein by acting as a ligand in affinity purification. In some embodiments, the expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carried out in E. coli using a vector containing a constitutive promoter or an inducible promoter that induces the expression of fusion or non-fusion polynucleotides and / or proteins. In some embodiments, the fusion expression vector can contain a protein cleavage site. The protein cleavage site can be introduced at the junction of the fusion vector backbone or other fusion part and the recombinant polynucleotide or protein, enabling the separation of the recombinant polynucleotide or protein from the fusion vector backbone or other fusion part after the purification of the fusion polynucleotide or protein. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase. Examples of fusion expression vectors include pGEX (Pharmacia Biotech Inc, Smith and Johnson, 1988 Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A to the target recombinant protein, respectively.Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0086] In some embodiments, one or more vectors that induce the expression of one or more elements of the engineered viral (e.g., AAV) capsid system or other compositions containing the n-amino acid length described herein are introduced into a host cell, whereby, by the expression of the elements of the engineered delivery system described herein, the formation of an engineered viral (e.g., AAV) capsid system or other composition (including, but not limited to, engineered gene delivery agent particles described in more detail elsewhere herein) containing the n-amino acid length motif described herein is induced. For example, different elements of an engineered viral (e.g., AAV) capsid system or other composition containing the n-amino acid length motif described herein can be operably linked to different regulatory elements of separate vectors. RNA(s) of different elements of the engineered delivery system described herein can be delivered to an animal or mammal or its cells to produce an animal or mammal or its cells that constitutively or inducibly or conditionally express different elements of an engineered viral (e.g., AAV) capsid system or other composition containing the n-amino acid length motif described herein. It either incorporates one or more elements of an engineered viral (e.g., AAV) capsid system or other composition containing the n-amino acid length motif described herein, or contains one or more cells that incorporate and / or express one or more elements of an engineered viral (e.g., AAV) capsid system or other composition containing the n-amino acid length motif described herein.

[0087] In some embodiments, two or more elements expressed from the same or different regulatory element(s) can be combined in a single vector, and one or more additional vectors can provide any components of the system not included in the first vector. The engineered polynucleotides of the invention combined in a single vector can be arranged in any suitable orientation, for example, one element can be arranged 5’ (“upstream”) or 3’ (“downstream”) relative to a second element. The coding sequence of one element can be arranged on the same or opposite strand of the coding sequence of the second element and oriented in the same or opposite direction. In some embodiments, a single promoter drives the expression of a transcript encoding one or more viral (e.g., AAV) capsid proteins or other compositions comprising the n-amino acid length motif described herein. It is incorporated within one or more intron sequences (e.g., within different introns respectively, two or more within at least one intron, or all within a single intron). In some embodiments, the engineered polynucleotides of the invention (including, but not limited to, engineered viral polynucleotides) are operably linked to the same promoter and can be expressed from the same promoter.

[0088] Vector Features Vectors can include additional features. Such features can impart one or more functionalities to the vector, the polynucleotide being delivered, the viral particles generated therefrom, or the polypeptides expressed therefrom. Such features include, but are not limited to, regulatory elements, selectable markers, molecular identifiers (e.g., molecular barcodes), stabilizing elements, and the like. As will be apparent to those skilled in the art, the design of expression vectors and the additional features included can depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like.

[0089] Regulatory Elements In embodiments, the polynucleotides and / or vectors described herein (including, but not limited to, the engineered AAV capsid polynucleotides of the invention) can include one or more regulatory elements that can be operably linked to the polynucleotide. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and polyU sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that induce constitutive expression of nucleotide sequences in many host cell types, and those that induce expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can predominantly induce expression in a desired tissue such as muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, brain), or a particular cell type (e.g., lymphocytes). Regulatory elements can also induce expression in a time-dependent manner such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not also be tissue-specific or cell-type specific. In some embodiments, the vector includes one or more pol III promoters (e.g., 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 promoter and the H1 promoter.Examples of pol II promoters include, but are not limited to, the Rous sarcoma virus (RSV) LTR promoter of retrovirus (optionally with RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with CMV enhancer) (see, for example, Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter. Further, the term "regulatory element" includes enhancer elements such as WPRE, CMV enhancer, the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988), SV40 enhancer, and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).

[0090] In some embodiments, the regulatory sequence can be the regulatory sequence described in U.S. Patent No. 7,776,321, U.S. Patent Publication No. 2011 / 0027239, and PCT International Publication No. 2011 / 028929. All of these contents are incorporated herein by reference. In some embodiments, the vector can include a minimal promoter. In some embodiments, the minimal promoter is the Mecp2 promoter, the tRNA promoter, or U6. In further embodiments, the minimal promoter is tissue-specific. In some embodiments, the lengths of the vector polynucleotide, the minimal promoter, and the polynucleotide sequence are less than 4.4 Kb.

[0091] To express a polynucleotide, the vector can include one or more transcriptional and / or translational start regulatory sequences (e.g., a promoter) that induce transcription of the gene and / or translation of the encoded protein in the cell. In some embodiments, a constitutive promoter may be used. Constitutive promoters suitable for mammalian cells are generally known in the art and include, but are not limited to, SV40, CAG, CMV, EF-1α, β-actin, RSV, and PGK. Constitutive promoters suitable for bacterial cells, yeast cells, and fungal cells are generally known in the art, such as the T-7 promoter for bacterial expression and the alcohol dehydrogenase promoter for yeast expression.

[0092] In some embodiments, the regulatory element can be a regulatory promoter. A "regulatory promoter" refers to a promoter that induces gene expression in a temporally and / or spatially controlled manner, rather than constitutively, and includes tissue-specific promoters, tissue-selective promoters, and inducible promoters. In some embodiments, the regulatory promoter is a tissue-specific promoter as described elsewhere herein above. Regulatory promoters include conditional promoters and inducible promoters. In some embodiments, a conditional promoter can be used to induce the expression of a polynucleotide in a specific cell type, under specific environmental conditions, and / or during a specific developmental stage. Suitable tissue-specific promoters include, but are not limited to, liver-specific promoters (e.g., APOA2, SERPIN A1 (hAAT), CYP3A4, and MIR122), pancreatic cell promoters (e.g., INS, IRS2, Pdx1, Alx3, Ppy), heart-specific promoters (e.g., Myh6 (alpha MHC), MYL2 (MLC-2v), TNI3 (cTnl), NPPA (ANF), Slc8a1 (Ncx1)), central nervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3 beta)), skin cell-specific promoters (e.g., FLG, K14, TGM3), immune cell-specific promoters (e.g., ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cell-specific promoters (e.g., Pbsn, Upk2, Sbp, Fer1l4), endothelial cell-specific promoters (e.g., ENG), pluripotent cell and germ layer cell-specific promoters (e.g., Oct4, NANOG, synthetic Oct4, T brachyury, NES, SOX17, FOXA2, MIR122), and muscle cell-specific promoters (e.g., desmin). Other tissue-specific promoters and / or cell-specific promoters are described elsewhere herein and are also generally known in the art and are within the scope of the present disclosure.

[0093] An inducible / conditional promoter can be a positive inducible / conditional promoter (e.g., a promoter that activates the transcription of a polynucleotide by appropriate interaction with an activated activator or inducer (compound, environmental condition, or other stimulus)). Alternatively, the inducible / conditional promoter can be a negative / conditionally inducible promoter (e.g., a promoter that is repressed until the repressor condition of the promoter is removed (e.g., bound by a repressor) (e.g., an inducer binds to the repressor bound to the promoter and stimulates the release of the promoter by the repressor or stimulates the removal of a chemical repressor from the promoter environment)). The inducer can be a compound, environmental condition, or other stimulus. Thus, the inducible / conditional promoter can respond to any suitable stimulus such as chemical, biological, or other molecular factors, temperature, light, and / or pH. Suitable inducible / conditional promoters include, but are not limited to, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn, XVE / OlexA, GVG, and pOp / LhGR.

[0094] In some embodiments, the vector or system thereof can include one or more elements capable of moving and / or expressing the engineered polynucleotide of the invention (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) in and / or at a particular cellular component or organelle. Such organelles include, but are not limited to, nucleus, ribosome, endoplasmic reticulum, Golgi apparatus, chloroplast, mitochondrion, vacuole, lysosome, cytoskeleton, cell membrane, cell wall, peroxisome, centrosome, etc.

[0095] Selectable Markers and Tags One or more of the engineered polynucleotides of the invention (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) can be operably linked, fused, or modified to include a polynucleotide that encodes a selectable marker or tag, or is a selectable marker or tag (the marker or tag can be a polynucleotide or a polypeptide). In some embodiments, a polynucleotide encoding a polypeptide selectable marker can be incorporated into an engineered polynucleotide of the invention (e.g., an engineered viral (e.g., AAV) capsid polynucleotide), such that upon translation, the selectable marker polypeptide is inserted between two amino acids between the N-terminus and C-terminus of the engineered polypeptide (e.g., an engineered AAV capsid polypeptide), or inserted at the N-terminus and / or C-terminus of the engineered polypeptide (e.g., an engineered AAV capsid polypeptide). In some embodiments, the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI).

[0096] As will be apparent, a polynucleotide encoding such a selectable marker or tag can be incorporated into a polynucleotide encoding one or more components of the engineered AAV capsid system described herein in a manner appropriate to enable expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be readily apparent to those of skill in the art in view of the present disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of the present disclosure.

[0097] Suitable selectable markers and tags include, but are not limited to, the following: affinity tags (e.g., chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag), solubilizing tags (e.g., thioredoxin (TRX) and poly(NANP), MBP, and GST), chromatographic tags (e.g., those consisting of polyanionic amino acids such as the FLAG tag), epitope tags (e.g., V5 tag, Myc tag, HA tag, and NE tag), protein tags that enable specific enzyme modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging), DNA and / or RNA segments containing restriction enzyme or other enzyme cleavage sites, DNA segments encoding products that confer resistance to otherwise toxic compounds including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT), DNA segments and / or RNA segments encoding products that are normally lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers), DNA segments and / or RNA segments encoding easily distinguishable products (e.g., phenotypic markers such as β-galactosidase, GUS, fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), luciferase, and cell surface proteins), polynucleotides that can generate one or more new primer sites for PCR (e.g., juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences that are not or are acted upon by restriction endonucleases or other DNA modifying enzymes, chemical substances, epitope tags (e.g., GFP, FLAG tag, His tag), and DNA sequences that make molecular barcodes or unique molecular identifiers (UMIs), DNA sequences required for specific modifications (e.g., methylation) that enable identification. Other suitable markers will be apparent to those skilled in the art.

[0098] Selectable markers and tags can be operably linked to one or more components of the engineered AAV capsid system or other compositions and / or systems described herein via a suitable linker (e.g., a short-length glycine linker or glycine-serine linker from GS or GG to (GGGGG)3 (SEQ ID NO: 20482) or (GGGGGS)3 (SEQ ID NO: 20483)). Other suitable linkers are described elsewhere in this specification.

[0099] A vector or vector system can include one or more polynucleotides encoding one or more n-amino acid lengths. In some embodiments, the polynucleotide encoding the n-amino acid length can be included in a vector or vector system, such as a viral vector system, such that the polynucleotide is expressed within and / or on the generated virus particle(s), and the virus particle(s) can target specific cells, tissues, organs, etc. In some embodiments, the polynucleotide encoding the n-amino acid length can be included in a vector or vector system such that the engineered polynucleotide(s) of the invention (e.g., the engineered virus (e.g., AAV) capsid polynucleotide(s)) and / or the product expressed therefrom includes the n-amino acid length and can target specific cells, tissues, organs, etc. In some embodiments, such as non-viral carriers, the n-amino acid length can be attached to a carrier (e.g., a polymer, lipid, inorganic molecule, etc.), and the carrier and any attached or bound engineered polynucleotide(s), engineered polypeptide, or other composition of the invention described herein can be targeted to specific cells, tissues, organs, etc. In some embodiments, the specific cell is a CNS cell.

[0100] Cell-free vectors and polynucleotide expression In some embodiments, the polynucleotide(s) encoding the n - amino - acid - long motif of the present invention can be expressed from a vector or an appropriate polynucleotide in a cell - free in vitro system. In some embodiments, the polynucleotide encoding one or more features of the engineered AAV capsid system can be expressed from a vector or an appropriate polynucleotide in a cell - free in vitro system. In other words, the polynucleotide can be transcribed in vitro and, if necessary, translated. In vitro transcription / translation systems and appropriate vectors are generally known in the art and are commercially available. Generally, in vitro transcription systems and in vitro translation systems each repeat the processes of RNA synthesis and protein synthesis outside of a cellular environment. Examples of vectors and appropriate polynucleotides for in vitro transcription include T7, SP6, T3 promoter regulatory sequences that can be recognized and acted upon by an appropriate polymerase to transcribe the polynucleotide or vector.

[0101] In vitro translation can be either stand-alone (e.g., translation of purified polynucleotides) or linked / coupled to transcription. In some embodiments, a cell-free (or in vitro) translation system can include extracts from rabbit reticulocytes, wheat germ, and / or Escherichia coli. The extracts can include various macromolecular components necessary for the translation of exogenous RNA (e.g., 70S or 80S ribosomes, tRNA, aminoacyl tRNA, synthetases, initiation factors, elongation factors, termination factors, etc.). During the translation reaction, other components can also be included or added. Such components include amino acids, energy sources (ATP, GTP), energy regeneration systems (creatine phosphate and creatine phosphokinase (eukaryotic)) (phosphoenolpyruvate and pyruvate kinase in the case of bacterial systems), and other cofactors (Mg2+, K+, etc.). As described above, in vitro translation can use RNA or DNA as starting materials. In some translation systems, an RNA template can be used as the starting material (e.g., reticulocyte lysate and wheat germ extract). In some translation systems, a DNA template can be used as the starting material (e.g., E. coli-based systems). In these systems, transcription and translation are coupled, where first DNA is transcribed into RNA, and then the RNA is translated. Appropriate standard and coupled cell-free translation systems are generally known in the art and are commercially available.

[0102] Codon Optimization of Vector Polynucleotides As described elsewhere in this specification, the polynucleotide encoding the n - amino acid - length motif of the invention and / or other polynucleotides described herein, or the transgene contained within the recombinant AAV genome, can be codon - optimized. In some embodiments, the polynucleotides of the engineered AAV capsid system described herein can be codon - optimized. In some embodiments, one or more polynucleotides contained in the vectors (“vector polynucleotides”) described herein can be codon - optimized, in addition to the polynucleotide encoding an optionally codon - optimized n - amino acid - length motif (including, but not limited to, the embodiments of the engineered AAV capsid system described herein). Generally, codon optimization refers to the process of altering a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit specific biases for particular codons of a particular amino acid. Codon bias (the difference in codon usage frequency among organisms) often correlates with the translational efficiency of messenger RNA (mRNA), which is thought to depend, inter alia, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of tRNAs selected within a cell usually reflects the codons most frequently used in peptide synthesis. Thus, based on codon optimization, genes can be adjusted for optimal gene expression in a particular organism. Codon usage frequency tables are readily available, for example, in the Codon Usage Database available at www.kazusa.or.jp / codon / , and those tables can be updated in various ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).Computer algorithms are also available for codon-optimizing specific sequences for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) within the sequence encoding the Cas protein targeting DNA / RNA correspond to the codons most frequently used for a particular amino acid. For codon usage frequencies in yeast, see the online Yeast Genome database available at www.yeastgenome.org / community / codon_usage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar 25;257(6):3026-31. For codon usage frequencies in plants, including algae, see Codon usage in higher plants, green algae, and cyanobacteria, Campbell and Gowri, Plant Physiol. 1990 Jan;92(1):1-11, and Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan 25;17(2):477-98, or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton BR, J Mol Evol. 1998 Apr;46(4):449-59.

[0103] Vector polynucleotides can be codon-optimized for expression in specific cell types, tissue types, organ types, and / or subject types. In some embodiments, the codon-optimized sequence is a sequence optimized for expression in eukaryotes, such as humans (i.e., a sequence optimized for expression in humans or human cells), or a sequence optimized for another eukaryote, such as another animal (e.g., a mammal or a bird) as described elsewhere herein. Such codon-optimized sequences are within the scope of those skilled in the art considering the description herein. In some embodiments, the polynucleotide is codon-optimized for a specific cell type. Such cell types include, but are not limited to, epithelial cells (including skin cells, inner layer cells of the digestive tract, inner layer cells of other hollow organs), nerve cells (nerves, brain cells, spinal cord cells, nerve support cells (e.g., astrocytes, glial cells, Schwann cells, etc.)), muscle cells (e.g., cardiac muscle, smooth muscle cells, skeletal muscle cells), connective tissue cells (fat and other soft tissue filling cells, bone cells, tendon cells, chondrocytes), blood cells, stem cells and other progenitor cells, immune system cells, germ cells, and combinations thereof. Such codon-optimized sequences are within the scope of those skilled in the art considering the description herein. In some embodiments, the polynucleotide is codon-optimized for a specific tissue type. Such tissue types include, but are not limited to, muscle tissue, connective tissue, nerve tissue, epithelial tissue, etc. Such codon-optimized sequences are within the scope of those skilled in the art considering the description herein. In some embodiments, the polynucleotide is codon-optimized for a specific organ. Such organs include, but are not limited to, muscle, skin, intestine, liver, spleen, brain, lung, stomach, heart, kidney, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinations thereof. Such codon-optimized sequences are within the scope of those skilled in the art considering the description herein.

[0104] In some embodiments, the vector polynucleotide is codon-optimized for expression in specific cells such as prokaryotic or eukaryotic cells. The eukaryotic cells may be cells derived from specific organisms such as plants or mammals, and such organisms include, but are not limited to, humans, or non-human eukaryotes, animals, or mammals (e.g., mice, rats, rabbits, dogs, livestock, or non-human mammals or primates) as shown herein.

[0105] Non-viral vectors and non-viral carriers In some embodiments, the vector is a non-viral vector or a non-viral carrier. In some embodiments, the non-viral vector may have the advantage(s) of being less toxic and / or immunogenic and / or having higher biosafety compared to viral vectors. As used in this context herein, the technical term "non-viral vector and non-viral carrier" refers to molecules and / or compositions (excluding nucleotides delivered and / or expressed by the non-viral vector) that are not based on one or more components of a virus or viral genome. Such molecules and / or compositions can attach to, incorporate into, bind to, and / or interact with the engineered capsid polynucleotides (e.g., engineered AAV capsid polynucleotides) or other compositions of the invention, and can transport polynucleotides into cells and / or express polynucleotides. Obviously, this does not exclude including virus-based polynucleotides to be delivered. For example, if the delivered gRNA targets viral components and it is inserted or bound to a non-viral vector or carrier, this vector is not a "viral vector". Non-viral vectors and carriers include naked polynucleotides, chemical-based carriers, polynucleotide (non-viral)-based vectors, and particle-based carriers. Obviously, when used with respect to non-viral vectors and carriers, the term "vector" refers to a vector of polynucleotides, and in this context, the term "carrier" refers to a non-nucleic acid or non-polynucleotide molecule or composition that attaches to or interacts with the polynucleotide to be delivered, such as the engineered AAV capsid polynucleotide of the invention.

[0106] Naked polynucleotide In some embodiments, one or more engineered AAV capsid polynucleotides or other polynucleotides of the invention described elsewhere herein can be included in a naked polynucleotide. As used herein, the technical term "naked polynucleotide" refers to a polynucleotide that is not associated with another molecule (e.g., a protein, lipid, and / or other molecule) that often serves to protect against environmental factors and / or degradation. As used herein, "associated" includes, but is not limited to, being linked, attached, adsorbed, encapsulated, entrapped within, mixed with, etc. A naked polynucleotide comprising one or more of the engineered AAV capsid polynucleotides or other polynucleotides of the invention described herein can be delivered directly to a host cell and optionally expressed therein. A naked polynucleotide can have any suitable two-dimensional and three-dimensional configuration. By way of non-limiting example, a naked polynucleotide can be a single-stranded molecule, a double-stranded molecule, a circular molecule (e.g., a plasmid and an artificial chromosome), a molecule comprising single-stranded and double-stranded portions (e.g., a ribozyme), etc. In some embodiments, the naked polynucleotide comprises only the engineered AAV capsid polynucleotide(s) or other polynucleotide(s) of the invention. In some embodiments, the naked polynucleotide can include certain other nucleic acids and / or polynucleotides in addition to the engineered AAV capsid polynucleotide(s) or other polynucleotide(s) of the invention described elsewhere herein. A naked polynucleotide can include one or more elements of a transposon system. Transposons and their systems are described in more detail elsewhere herein.

[0107] Non-viral polynucleotide vector In some embodiments, one or more of the engineered AAV capsid polynucleotides or other polynucleotides of the invention can be included in a non-viral polynucleotide vector. Suitable non-viral polynucleotide vectors include transposon vectors and vector systems, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, antibiotic resistance (AR)-free plasmids and miniplasmids, covalently closed circular vectors (e.g., minicircles, minivectors, mininots), linear covalently closed circular vectors (“dumbbell type”), MIDGE (minimalistic immunologically defined gene expression) vectors, MiLV (microlinear vector) vectors, ministrings, mini-intron plasmids, PSK (post-segregationally killing) systems, ORT (operator-repressor titration) plasmids, and the like, but are not limited thereto. See, e.g., Hardee et al. 2017. Genes. 8(2):65.

[0108] In some embodiments, the non-viral polynucleotide vector can have a conditional origin of replication. In some embodiments, the non-viral polynucleotide vector can be an ORT plasmid. In some embodiments, the non-viral polynucleotide vector can have minimal immunologically defined gene expression. In some embodiments, the non-viral polynucleotide vector can have one or more post-separation killing system genes. In some embodiments, the non-viral polynucleotide vector is AR-free. In some embodiments, the non-viral polynucleotide vector is a minivector. In some embodiments, the non-viral polynucleotide vector contains a nuclear localization signal. In some embodiments, the non-viral polynucleotide vector can contain one or more CpG motifs. In some embodiments, the non-viral polynucleotide vector can contain one or more scaffold / matrix attachment regions (S / MARs). See, for example, Mirkovitch et al. 1984. Cell. 39:223-232, Wong et al. 2015 Adv. Genet. 89:113-152. The techniques and vectors can be adapted for use in the present invention. S / MARs are AT-rich sequences that play a role in the spatial arrangement of chromosomes through attachment of DNA loop bases to the nuclear matrix. S / MARs are often present near regulatory elements such as promoters, enhancers, and origins of DNA replication. Inclusion of one or more S / MARs promotes replication once per cell cycle and the non-viral polynucleotide vector is maintained as an episome within daughter cells. In embodiments, the S / MAR sequence is located downstream of a polynucleotide that is actively transcribed and included in the non-viral polynucleotide vector (e.g., one or more engineered AAV capsid polynucleotides or other polynucleotides or molecules of the present invention). In some embodiments, the S / MAR can be an S / MAR from the beta interferon gene cluster.See, for example, Verghese et al. 2014. Nucleic Acid Res. 42: e53; Xu et al. 2016. Sci. China Life Sci. 59: 1024-1033, Jin et al. 2016. 8: 702-711, Koirala et al. 2014. Adv. Exp. Med. Biol. 801: 703-709, and Nehlsen et al. 2006. Gene Ther. Mol. Biol. 10: 233-244. These techniques and vectors can be adapted for use in the present invention.

[0109] In some embodiments, the non-viral vector is a transposon vector or a system thereof. As used herein, a "transposon" (also referred to as a transposable element) refers to a polynucleotide sequence that can move from one location to another within a genome. There are several classes of transposons. Transposons include retrotransposons and DNA transposons. Retrotransposons require transcription of the polynucleotide being moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. DNA transposons do not require reverse transcription of the polynucleotide being moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. In some embodiments, the non-viral polynucleotide vector can be a retrotransposon vector. In some embodiments, the retrotransposon vector includes long terminal repeats. In some embodiments, the retrotransposon vector does not include long terminal repeats. In some embodiments, the non-viral polynucleotide vector can be a DNA transposon vector. The DNA transposon vector can include a polynucleotide sequence encoding a transposase. In some embodiments, the transposon vector is configured as a non-autonomous transposon vector, which means that transposition does not occur spontaneously by itself. In some of these embodiments, the transposon vector lacks one or more polynucleotide sequences encoding the proteins necessary for transposition. In some embodiments, the non-autonomous transposon vector lacks one or more Ac elements.

[0110] In some embodiments, a non-viral polynucleotide transposon vector system can include a first polynucleotide vector comprising an engineered AAV capsid polynucleotide(s) of the invention described herein or other polynucleotide or molecule, and transposon terminal inverted repeat sequences (TIRs) flanking it at the 5' and 3' ends, and a second polynucleotide vector comprising a polynucleotide capable of encoding a transposase bound to a promoter to drive expression of the transposase. When both are expressed in the same cell, the transposase can be expressed from the second vector and transpose a material (e.g., an engineered AAV capsid polynucleotide of the invention or other polynucleotide or molecule) between the TIRs on the first vector and integrate it into one or more positions within the genome of the host cell. In some embodiments, the transposon vector or system thereof can be configured as a gene trap. In some embodiments, the TIR can be flanked by a strong splice acceptor site followed by a reporter and / or other gene (e.g., one or more engineered AAV capsid polynucleotides of the invention or other polynucleotide or molecule) and a strong polyA tail. When transposition occurs during use of this vector or system thereof, the transposon can enter the intron of a gene, and the inserted reporter or other gene can cause a mis-splicing process, resulting in activation of the trapped gene.

[0111] Any suitable transposon system can be used. Suitable transposons and their systems include the Sleeping Beauty transposon system (Tc1 / mariner superfamily) (see, for example, Ivics et al. 1997. Cell. 91(4):501-510), piggyBac (piggyBac superfamily) (see, for example, Li et al. 2013 110(25):E2279-E2287 and Yusa et al. 2011. PNAS. 108(4):1531-1536), Tol2 (superfamily hAT), Frog Prince (Tc1 / mariner superfamily) (see, for example, Miskey et al. 2003 Nucleic Acid Res. 31(23):6873-6881), and variants thereof.

[0112] Chemical carrier In some embodiments, the engineered AAV capsid polynucleotides or other polynucleotides or other molecules described herein can be conjugated to a chemical carrier. Chemical carriers suitable for polynucleotide delivery can be broadly classified into (i) inorganic particles, (ii) lipid-based, (iii) polymer-based, and (iv) peptide-based classes. These can be classified as (1) those that can form a condensed complex with a polynucleotide (such as the engineered AAV capsid polynucleotide(s) of the present invention), (2) those that can target specific cells, (3) those that can increase delivery of the polynucleotide or other molecule of the present invention (such as the engineered AAV capsid polynucleotide(s)) to the nucleus or cytoplasm of a host cell, (4) those that can separate from DNA / RNA within the cytoplasm of a host cell, and (5) those that enable sustained or controlled release. Obviously, any one chemical carrier can include functions of multiple categories. As used herein, the term "particle" refers to particles of any size suitable for delivering the compositions of the present invention described herein (including the particles, polypeptides, polynucleotides, and other compositions described herein). Suitable sizes include macro-sized, micro-sized, and nano-sized particles.

[0113] In some embodiments, the non-viral carrier can be an inorganic particle. In some embodiments, the inorganic particle can be a nanoparticle. The inorganic particle can be configured and optimized by varying its size, shape, and / or porosity. In some embodiments, the inorganic particle is optimized to escape from the reticuloendothelial system. In some embodiments, the inorganic particle can be optimized to protect the captured molecule from degradation. In this regard, suitable inorganic particles that can be used as non-viral carriers include, but are not limited to, calcium phosphate, silica, metals (e.g., gold, platinum, silver, palladium, rhodium, osmium, iridium, ruthenium, mercury, copper, rhenium, titanium, niobium, tantalum, and combinations thereof), magnetic compounds, particles, and materials (e.g., superparamagnetic iron oxide and magnetite), quantum dots, fullerenes (e.g., carbon nanoparticles, nanotubes, nanowires, etc.), and combinations thereof. Other suitable inorganic non-viral carriers are described elsewhere in this specification.

[0114] In some embodiments, the non-viral carrier can be lipid-based. Suitable lipid-based carriers are described in more detail herein. In some embodiments, the lipid-based carrier comprises a cationic lipid or amphiphilic lipid that can bind or interact with the negative charge on the polynucleotide to be delivered (e.g., the engineered AAV capsid polynucleotide of the invention). In some embodiments, the chemical non-viral carrier system can comprise a polynucleotide (e.g., the engineered AAV capsid polynucleotide(s) of the invention or other compositions or molecules) and a lipid (e.g., a cationic lipid). These are also referred to in the art as lipoplexes. Other embodiments of lipoplexes are described elsewhere herein. In some embodiments, the non-viral lipid-based carrier can be a lipid nanoemulsion. A lipid nanoemulsion can be formed by dispersing a liquid that is immiscible in another stabilizing emulsifier and can have particles of about 200 nm. The particles are composed of a lipid, water, and a surfactant that can comprise the polynucleotide to be delivered (e.g., the engineered AAV capsid polynucleotide(s) of the invention). In some embodiments, the lipid-based non-viral carrier can be solid lipid particles or solid lipid nanoparticles.

[0115] In some embodiments, the non-viral carrier can be peptide-based. In some embodiments, the peptide-based non-viral carrier can include one or more cationic amino acids. In some embodiments, 35-40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% of the amino acids are cationic. In some embodiments, the peptide carrier can be used in combination with other types of carriers (e.g., polymer-based carriers and lipid-based carriers for enabling the function of the carrier). In some embodiments, the function is to target host cells. Suitable polymers that can be included in the polymer-based non-viral carrier include, but are not limited to, polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA), poly(DL-lactide-co-glycoside) (PLGA), dendrimers (see, e.g., U.S. Patent Publication 2017 / 0079916, the techniques and compositions of which can be adapted for use with the engineered AAV capsid polynucleotides of the present invention), polymethacrylates, and combinations thereof.

[0116] In some embodiments, the non-viral carrier can be configured to release the engineered delivery system polynucleotide bound or attached to the non-viral carrier in response to external stimuli such as pH, temperature, osmotic pressure, concentration of a particular molecule or composition (e.g., calcium, NaCl, etc.), pressure, etc. In some embodiments, the non-viral carrier can be in the form of particles, and the particles are configured to include one or more of the engineered AAV capsid polynucleotides or other compositions of the present invention described herein, an environmental trigger-responsive element, and optionally a trigger. In some embodiments, the particles can include a polymer that can be selected from the group of polymethacrylates and polyacrylates. In some embodiments, the non-viral particles can include one or more embodiments of the composition microparticles described in U.S. Patent Publications 20150232883 and 20050123596, the techniques and compositions of which can be adapted for use in the present invention.

[0117] In some embodiments, the non-viral carrier can be a polymer-based carrier. In some embodiments, the polymer is cationic or predominantly cationic, such that the polymer can interact charge-dependently with the negatively charged polynucleotide to be delivered (such as the engineered AAV capsid polynucleotide(s) of the present invention). Polymer-based systems are described in more detail elsewhere in this document.

[0118] Viral vector In some embodiments, the vector is a viral vector. As used herein in this context, the technical term "viral vector" refers to a polynucleotide-based vector that contains one or more elements derived from or based on one or more of the elements of a virus. The vector, alone or when used with one or more other viral vectors (such as vectors within a viral vector system), can express and package the polynucleotides of the present invention (such as the engineered AAV capsid polynucleotides of the present invention), cargo, or other compositions or molecules into viral particles and can generate viral particles. Viral vectors and their systems can be used to produce viral particles for delivering and / or expressing and / or generating one or more compositions of the present invention described herein (including, but not limited to, any viral particles and associated cargo). The viral vector can be part of a viral vector system that includes multiple vectors. In some embodiments, a system incorporating multiple viral vectors can enhance the safety of these systems. Suitable viral vectors can include adenovirus-based vectors, adeno-associated vectors, helper-dependent adenovirus (HdAd) vectors, and hybrid adenovirus vectors. Other embodiments of viral vectors and the viral particles generated therefrom are described elsewhere in this specification. In some embodiments, the viral vector is configured to generate replication-incompetent viral particles to improve the safety of the system.

[0119] Adenovirus vectors, helper-dependent adenovirus vectors, and hybrid adenovirus vectors In some embodiments, the vector can be an adenovirus vector. In some embodiments, the adenovirus vector can include elements such that the virus particles generated using the vector or its system are of serotype 2, 5, or 9. In some embodiments, the polynucleotide delivered via the adenovirus particle can be up to about 8 kb. Thus, in some embodiments, the adenovirus vector can include a delivery DNA polynucleotide having a size in the range of about 0.001 kb to about 8 kb. Adenovirus vectors have been effectively used in some situations (see, for example, Teramato et al. 2000. Lancet. 355:1911-1912, Lai et al. 2002. DNA Cell. Biol. 21:895-913, Flotte et al., 1996. Hum. Gene. Ther. 7:1145-1159, and Kay et al. 2000. Nat. Genet. 24:257-261). The vector can encode a processed AAV capsid that forms the adenovirus particle.

[0120] In some embodiments, the vector can be a helper-dependent adenovirus vector or a system thereof. Such vectors are also referred to in the art as gutless vectors or gutted vectors and are a modified generation of adenovirus vectors (see, e.g., Thrasher et al. 2006. Nature. 443:E5-7). In embodiments of the helper-dependent adenovirus vector system, one vector (the helper) can contain all the viral genes necessary for replication but contains a conditional genetic defect in the packaging domain. The second vector of this system can contain only the termini of the viral genome, one or more engineered AAV capsid polynucleotides, and the wild-type packaging recognition signal, which can enable selective packaging and release from the cell (see, e.g., Cideciyan et al. 2009. N Engl J Med. 361:725-727). The helper-dependent adenovirus vector system has been successful in gene delivery in some situations (see, e.g., Simonelli et al. 2010. J Am Soc Gene Ther. 18:643-650, Cideciyan et al. 2009. N Engl J Med. 361:725-727, Crane et al. 2012. Gene Ther. 19(4):443-452, Alba et al. 2005. Gene Ther. 12:18-S27, Croyle et al. 2005. Gene Ther. 12:579-587, Amalfitano et al. 1998. J.Virol. 72:926-933, and Morral et al. 1999. PNAS. 96:12816-12821). The techniques and vectors described in these publications can be adapted for the incorporation and delivery of the engineered AAV capsid polynucleotides described herein. In some embodiments, the polynucleotide delivered via viral particles generated from a helper-dependent adenovirus vector or a system thereof can be up to about 38 kb.Thus, in some embodiments, the adenovirus vector can comprise a delivery DNA polynucleotide that can range in size from about 0.001 kb to about 37 kb (see, e.g., Rosewell et al. 2011. J. Genet. Syndr. Gene Ther. Suppl. 5:001).

[0121] In some embodiments, the vector is a hybrid adenovirus vector or a system thereof. The hybrid adenovirus vector consists of the high transduction efficiency of the gene-deleted adenovirus vector and the long-term genomic integration potential of adeno-associated virus, retrovirus, lentivirus, and transposon-based gene transfer. In some embodiments, such a hybrid vector system can provide stable transduction and limited integration sites. See, for example, Balague et al. 2000. Blood. 95:820-828, Morral et al. 1998. Hum. Gene Ther. 9:2709-2716, Kubo and Mitani. 2003. J. Virol. 77(5):2964-2971, Zhang et al. 2013. PloS One. 8(10) e76771, and Cooney et al. 2015. Mol. Ther. 23(4):667-674. The techniques and vectors described therein can be modified and adapted for use with the engineered AAV capsid systems of the present invention. In some embodiments, the hybrid adenovirus vector can include one or more characteristics of a retrovirus and / or an adeno-associated virus. In some embodiments, the hybrid adenovirus vector can include one or more characteristics of a spumaretrovirus or foamy virus (FV). See, for example, Ehrhardt et al. 2007. Mol. Ther. 15:146-156 and Liu et al. 2007. Mol. Ther. 15:1834-1841. The techniques and vectors described therein can be modified and adapted for use with the engineered AAV capsid systems of the present invention. Advantages of using one or more characteristics of FV in a hybrid adenovirus vector or a system thereof include the ability of the virus particles generated therefrom to infect a wide range of cells, a large packaging capacity compared to other retroviruses, and the ability to persist within quiescent (non-dividing) cells.See, e.g., Ehrhardt et al. 2007. Mol. Ther. 156:146-156 and Shuji et al. 2011. Mol. Ther. 19:76-82. The techniques and vectors described therein can be modified and adapted for use with the engineered AAV capsid systems of the present invention.

[0122] Adeno-associated vector In one embodiment, the engineered vector or system thereof can be an adeno-associated vector (AAV). See, e.g., West et al., Virology 160:38-47 (1987), U.S. Patent No. 4,797,368, WO 93 / 24641, Kotin, Human Gene Therapy 5:793-801 (1994), and Muzyczka, J. Clin. Invest. 94:1351 (1994). AAV is similar to adenoviral vectors in some characteristics, but may be safer than adenoviral vectors because of some defect in its replication and / or pathogenicity. In some embodiments, AAV can integrate into a specific site on chromosome 19 of human cells without obvious side effects. In some embodiments, the capacity of the AAV vector, system thereof, and / or AAV particles can be up to about 4.7 kb. The AAV vector or system thereof can include one or more of the engineered capsid polynucleotides described herein.

[0123] An AAV vector or a system thereof can be operably linked to a regulatory sequence, and the regulatory sequence encodes one or more regulatory molecules. In some embodiments, the regulatory molecules are promoters, enhancers, repressors, etc., which are described in more detail elsewhere in this specification. In some embodiments, the AAV vector or a system thereof can include one or more polynucleotides that can encode one or more regulatory proteins. In some embodiments, the promoter can be a tissue-specific promoter as described above. In some embodiments, the tissue-specific promoter can promote the expression of the engineered capsid AAV capsid polynucleotide described herein.

[0124] An AAV vector or a system thereof can include one or more polynucleotides that can encode one or more capsid proteins such as the engineered AAV capsid proteins described elsewhere in this specification. The engineered capsid proteins can assemble into the protein shell (engineered capsid) of the AAV viral particle. The engineered capsid can have cell-specific, tissue-specific, and / or organ-specific tropism.

[0125] In some embodiments, the AAV vector or a system thereof can include one or more adenovirus helper factors, or can include a polynucleotide that can encode one or more adenovirus helper factors. Examples of such adenovirus helper factors include, but are not limited to, E1A, E1B, E2A, E4ORF6, and VARNA. In some embodiments, the production host cell line can express one or more adenovirus helper factors.

[0126] An AAV vector or a system thereof can be configured to generate AAV particles having a specific serotype. In some embodiments, the serotype can be AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combination thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9, or any combination thereof. Depending on the target cell, the AAV of the AAV can be selected. For example, when targeting brain cells and / or nerve cells, AAV serotypes 1, 2, 5, 9 or hybrid capsid AAV-1, AAV-2, AAV-5, AAV-9, or any combination thereof can be selected. When targeting heart tissue, AAV-4 can be selected. When delivering to the liver, AAV-8 can be selected. Thus, in some embodiments, an AAV vector or a system thereof that can generate AAV particles capable of targeting brain cells and / or nerve cells can be configured to generate AAV particles having serotype 1, 2, 5 or hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof. In some embodiments, an AAV vector or a system thereof that can generate AAV particles capable of targeting heart tissue can be configured to generate AAV particles having the AAV-4 serotype. In some embodiments, an AAV vector or a system thereof that can generate AAV particles capable of targeting the liver can be configured to generate AAV particles having the AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21:75-80.

[0127] As is apparent, while different serotypes can provide a degree of cell specificity, tissue specificity, and / or organ specificity, each serotype remains polyvalent, such that using that serotype to target tissues where it is less efficient at transduction can result in tissue toxicity. Thus, in addition to achieving a degree of tissue targeting ability by selecting a particular serotype of AAV, it is of course possible to modify the tropism of the AAV serotype with the engineered AAV capsids described herein. As described elsewhere herein, variants of wild-type AAV of any serotype can be generated by the methods described herein and can be determined to have a particular cell-specific tropism. That tropism can be the same as or different from the reference wild-type AAV serotype. In some embodiments, the cell specificity, tissue specificity, and / or tropism of the wild-type serotype can be enhanced (e.g., made more selective or specific for a particular cell type that the serotype is already in that direction). For example, wild-type AAV-9 can be directed towards human muscle and brain (see, e.g., Srivastava. 2017. Curr. Opin. Virol. 21:75-80). By including engineered AAV capsids and / or capsid protein variants of wild-type AAV-9 as described herein, for example, tropism for the brain can be reduced or eliminated, and / or tropism can be enhanced such that brain specificity is reduced by comparison and, as a result, specificity for muscle is improved compared to wild-type AAV-9. As described above, including engineered capsids and / or capsid protein variants of wild-type AAV serotypes can result in having a tropism different from the wild-type reference AAV serotype. For example, engineered AAV capsids and / or capsid protein variants of AAV-9 can have specificity for tissues other than human muscle and brain.

[0128] In some embodiments, the AAV vector is a hybrid AAV vector or a system thereof. A hybrid AAV contains a genome having multiple elements of one serotype, where the multiple elements are packaged into a capsid derived from at least one different serotype. For example, if the one to be produced is rAAV2 / 5 and the production method is based on the helper-free transient transfection method described below, the first plasmid and the third plasmid (adeno helper plasmid) will be the same as those described for the production of rAAV2. However, the second plasmid, pRepCap, is different. In this plasmid, called pRep2 / Cap5, the Rep gene is still derived from AAV2, but the Cap gene is derived from AAV5. The production scheme is the same as the aforementioned AAV2 production approach. The resulting rAAV is called rAAV2 / 5, where the genome is based on recombinant AAV2 and the capsid is based on AAV5. The cell tropism or tissue tropism exhibited by this AAV2 / 5 hybrid virus is assumed to be the same as that of AAV5. As is clear, wild-type hybrid AAV particles, like the aforementioned non-hybrid wild-type serotypes, have problems with specificity.

[0129] The advantages obtained by wild-type-based hybrid AAV systems can be combined with the high and customizable cell specificity achievable with engineered AAV capsids, and can be combined by generating hybrid AAVs that can include engineered AAV capsids as described elsewhere herein. As is apparent, hybrid AAVs can include engineered AAV capsids that contain a genome with elements of a serotype different from the reference wild-type serotype. The engineered AAV capsid is a variant of the reference wild-type serotype. For example, a hybrid AAV can be generated that includes an engineered AAV capsid that is a variant of the AAV-9 serotype used to package a genome that includes components of the AAV-2 serotype (such as AAV2 ITRs). Similar to the wild-type-based hybrid AAVs described above, the tropism of the resulting AAV particles will be the same as the tropism of the engineered AAV capsid.

[0130] A list of specific wild-type AAV serotypes for these cells can be found in Grimm, D. et al, J. Virol. 82:5887-5911 (2008), and is reproduced below as Table A. Further details regarding tropism can be found in Srivastava. 2017. Curr. Opin. Virol. 21:75-80, as described above.

Table 1

[0131] In an exemplary embodiment, the AAV vector or system thereof is AAVrh.74 or AAVrh.10.

[0132] In an exemplary embodiment, the AAV vector or a system thereof is configured as a gutless vector similar to that described in connection with the retroviral vector. In some embodiments, the gutless AAV vector or a system thereof can have cis-acting viral DNA elements involved in genome amplification and packaging ligated to a heterologous sequence of interest (e.g., a transgene encoding a therapeutic protein or nucleic acid of interest).

[0133] Vector encoding a transgene In one exemplary embodiment, a vector encoding a transgene (also referred to as an “artificial genome”) includes the transgene to be delivered and AAV ITRs flanking both sides thereof. For the propagation of recombinant AAV (rAAV), only about 145 bp of the AAV ITR is required. This is because the AAV ITR is involved in vector production, induces transgene expression, and ensures continuous transduction. Thus, for gene therapy, about 96% of the AAV genome can be removed. For example, the rep gene and the cap gene can be used in place of an expression cassette (which forms an essential part of all AAV vectors) including a promoter (such as those described herein), a therapeutic transgene (e.g., IDS), and a poly(A) tail.

[0134] In exemplary embodiments, additional modifications can be made to further enhance the effectiveness of AAV. For example, the AAV ITR can be modified to increase the expression of the rAAV vector upon transduction. This can enable the expression of the transgene without second-strand DNA synthesis. Also, the promoter can be modified to increase transcription, or the codons within the transgene can be manipulated to modify mRNA production and translation.

[0135] In an exemplary embodiment, the ITR is modified to overcome second-strand synthesis after infection. The transduction rate of AAV is limited by the synthesis of dsDNA from the single-stranded AAV genome. The ITR initiates second-strand synthesis. In one exemplary embodiment, the modified ITR is no longer an appropriate substrate for the Rep68 and Rep78 proteins. As a result, terminal resolution of replication is avoided, and certain self-complementary AAV (scAAV) replication intermediates are generated. The scAAV intermediate is composed of a plus strand and a minus strand of DNA that are fused by the modified ITR encapsulated in the capsid of the viral particle. Wild-type AAV packages a single plus-strand DNA or minus-strand DNA. The modified scAAV intermediate is delivered to the nucleus, and these plus and minus strands anneal instantaneously to form dsDNA.

[0136] In an exemplary embodiment, the cis element is optimized for target delivery. Since the packaging capacity of AAV is limited, the cis element is optimized. In one exemplary embodiment, a small cis element is used instead of a long promoter sequence to deliver a large therapeutic transgene (e.g., 4.4 - 4.5 kbs).

[0137] In an exemplary embodiment, several strategies can be used to deliver a transgene using an AAV vector. In exemplary approach 1, an AAV genome concatemerized by homologous recombination of the ITR sequence is utilized. In this approach, the transgene cassette can be divided into two or more vectors, which can be delivered to the same cell. After viral uncoating, an intact transgene is formed by homologous recombination between two or more fragments.

[0138] In exemplary approach 2, truncated transgene fragments of different lengths are packaged into different AAV virions at undetermined positions on the vector genome. The transgene cassette is generated by homologous recombination of overlapping regions of different AAV vector genomes, or annealing in complementary regions of different AAV vector genomes via a single-stranded template. In an exemplary embodiment, overlapping fragments can be added to the ends of individual AAV vectors to facilitate homologous recombination.

[0139] In exemplary approach 3, a region overlapping with the intron splice site within the split vector transgene is incorporated into the hybrid dual vector. Approach 3 utilizes the concatemerization activity of the AAV genome to combine independent AAV vector genomes. Recombination (e.g., the starting vector is separated into two parts, each carrying a 5' splicing element and a 3' splicing element) and splicing result in the production of the appropriate transgene protein. This strategy can increase the expression of a fully functional protein.

[0140] In exemplary approach 4, the AAV genome is cross-packaged into the capsid of another parvovirus to generate a chimeric vector. In exemplary approach 5, protein trans-splicing via an intein is used. The intein catalyzes protein splicing and results in the ligation of two polypeptides via trans-splicing (this approach is similar to RNA splicing via an intron). Multiple AAV vectors are delivered to the same cell. Each of the AAV vectors encodes one of the fragments of the target protein, and a short split intein is adjacent to the fragment. After protein trans-splicing, the full-length protein is formed. See, for example, Li, C., Samulski, R. J. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet 21, 255-272 (2020) (which is incorporated herein by reference).

[0141] Construction of Vectors

[0142] The vectors described herein can be constructed using any suitable process or technique. In some embodiments, one or more suitable recombination and / or cloning methods or techniques can be used for the vectors (s) described herein. Suitable recombination and / or cloning techniques and / or methods include, but are not limited to, those described in US Patent Application Publication US2004-0171156A1. Other suitable methods and techniques are described elsewhere in this specification.

[0143] The construction of recombinant AAV vectors has been described in several publications. Such publications include U.S. Patent No. 5,173,414, Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:6466-6470 (1984), and Samulski et al., J. Virol. 63:03822-3828 (1989). Any technique and / or method can be used and / or adapted to construct the AAV or other vectors described herein. AAV vectors are described elsewhere in this book.

[0144] In some embodiments, the vector can have one or more insertion sites, such as restriction enzyme recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors.

[0145] Delivery vehicles, vectors, particles, nanoparticles, formulations, and components thereof for the expression of one or more elements of the engineered AAV capsid systems described herein are those used in the above-mentioned literature, such as International Patent Application Publication No. WO2014 / 093622 (PCT / US2013 / 074667), and are described in more detail herein. Generation of viral particles from viral vectors

[0146] Generation of AAV particles To generate AAV particles from AAV vectors and their systems as described herein, there are two main strategies. These differ in how adenoviral helper factors are provided (helper vs. helper-free). In some embodiments, methods for generating AAV particles from AAV vectors and their systems can include infection of a cell line with adenovirus, the cell line being one that stably retains polynucleotides encoding AAV replication and capsid along with the AAV vector, the AAV vector including a polynucleotide (e.g., an engineered AAV capsid polynucleotide(s)) to be packaged and delivered by the resulting AAV particles. In some embodiments, methods for generating AAV particles from AAV vectors and their systems can be "helper-free" methods. Helper-free methods include co-transfecting a suitable production cell line with three vectors (e.g., plasmid vectors). The three vectors are: (1) an AAV vector including a polynucleotide of interest (e.g., a transgene encoding a therapeutic protein or nucleic acid operably linked to regulatory elements that promote expression in a target tissue) between two ITRs, (2) a vector carrying a polynucleotide encoding an AAV Rep-Cap including the engineered capsid protein described herein, and a helper polynucleotide. The various methods, both helper and helper-free, and their variations, as well as the various advantages of each system, will be apparent to those of skill in the art.

[0147] The processed AAV vectors and systems described herein can be generated by any of these methods.

[0148] Delivery of Vectors and Virus Particles Introduce the vectors (including non-viral carriers) described herein into host cells to generate transcripts, proteins, or peptides containing the fusion proteins or peptides encoded by the nucleic acids described herein (e.g., transcripts, proteins, enzymes, variants thereof, fusion proteins thereof, etc. of the processed AAV capsid system), and virus particles (e.g., those derived from viral vectors and their systems).

[0149] AAV capsids prepared from one or more processed AAV capsid polynucleotides can be used to deliver recombinant AAV genomes encoding a therapeutic protein or nucleic acid of interest. Alternatively, the aforementioned adenoviruses or other plasmid or viral vector types can be used, where specifically, for example, the formulations and dosages from U.S. Patent Nos. 8,454,972 (adenovirus formulation, dosage), 8,404,658 (AAV formulation, dosage), and 5,846,946 (DNA plasmid formulation, dosage), as well as the formulations and dosages from clinical trials related to lentivirus, AAV, and adenovirus and publications related to such clinical trials can be used. For example, in the case of AAV, the route of administration, formulation, and dosage can be made the same as those in U.S. Patent No. 8,454,972 and clinical trials related to AAV. In the case of adenovirus, the route of administration, formulation, and dosage can be made the same as those in U.S. Patent No. 8,404,658 and clinical trials related to adenovirus.

[0150] In the case of plasmid delivery, the route of administration, formulation, and dosage can be made similar to those in U.S. Patent No. 5,846,946 and clinical studies regarding the plasmid. In some embodiments, the dosage can be based on, or extrapolated for, an individual of average 70 kg (e.g., adult male), and can be adjusted for patients, subjects, mammals of different weights and species. The frequency of administration is within the discretion of a medical or veterinary practitioner (e.g., physician, veterinarian), which may vary depending on normal factors such as the age, sex, general health, other conditions of the patient or subject, and the particular disease or condition being addressed. The viral vector can be injected into the target tissue or cells or delivered by other means.

[0151] Regarding in vivo delivery, AAV is advantageous over other viral vectors for several reasons. Such reasons include, for example, low toxicity (which is thought to be due to a purification method that does not require ultracentrifugation of cellular particles that can activate an immune response), and a low likelihood of causing insertional mutations because it does not integrate into the host genome.

[0152] The vector(s) and virus particles described in this specification can be delivered to host cells in vitro, in vivo, or ex vivo. Delivery can be performed by any suitable method, including but not limited to physical methods, chemical methods, and biological methods. Physical delivery methods are methods that utilize physical forces to counter the cell membrane barrier and facilitate the intracellular delivery of the vector. Suitable physical methods include, but are not limited to, needles (e.g., injection), ballistic polynucleotides (e.g., particle bombardment, gene gun transfection, gene gun), electroporation, sonoporation, photoporation, magnetofection, hydrodynamic poration, mechanical massage, etc. Chemical methods are methods that use chemicals to cause changes in cell membrane permeability and other property(ies) and facilitate the entry of the vector into the cell. For example, changing the pH of the environment can cause a change in cell membrane permeability. Biological methods are methods that rely on and utilize the biological processes or biological properties of host cells to facilitate the intracellular transport of the vector (with or without a carrier). For example, the vector and / or its carrier can stimulate endocytosis or similar processes within the cell to facilitate the uptake of the vector by the cell.

[0153] Delivery of Engineered AAV Capsid-Based Components (e.g., Engineered AAV Capsids and / or Polynucleotides Encoding Capsid Proteins) to Cells via Particles As used herein, the term "particle" refers to particles of any size suitable for delivering the engineered AAV capsid-based components described herein. Suitable sizes include macro-sized, micro-sized, and nano-sized particles. In some embodiments, any of the engineered AAV capsid-based components described herein (e.g., polypeptides, polynucleotides, vectors, and combinations thereof) can be attached, bound, integrated, or associated with one or more particles or components thereof. The particles described herein can then be administered to cells or organisms by appropriate routes and / or methods. In some embodiments, particle delivery can be selected, which can be advantageous for the delivery of polynucleotide or vector components. As will be apparent in the embodiments, particle delivery can also be advantageous for other engineered capsid-based molecules and formulations described elsewhere herein.

[0154] Engineered Viral Particles Comprising Engineered Viruses (e.g., AAV) Capsids In addition, this specification describes engineered viral particles (engineered viral particles are referred to as "engineered viral particles" in this chapter and other parts of this specification, and can include engineered viral capsids (e.g., AAV capsids referred to as "engineered AAV particles") as described in detail elsewhere in this specification). Obviously, as described above, engineered AAV particles can be adenovirus-based particles, helper adenovirus-based particles, AAV-based particles, or hybrid adenovirus-based particles that contain at least one engineered AAV capsid protein. Engineered AAV capsids are capsids that contain one or more engineered AAV capsid proteins as described elsewhere in this specification. In some embodiments, engineered AAV particles can contain 1 to 60 engineered AAV capsid proteins described herein. In some embodiments, engineered AAV particles can contain 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, engineered AAV particles can contain 0 to 59 wild-type AAV capsid proteins. In some embodiments, engineered AAV particles can contain 0, 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type AAV capsid proteins. Thus, engineered AAV particles can contain one or more n-amino acid long motifs as described above.

[0155] The engineered AAV particles can contain one or more cargo polynucleotides, which will be described in more detail elsewhere in this specification. Methods for producing engineered AAV particles from viral vectors and non-viral vectors will be described elsewhere in this specification. Formulations containing engineered viral particles will be described elsewhere in this specification.

[0156] Examples of Cargo An n-amino acid length can be attached or associated with the cargo. The cargo can include any molecule that can be attached or associated with the n-amino acid lengths described herein. Examples of cargo include, but are not limited to, nucleotides, oligonucleotides, polynucleotides, amino acids, peptides, polypeptides, ribonucleoproteins, lipids, sugars, pharmaceutically active agents (such as drugs, imaging diagnostics, and other diagnostics), compounds, and combinations thereof. In some embodiments, the cargo is DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of important tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, antihistamines, anti-infectives, radiosensitizers, chemotherapeutic agents, radioactive compounds, contrast agents, and combinations thereof. In an embodiment, the cargo is a recombinant AAV genome containing a transgene encoding, for example, a therapeutic protein or nucleic acid, wherein the transgene is operably linked to regulatory sequences that induce expression of the therapeutic protein or nucleic acid in a target tissue and is flanked by AAV ITR sequences.

[0157] In some embodiments, the cargo can treat or prevent a neurological disease or disorder, the details of which are described herein.

[0158] In some embodiments, the cargo is a morpholino, a peptide - linked morpholino, an antisense oligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a PPMO, one or more peptides, a CRISPR - Cas protein, a guide RNA, or one or more polynucleotides encoding both, a ribonucleoprotein, where the ribonucleoprotein is a CRISPR - Cas system molecule, a therapeutic transgene RNA, or other gene - modifying or therapeutic RNA and / or protein, or any combination thereof.

[0159] In some embodiments, one or more of the n - amino - acid lengths described herein are directly attached to the cargo. In some embodiments, one or more of the n - amino - acid lengths described herein are indirectly attached to the cargo, such as via a linker molecule. In some embodiments, one or more of the n - amino - acid lengths described herein are bound to, attached to, a polypeptide or other particle that binds to the cargo and / or a polypeptide or other particle that encapsulates, contains the cargo.

[0160] Exemplary particles include, but are not limited to, virus particles (e.g., virus capsids including bacteriophage capsids), polysomes, liposomes, nanoparticles, microparticles, exosomes, micelles, etc. As used herein, the term "nanoparticle" encompasses nanoscale deposits of homogeneous or heterogeneous materials. Nanoparticles can have regular or irregular shapes and can be formed from multiple co - deposited particles that form composite nanoscale particles. Nanoparticles can have a generally spherical shape or a composite shape formed from multiple co - deposited generally spherical particles. Exemplary shapes of nanoparticles include, but are not limited to, spherical, rod - shaped, ellipsoidal, cylindrical, discoidal, etc. In some embodiments, the nanoparticles have a substantially spherical shape.

[0161] Cargo polynucleotide The cargo is described elsewhere in this book. In some embodiments, the cargo is a cargo polynucleotide that can be packaged into engineered virus particles and then delivered to cells. In some embodiments, the delivery is cell-selective, for example, selective for neuronal and glial cells of the central nervous system. In some embodiments, one or more cargo polynucleotides are part of the engineered virus (e.g., AAV) genome of a virus (e.g., AAV) system and are packaged within an engineered capsid containing the targeting moiety of the invention. The cargo polynucleotide can be packaged into engineered virus (e.g., AAV) particles, which can be delivered, for example, to cells. In some embodiments, the cargo polynucleotide can modify the polynucleotide (e.g., gene or transcript) of the cell to which it is delivered. As used herein, "gene" can refer to a genetic unit corresponding to a DNA sequence that occupies a specific position on a chromosome and contains genetic instructions for a biological characteristic(s) or trait(s). The term gene can refer to the translated and / or untranslated regions of the genome. A "gene" can refer to a specific DNA sequence that is transcribed into an RNA transcript that can be translated into a polypeptide, or a specific DNA sequence that is transcribed into an RNA transcript that can be a catalytic RNA molecule including, but not limited to, tRNA, siRNA, piRNA, miRNA, long non-coding RNA, and shRNA. Modification of polynucleotides, genes, transcripts, etc. includes any genetic engineering technique, such techniques including, but not limited to, gene editing, as well as conventional recombinant gene modification techniques (e.g., techniques for insertion, deletion, and mutagenesis (e.g., insertion and deletion mutagenesis) of all or part of a gene).

[0162] In an exemplary embodiment, the cargo molecule is a vaccine or a polynucleotide that can encode a vaccine. In an exemplary embodiment, the cargo molecule is a polynucleotide that encodes an antibody.

[0163] Interfering RNA In certain exemplary embodiments, one or more polynucleotides may encode one or more interfering RNAs. Interfering RNAs are RNA molecules that can suppress gene expression. Examples of interfering RNAs include small interfering RNAs (siRNAs), microRNAs (miRNAs), and short hairpin RNAs (shRNAs).

[0164] In certain exemplary embodiments, the interfering RNA can be an siRNA. Small interfering RNA (siRNA) molecules can inhibit the expression of a target gene by interfering with RNA. siRNAs can be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo within target cells. siRNAs can be composed of double-stranded RNAs that are 15 to 40 nucleotides in length and can include 3' and / or 5' overhang regions that are 1 to 6 nucleotides in length. The length of the overhang region is independent of the total length of the siRNA molecule. siRNAs can act by silencing the target messenger or post-transcriptional degradation. In some cases, an exogenous polynucleotide encodes an shRNA. In shRNAs, the antiparallel strands that form the siRNA are connected by a loop or hairpin region.

[0165] Interfering RNAs (e.g., siRNAs) can suppress gene expression and promote the long-term survival and functionality of cells after transplantation into a subject. In some examples, the interfering RNAs suppress genes of the TGFβ pathway, such as TGFβ, TGFβ receptors, and SMAD proteins. In some examples, the interfering RNAs suppress genes of the colony stimulating factor 1 (CSF1) pathway, such as CSF1 and CSF1 receptor. In certain embodiments, one or more interfering RNAs suppress genes of both the CSF1 pathway and the TGFβ pathway. TGFβ pathway genes can include one or more of ACVR1, ACVR1C, ACVR2A, ACVR2B, ACVRL1, AMH, AMHR2, BMP2, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMPR1A, BMPR1B, BMPR2, CDKN2B, CHRD, COMP, CREBBP, CUL1, DCN, E2F4, E2F5, EP300, FST, GDF5, GDF6, GDF7, ID1, ID2, ID3, ID4, IFNG, INHBA, INHBB, INHBC, INHBE, LEFTY1, LEFTY2, LOC728622, LTBP1, MAPK1, MAPK3, MYC, NODAL, NOG, PITX2, PPP2CA, PPP2CB, PPP2R1A, PPP2R1B, RBL1, RBL2, RBX1, RHOA, ROCK1, ROCK2, RPS6KB1, RPS6KB2, SKP1, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD9, SMURF1, SMURF2, SP1, TFDP1, TGFB1, TGFB2, TGFB3, TGFBR1, TGFBR2, THBS1, THBS2, THBS3, THBS4, TNF, ZFYVE16, and / or ZFYVE9.

[0166] In some embodiments, the cargo polynucleotide is, or encodes, an RNAi molecule, an antisense molecule, and / or a gene silencing oligonucleotide.

[0167] As used herein, "gene silencing oligonucleotide" refers to any oligonucleotide that, alone or together with other gene silencing oligonucleotides, utilizes endogenous mechanisms, molecules, proteins, enzymes, and / or other cellular machinery in a cell, or exogenous molecules, factors, proteins, enzymes, and / or polynucleotides, to cause an overall or specific reduction or elimination of gene expression, RNA level(s), RNA translation, RNA transcription, resulting in a reduction or effective loss of protein expression and / or function of non-coding RNA as compared to wild type or appropriate controls. This is synonymous with the expression "gene knockdown". The reduction of gene expression, RNA level(s), RNA translation, RNA transcription, and / or protein expression can be a reduction from about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2% to 1% or less. Examples of "gene silencing oligonucleotides" include, but are not limited to, antisense oligonucleotides, ribozymes, any oligonucleotide (single-stranded or double-stranded) used to stimulate the intracellular RNA interference (RNAi) pathway (collectively referred to as RNAi oligonucleotides), small interfering RNA (siRNA), microRNA, and short hairpin RNA (shRNA). Based on gene sequences and other information available to those skilled in the art, commercially available programs and tools can be used to design the nucleotide sequences of gene silencing oligonucleotides for a desired gene.

[0168] In some embodiments, a cargo polynucleotide, such as a coding polynucleotide, has at least adjacent to it a polynucleotide 3’UTR or a portion thereof that encodes a retroelement polypeptide, such as the proximal region of about 500 base pairs of the 3’UTR. In some embodiments, a cargo polynucleotide, such as a coding polynucleotide, has adjacent to it a 5’UTR of a (e.g., endogenous or modified) retroelement polypeptide (such as a retroviral gag protein or a gag homolog). In some embodiments, a cargo polynucleotide, such as a coding polynucleotide, has adjacent to it a 5’UTR and a 3’UTR of a polynucleotide that encodes a (e.g., endogenous or modified) retroelement polypeptide. In some embodiments, the UTR(s) encoding an adjacent retroelement polypeptide is / are derived from PNMA, Arc, PEG10, or other Sushi class polypeptides. In some embodiments, including the 3’UTR, 5’UTR, or both can increase the packaging and / or delivery of the cargo to which they are adjacent. These and other packaging elements are described in more detail elsewhere in this document.

[0169] Genetically modified cargo polynucleotide In some embodiments, a cargo molecule is a polynucleotide, polypeptide, or polynucleotide encoding a polypeptide that, when delivered alone or as part of a system, can be engineered to modify the genome, epigenome, and / or transcriptome of the target cell, regardless of whether it is delivered together with other components of the system. Such systems include, but are not limited to, CRISPR-Cas systems. Other gene editing systems, such as TALEN, zinc finger nucleases, Cre-Lox, morpholino, etc., are other non-limiting examples of gene editing systems that can deliver one or more components by the engineered viral (e.g., AAV) particles described herein.

[0170] In some embodiments, the cargo molecule is, or encodes, a gene editing system or a component thereof. In some embodiments, the cargo molecule is, or encodes, a CRISPR-Cas system molecule or a component thereof. In some embodiments, the cargo molecule is a polynucleotide encoding one or more components of a gene modification system (such as the CRISPR-Cas system). In some embodiments, the cargo molecule is a gRNA or encodes a gRNA. As used herein, the CRISPR-Cas system is intended to include class 1 and class 2 CRISPR-Cas systems, and derivatives of the CRISPR-Cas system (e.g., base editors, prime editors, CRISPR-associated transposase (CAST) systems).

[0171] In some embodiments, the cargo molecule, whether delivered alone or as part of a system, can be engineered to modify the genome, epigenome, and / or transcriptome of the target cell, regardless of whether it is delivered together with other components of the system, and can be a polynucleotide, polypeptide, or polynucleotide encoding a polypeptide that treats or prevents a neurological disease or disorder and / or a disease, disorder, or symptom thereof of a virus (such as a single-stranded RNA virus). In some embodiments, the cargo molecule, regardless of whether it is delivered together with other components of the system, is engineered to modify the genome, epigenome, and / or transcriptome of the target cell and is one that treats or prevents a neurological disease or disorder further described herein.

[0172] In some embodiments, the cargo molecule is operative to modify the genome, epigenome, and / or transcriptome of the destination cell, whether or not it is delivered together with other components of the system, and is capable of modifying the GAA gene (e.g., as described in U.S. Patent Application Publication No. 20190284555). The content of the said publication is incorporated by reference in its entirety as if set forth herein in full and can be adapted for use in the present invention.

[0173] In some embodiments, the cargo molecule is an antisense oligomer or RNA molecule such as those described in U.S. Patent Application Publication Nos. 20160251398, 20150267202, and 20180216111, or encodes such an antisense oligomer or RNA molecule. The content of these publications is incorporated by reference in its entirety as if set forth herein in full and can be adapted for use in the present invention.

[0174] In some embodiments, the cargo molecule can be a peptide oligomer complex such as that described in International Patent Application Publication WO2017106304A1, the content of which is incorporated by reference as if set forth herein in full and can be adapted for use in the present invention.

[0175] An exemplary embodiment of the present invention includes a method of modifying a genomic locus of interest to alter gene expression in a cell by introducing any of the compositions described herein into the cell.

[0176] One embodiment of the present invention is that the above elements are included in a single composition or in individual compositions. These compositions can be advantageously applied to a host and can produce a functional effect at the genomic level.

[0177] Polypeptide In certain exemplary embodiments, the cargo molecule may be one or more polypeptides or nucleic acids encoding polypeptides. The polypeptide may be a full-length protein or a functional fragment or domain thereof that is a fragment or domain that maintains the desired functionality of the full-length protein. As used within this section, the term "protein" refers to full-length proteins and their functional fragments and domains. Using the engineered delivery vehicles described herein, a variety of polypeptides can be delivered. Such polypeptides include, but are not limited to, secreted proteins, immunomodulatory proteins, antifibrotic proteins, proteins that promote tissue regeneration and / or graft survival functions, hormones, antibacterial proteins, antifibrotic polypeptides, and antibodies. One or more polypeptides can also include combinations of the exemplary classes of polypeptides described above. Obviously, any of the polypeptides described herein can also be delivered via the delivery of the corresponding coding polynucleotide through the engineered delivery vehicles and systems described herein.

[0178] antibody In certain embodiments, one or more polypeptides can include one or more antibodies. The term "antibody" is used herein synonymously with the term "immunoglobulin" and includes intact antibodies, antibody fragments such as Fab, F(ab')2 fragments, scFv, and mutants in either their constant and / or variable regions (e.g., mutations to produce chimeric antibodies, partially humanized antibodies, or fully humanized antibodies, as well as mutations to produce antibodies having desired properties (e.g., enhanced binding and / or reduced FcR binding)), intact antibodies and fragments. The term "fragment" refers to a part or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained through chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, VHH and includes scF and / or Fv fragments. As used herein, a preparation of an antibody protein in which "non-antibody proteins (also referred to herein as "contaminating proteins") or chemical precursors are less than about 50%" is considered to be "substantially free". Preparations in which the non-antibody protein or chemical precursor is 40%, 30%, 20%, 10%, more preferably 5% (by dry weight) are considered to be substantially free. When the antibody protein or a biologically active portion thereof is produced recombinantly, it is preferably substantially free of culture medium, i.e., the culture medium is less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume or mass of the protein preparation.

[0179] In an exemplary embodiment, the antibody is a fragment or a portion thereof. In one exemplary embodiment, the antibody is an epitope-binding protein or a portion thereof. The term "binding portion" of an antibody (or "antibody portion") encompasses one or more complete domains (e.g., a pair of complete domains, and fragments of an antibody that retain the ability to specifically bind to a target molecule). It has been demonstrated that the binding function of an antibody can be provided by a fragment of a full-length antibody. Binding fragments are produced by recombinant DNA methods or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab’, F(ab’)2, Fabc, Fd, dAb, Fv, single-chain, single-chain antibodies such as scFv, and single-domain antibodies.

[0180] In some embodiments, the cargo or antibody is an antibody fragment or antibody portion. In some embodiments, the cargo is an epitope-binding protein. Examples of portions of antibodies or epitope-binding proteins included by this definition include the following. (i) V L 、C L 、V H 、and C H Fab fragments having 1 domain, (ii) C HA Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the domain, (iii) V H and C H An Fd fragment having a domain, (iv) V H and C H An Fd' fragment having a domain and one or more cysteine residues at the C-terminus of the CHI domain, (v) V of a single arm of an antibody L and V H An Fv fragment having domains, (vi) V that binds to an antigen H domain or V L A dAb fragment consisting of a domain (Ward et al., 341 Nature 544 (1989)), (vii) An isolated CDR region or an isolated CDR region present in a functional framework, (viii) An F(ab')2 fragment, which is a bivalent fragment containing two Fab' fragments linked by a disulfide bond in the hinge region, (ix) A single-chain antibody molecule (e.g., single-chain Fv, scFv) (Bird et al., 242 Science 423 (1988), and Huston et al., 85 PNAS 5879 (1988)), (x) A "diabody" (having two antigen-binding sites and containing a heavy-chain variable domain (V L ) bound to a light-chain variable domain (V H ) within the same polypeptide chain) (see, e.g., EP404,097, WO93 / 11161, Hollinger et al., 90 PNAS 6444 (1993)), (xi) A "linear antibody" containing a pair of tandem Fd segments (V H -C h 1-V H -C h 1), which forms a pair of "antibody-binding regions" together with complementary light-chain oligopeptides (Zapata et al., Protein Eng. 8(10):1057-62 (1995), and U.S. Patent No. 5,641,870).

[0181] The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which they are derived) for antigen binding (i.e., specific binding). Thus, these antibodies or fragments thereof are included within the scope of the present invention when the antibody or fragment specifically binds to a target molecule.

[0182] In some embodiments, the antibody is a single-chain antibody (scFv). As used herein, the term "single-chain variable fragment" refers to a fusion protein comprising the variable regions (s) of the heavy chain (V H ) and light chain (V L ) of an immunoglobulin linked via a linker peptide. The linker peptide is typically in the range of about 10 to about 25 amino acids. The linker can be flexible and can contain one or more glycine residues for flexibility. The linker can contain one or more serine residues or threonine residues to increase or alter solubility. V H and the light chain (V L ) can be joined in either order via the linker. In some embodiments, the N-terminus of V H is joined via the linker to the C-terminus of (V L ). In some embodiments, the C-terminus of V H is joined via the linker to the (V L) is bound to the N-terminus of. In some embodiments, the scFV is a bivalent or trivalent scFv. In some embodiments, the bivalent or trivalent scFv is bispecific or trispecific, which means that it can target 2 or 3 different epitopes respectively. Further, for example, Hollinger, Philipp; Prospero, T; Winter, G (July 1993). “Diabodies”: small bivalent and bispecific antibody fragments”. Proceedings of the National Academy of Sciences of the United States of America. 90(14):6444 - 8, incq, S; Bosman, F; Buyse, MA; Degrieck, R; Celis, L; De Boer, M; Van Doorsselaere, V; Sablon, E (2001). “Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80 / CD86 - CD28 costimulatory pathway”. Protein Expression and Purification.22 (1):11 - 24.doi:10.1006 / prep.2001.1417, Le gall, F.; Kipriyanov, SM; Moldenhauer, G; Little, M(1999). “Di-, Tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding”. FEBS Letters.453(1):164 - 168.doi:10.1016 / S0014 - 5793(99)00713 - 9, Huston, J.S.; Levinson, D.; Mudgett - Hunter, M.; Tai, M. S.; Novotny, J.; Margolies, M.N.; Crea, R.(1988). “Protein engineering of antibody binding sites:recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli”. Proceedings of the National Academy of Sciences of the United States of America. 85(16):5879-5883, see de Graaf et al., Methods Mol Biol. 2002;178:379-87.doi:10.1385 / 1-59259-240-6:379, Zhou, H.X., J Mol Biol. 2003 May 23;329(1):1-8.doi:10.1016 / s0022-2836(03)00372-3, Bird and Walker. Trends Biotechnol. 1991 Apr;9(4):132-7.doi:10.1016 / 0167-7799(91)90044-I, Worn et al., J Mol Biol. 2001 Feb 2;305(5):989-1010.doi:10.1006 / jmbi.2000.4265.

[0183] As used herein, the terms "heavy chain antibody", "VHH", or "single domain antibody" (sdAb) refer to an antibody composed of only two heavy chains and lacking the two light chains normally included in an antibody (see, for example, Henry and MacKenzie, Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs. 2018 Aug-Sep;10(6):815-826). VHH can refer to an antibody or a VHH domain. A single domain antibody (sdAb), also referred to as a "nanobody", is defined herein as an antibody fragment composed of a single monomeric variable antibody domain. As used herein, "VHH" is used synonymously with "nanobody". The variable domains (VHH and VNAR) of these antibodies, which are approximately 12 - 15 kDa, can be produced recombinantly and can recognize antigens even in the absence of the remaining portion of the antibody heavy chain. In a general antibody, the antigen-binding region is composed of the variable domains of the heavy and light chains (VH and VL). Heavy chain antibodies can bind antigens despite having only the VH domain. In certain embodiments, the heavy chain antibody is an antibody derived from cartilaginous fish (immunoglobulin new antigen receptor (IgNAR)) or camelid ungulates. Non-limiting examples of camelid ungulates include dromedary camels, camels, llamas, and alpacas.

[0184] The term "antibody" is intended to encompass any Ig class or any Ig subclass (e.g., the IgG1, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any origin (e.g., human and non-human primates, as well as rodents, rabbits, goats, cows, horses, sheep, etc.).

[0185] As used herein, the terms "Ig class" or "immunoglobulin class" refer to the five classes of immunoglobulins identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE. The term "Ig subclass" refers to two subclasses of IgM (H and L), three subclasses of IgA (IgA1, IgA2, and secretory IgA), and four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4) identified in humans and higher mammals. Antibodies can exist in monomeric or polymeric forms; for example, IgM antibodies exist in pentameric form and IgA antibodies exist in monomeric, dimeric, or multimeric forms.

[0186] The term "IgG subclass" refers to each of the four subclasses of immunoglobulin class IgG (IgG1, IgG2, IgG3, and IgG4) that are identified in humans and higher mammals by the heavy chains (V1-γ4) of immunoglobulins. The term "single-chain immunoglobulin" or "single-chain antibody" (used interchangeably herein) refers to a protein having a two-polypeptide chain structure consisting of a heavy chain and a light chain, the chains being stabilized, for example, by an interchain peptide linker having the ability to specifically bind to an antigen. The term "domain" refers to a globular region of a heavy or light chain polypeptide that includes, for example, a peptide loop stabilized by β-pleated sheets and / or intrachain disulfide bonds (e.g., includes 3-4 peptide loops). Domains are further referred to herein as "constant" or "variable." A "constant" domain is so called based on the relative lack of sequence variation within the domains of various class members, and a "variable" domain is so called based on the significant variation within the domains of various class members. The "domain" of an antibody or polypeptide is often synonymously referred to in the art as the "region" of the antibody or polypeptide. The "constant" domain of an antibody light chain is synonymously referred to as the "light chain constant region," "light chain constant domain," "CL" region, or "CL" domain. The "constant" domain of an antibody heavy chain is synonymously referred to as the "heavy chain constant region," "heavy chain constant domain," "CH" region, or "CH" domain. The "variable" domain of an antibody light chain is synonymously referred to as the "light chain variable region," "light chain variable domain," "VL" region, or "VL" domain. The "variable" domain of an antibody heavy chain is synonymously referred to as the "heavy chain constant region," "heavy chain constant domain," "VH" region, or "VH" domain.

[0187] The term "region" can also refer to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain as defined herein, or a part or portion of a constant or variable domain), and to more discrete parts or portions of such chain or domain. For example, light and heavy chains or light and heavy chain variable domains contain "complementary determining regions" or "CDRs" that are interspersed among "framework regions" or "FRs" as defined herein.

[0188] The term "conformation" refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, its antibody chain, domain, or region). For example, the term "light (or heavy) chain conformation" refers to the tertiary structure of the light (or heavy) chain variable region, and the terms "antibody conformation" or "antibody fragment conformation" refer to the tertiary structure of an antibody or its fragment.

[0189] The term "antibody-like protein scaffold" or "engineered protein scaffold" broadly encompasses proteinaceous non-immunoglobulin specific binding factors typically obtained by combinatorial engineering (e.g., site-directed random mutagenesis combined with phage display or other molecular selection techniques). Generally, such scaffolds are derived from a stable, soluble small monomeric protein (such as a kunitz inhibitor or lipocalin) or from a stably folded extracellular domain of a cell surface receptor (such as protein A, fibronectin, or ankyrin repeats).

[0190] Such scaffolds are widely outlined below. Binz et al. (Engineering novel binding proteins from non-immunoglobulin domains. Nat Biotechnol 2005,23:1257-1268), Gebauer and Skerra (Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol.2009,13:245-55), Gill and Damle (Biopharmaceutical drug discovery using novel protein scaffolds. Curr Opin Biotechnol 2006,17:653-658), Skerra (Engineered protein scaffolds for molecular recognition. J Mol Recognit 2000,13:167-187), and Skerra (Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol 2007,18:295-304). Such scaffolds include, without limitation, the following. Affibodies based on the Z domain of staphylococcal protein A, a 58-residue three-helix bundle that provides interfaces to two of the α-helices (Nygren, Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 2008,275:2668-2676), engineered knotted domains based on a 58-residue small polypeptide and a robust disulfide-bridged serine protease inhibitor, usually of human origin (e.g., LACI-D1), which can be engineered to match various protease specificities (Nixon and Wood, Engineered protein inhibitors of proteases.Curr Opin Drug Discov Dev 2006,9:261-268), monobodies or adnectins based on the tenth extracellular domain of human fibronectin type III (10Fn3), which adopt an Ig-like beta-sandwich fold (94 residues) with two to three exposed loops but lack a central disulfide bridge (Koide and Koide, Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain. Methods Mol Biol 2007,352:95-109), anticalins derived from the lipocalin family, a diverse family of eight-stranded beta-barrel proteins (approx. 180 residues) that naturally form binding sites for small ligands by four structurally variable loops at the open ends (which are abundant in humans, insects, and many other organisms) (Skerra, Alternative binding proteins: Anticalins - harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities. FEBS J 2008,275:2677-2683), DARPins (designed ankyrin repeat domains (166 residues)), which usually result in a rigid interface arising from three repeating beta-turns (Stumpp et al., DARPins: a new generation of protein therapeutics. Drug Discov Today 2008,13:695-701), avimers (multimerized LDLR-A modules) (Silverman et al., Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains.(Nat Biotechnol 2005, 23: 1556-1561), and cystine-rich Notch peptides (Kolmar "Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins. FEBS J 2008, 275: 2684-2690).

[0191] "Specific binding" of an antibody means that the antibody exhibits a recognizable affinity for a specific antigen or epitope and generally does not exhibit significant cross-reactivity. "Recognizable" binding includes binding with an affinity of at least 25 μM. An antibody with an affinity greater than 1×10 7 M -1 (or a dissociation coefficient of 1 μM or less or a dissociation coefficient of 1 nM or less) typically binds with a correspondingly greater specificity. Intermediate values of the values shown herein are also within the scope of the present invention, and the antibodies of the present invention can bind, for example, within an affinity range of 100 nM or less, 75 nM or less, 50 nM or less, 25 nM or less, for example, 10 nM or less, 5 nM or less, 1 nM or less, or in an embodiment, 500 pM or less, 100 pM or less, 50 pM or less, or 25 pM or less. An antibody that "does not exhibit significant cross-reactivity" is an antibody that does not recognizably bind to something other than its target (e.g., a different epitope or another molecule). For example, an antibody that specifically binds to a target molecule recognizably binds to the target molecule but does not significantly react with a non-target molecule or peptide. An antibody specific for a particular epitope, for example, does not significantly cross-react with a distant epitope on the same protein or peptide. Specific binding can be determined by any means recognized in the art for determining such binding. Specific binding is preferably determined by Scatchard analysis and / or competitive binding assays.

[0192] As used herein, the term "affinity" refers to the strength of the binding between a single antigen-binding site and an epitope. Affinity depends on, among other things, the closeness of the stereochemical fit between the antibody-binding site and the epitope, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Antibody affinity can be measured by equilibrium dialysis or by the kinetic BIACORE™ method. The dissociation constant Kd and the association constant Ka are quantitative measures of affinity.

[0193] As used herein, the term "monoclonal antibody" refers to an antibody derived from a cloned population of antibody-producing cells (e.g., B lymphocytes or B cells) that are uniform in structure and antigen specificity. The term "polyclonal antibody" refers to a plurality of antibodies of different cloned populations of antibody-producing cells that are non-uniform in structure and epitope specificity but recognize a common antigen. Monoclonal and polyclonal antibodies may be present as crude preparations in body fluids or may be purified as described herein. A "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody that contains minimal sequences derived from non-human immunoglobulins. In most parts, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable regions of the recipient are replaced by residues from the hypervariable regions of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate that has the desired specificity, affinity, and capacity. In some cases, FR residues of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody can contain residues that are not present in either the recipient antibody or the donor antibody. Such modifications are made to further improve the performance of the antibody. Generally, humanized antibodies contain substantially all or at least one, typically two variable domains, in which all or substantially all of the hypervariable regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin sequence. A humanized antibody optionally also contains at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin.

[0194] As used herein, a "blocking" antibody or antibody "antagonist" is one that inhibits or reduces the biological activity of the antigen(s) to which it binds. In certain embodiments, the blocking antibodies or antagonist antibodies or fragments thereof described herein completely inhibit the biological activity of the antigen(s).

[0195] Antibodies can act as agonists or antagonists of the polypeptides they recognize. For example, the present invention includes antibodies that partially or completely interfere with receptor / ligand interactions. The present invention features both receptor-specific antibodies and ligand-specific antibodies. The present invention also features receptor-specific antibodies that do not block ligand binding but block receptor activation. Receptor activation (i.e., signal transduction) can be determined by the methods described herein or methods known in the art. For example, receptor activation can be determined by detecting phosphorylation (e.g., tyrosine or serine / threonine) of one of the receptor or its downstream substrates by Western blot analysis performed after immunoprecipitation. In certain embodiments, an antibody is provided that inhibits ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in the absence of the antibody.

[0196] The present invention also features receptor-specific antibodies that block both ligand binding and receptor activation, as well as antibodies that recognize receptor-ligand complexes. Similarly, the present invention encompasses neutralizing antibodies that bind to a ligand and prevent the ligand from binding to its receptor, and the present invention also encompasses antibodies that bind to a ligand and thereby prevent receptor activation, but do not prevent the ligand from binding to the receptor. Antibodies that activate the receptor are further included in the present invention. These antibodies can act as receptor agonists. That is, these antibodies can enhance or activate all or a subset of the biological activities of ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. Antibodies can be identified as agonists, antagonists, or inverse agonists to biological activities including the specific biological activities of the peptides disclosed herein. See, for example, PCT Publication No. WO96 / 40281, U.S. Patent No. 5,811,097, Deng et al., Blood 92(6):1981-1988(1998), Chen et al., Cancer Res. 58(16):3668-3678(1998), Harrop et al., J. Immunol. 161(4):1786-1794(1998), Zhu et al., Cancer Res. 58(15):3209-3214(1998), Yoon et al., J. Immunol. 160(7):3170-3179(1998), Prat et al., J. Cell. Sci. III(Pt2):237-247(1998), Pitard et al., J. Immunol. Methods 205(2):177-190(1997), Liautard et al., Cytokine 9(4):233-241(1997), Carlson et al., J. Biol. Chem. 272(17):11295-11301(1997), Taryman et al., Neuron 14(4):755-762(1995), Muller et al., Structure 6(9):1153-1167(1998), Bartunek et al., Cytokine 8(1):14-20(1996).

[0197] Antibodies as defined in the present invention include modified derivatives (i.e., derivatives modified by covalently bonding any type of molecule to the antibody, and the covalent bond prevents the antibody from generating an anti-idiotype response). For example, without limitation, antibody derivatives include antibodies modified by, for example, glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cell ligands or other proteins, etc. Any of a number of chemical modifications can be carried out by known techniques. Such techniques include, but are not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative may contain one or more non-classical amino acids.

[0198] The engineered cells and engineered organisms expressing the engineered AAV capsid As used herein, engineered cells are described. The engineered cells can include one or more of an engineered AAV capsid polynucleotide, polypeptide, vector, and / or vector system. In some embodiments, one or more of the engineered AAV capsid polynucleotides can be expressed within the engineered cells. In some embodiments, the engineered cells can generate the engineered AAV capsid protein and / or engineered AAV capsid particles as described elsewhere herein. Also as used herein, a modified organism or engineered organism is described. The modified organism or engineered organism can include one or more of the engineered cells described herein. The engineered cells can be engineered to express a cargo molecule (e.g., a cargo polynucleotide) either dependent or independent of the engineered AAV capsid polynucleotide as described elsewhere herein (e.g., packaging into an engineered AAV capsid as described herein).

[0199] A variety of animals, plants, algae, fungi, yeasts, etc., and systems of animal, plant, algal, fungal, yeast cells, or tissues can be engineered to express one or more nucleic acid constructs of the engineered AAV capsid systems described herein using the various transformation methods referred to elsewhere in this specification. This can result in obtaining organisms that can generate engineered AAV capsid particles for production purposes, the design and / or generation of engineered AAV capsids, and / or for model organisms. In some embodiments, a polynucleotide(s) encoding one or more components of the engineered AAV capsid systems described herein can be stably or transiently incorporated into one or more cells of a plant, animal, algae, fungus, and / or yeast or tissue system. In some embodiments, one or more of the engineered AAV capsid system polynucleotides can be incorporated into the genome of one or more cells of a plant, animal, algae, fungus, and / or yeast or tissue system. Further embodiments of the engineered organisms and systems are described elsewhere in this specification. In some embodiments, one or more components of the engineered AAV capsid systems described herein are expressed in one or more cells of a plant, animal, algae, fungus, yeast, or tissue system.

[0200] Engineered cell This specification describes various embodiments of engineered cells. The engineered cells can include one or more of the engineered AAV capsid-based polynucleotides, polypeptides, vectors, and / or vector systems described elsewhere in this specification. In some embodiments, the cells can express one or more of the engineered AAV capsid polynucleotides and can generate one or more engineered AAV capsid particles, which will be described in more detail herein. Such cells are also referred to herein as "producer cells." Clearly, these engineered cells are different from the "modified cells" described elsewhere in this specification. This is because a modified cell is not necessarily a producer cell unless it includes one or more of the engineered AAV capsid polynucleotides, the engineered AAV capsid vectors described herein, or other vectors that confer on the cell the ability to produce engineered AAV capsid particles. A modified cell can be a recipient cell for the engineered AAV capsid particles and, in some embodiments, can be modified by the engineered AAV capsid particles (s) and / or the cargo polynucleotide delivered to the recipient cell. Modified cells will be described in more detail elsewhere in this specification. The term "modified" may be used in the context of modifying cells that do not depend on being recipient cells. For example, isolated cells can be modified before receiving the engineered AAV capsid molecules.

[0201] In one embodiment, the invention provides a non-human eukaryote, such as a multicellular eukaryote, that includes a eukaryotic host cell that includes one or more components of the engineered delivery system described herein according to any of the described embodiments. In other embodiments, the invention provides a eukaryote, preferably a multicellular eukaryote, that includes a eukaryotic host cell that includes one or more components of the engineered delivery system described herein according to any of the described embodiments. In some embodiments, the organism is a host for AAV.

[0202] In certain embodiments, the resulting cells or parts thereof, such as plants, algae, fungi, yeast, etc., are transgenic plants that contain exogenous DNA sequences integrated into all or part of the genome of the cells.

[0203] The engineered cells can be prokaryotic cells. The prokaryotic cells can be bacterial cells. The prokaryotic cells can be archaeal cells. The bacterial cells can be any suitable bacterial cells. Suitable bacterial cells can be those of the genera Escherichia, Bacillus, Lactobacillus, Rhodococcus, Rhodobacter, Synechococcus, Synechocystis, Pseudomonas, Psedoaltermonas, Stenotrophamonas, and Streptomyces. Suitable bacterial cells include, but are not limited to, Escherichia coli cells, Caulobacter crescentus cells, Rhodobacter sphaeroides cells, and Psedoaltermonas haloplanktis cells. Suitable bacterial strains include, but are not limited to, BL21(DE3), DL21(DE3)-pLysS, BL21Star-pLysS, BL21-SI, BL21-AI, Tuner, Tuner pLysS, Origami, Origami B pLysS, Rosetta, Rosetta pLysS, Rosetta-gami-pLysS, BL21 CodonPlus, AD494, BL2trxB, HMS174, NovaBlue(DE3), BLR, C41(DE3), C43(DE3), Lemo21(DE3), Shuffle T7, ArcticExpress, and ArcticExpress(DE3).

[0204] The manipulated cells can be eukaryotic cells. The eukaryotic cells may be cells derived from specific organisms such as plants and mammals, and such organisms include, but are not limited to, humans, or non-human eukaryotes, animals, or mammals (e.g., mice, rats, rabbits, dogs, livestock, or non-human mammals or primates) as shown herein. In some embodiments, the manipulated cells can be a cell line. Examples of cell lines include C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial cells, BALB / 3T3 mouse fetal fibroblasts, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts, 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr- / - , COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CMLT1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa 1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR / 0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell lines, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic forms thereof, including but not limited to. Cell lines are available from a variety of sources known to those skilled in the art (see, for example, American Type Culture Collection (ATCC), Manassas, Virginia).

[0205] In some embodiments, the engineered cells are muscle cells (e.g., cardiac, skeletal, and / or smooth muscle), bone cells, blood cells, immune cells (including but not limited to B cells, macrophages, T cells, CAR-T cells, etc.), kidney cells, bladder cells, lung cells, heart cells, liver cells, brain cells, neurons, skin cells, stomach cells, nerve support cells, intestinal cells, epithelial cells, endothelial cells, stem cells or other progenitor cells, adrenal cells, chondrocytes, and combinations thereof.

[0206] In some embodiments, the engineered cells can be fungal cells. As used herein, "fungal cell" refers to any type of eukaryotic cell within the fungal kingdom. Phyla within the fungal kingdom include Ascomycota, Basidiomycota, Glomeromycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. Examples of fungal cells include yeast, mold, and filamentous fungi. In some embodiments, the fungal cells are yeast cells.

[0207] As used herein, the term "yeast cell" refers to any fungal cell within the phyla Ascomycota and Basidiomycota. Yeast cells include budding yeast cells, fission yeast cells, and mold cells. Without being limited to these organisms, many types of yeast used in the laboratory and industrial settings belong to the phylum Ascomycota. In some embodiments, the yeast cell is a cell of S. cerevisiae, Kluyveromyces marxianus, or Issatchenkia orientalis. Other yeast cells include, but are not limited to, cells of the genus Candida (e.g., Candida albicans), the genus Yarrowia (e.g., Yarrowia lipolytica), the genus Pichia (e.g., Pichia pastoris), the genus Kluyveromyces (e.g., Kluyveromyces lactis and Kluyveromyces marxianus), the genus Neurospora (e.g., Neurospora crassa), the genus Fusarium (e.g., Fusarium oxysporum), and the genus Issatchenkia (e.g., Issatchenkia orientalis, a.k.a. Pichia kudriavzevii, and Candida acidothermophilum). In some embodiments, the fungal cell is a filamentous fungal cell. As used herein, the term "filamentous fungal cell" refers to any type of fungal cell that grows in a filamentous form, i.e., hyphae or mycelium. Examples of filamentous fungal cells include, but are not limited to, cells of the genus Aspergillus (e.g., Aspergillus niger), the genus Trichoderma (e.g., Trichoderma reesei), the genus Rhizopus (e.g., Rhizopus oryzae), and the genus Mortierella (e.g., Mortierella isabellina).

[0208] In some embodiments, the fungal cells are industrial strains. As used herein, "industrial strain" refers to any strain of fungal cells that is used in an industrial process (e.g., the manufacture of products on a commercial or industrial scale) or that is isolated from an industrial process. An industrial strain may typically refer to a fungal species used in an industrial process, or may refer to an isolate of a fungal species that can also be used for non-industrial purposes (such as laboratory research). Examples of industrial processes include fermentation (e.g., the manufacture of food and beverage products), distillation, the manufacture of biofuels, the manufacture of compounds, and the manufacture of polypeptides. Examples of industrial strains include, but are not limited to, JAY270 and ATCC4124.

[0209] In some embodiments, the fungal cells are polyploid cells. As used herein, "polyploid" cells can refer to any cells in which the genome is present in multiple copies. Polyploid cells may refer to cell types that naturally exist in a polyploid state, or may refer to cells that have been induced to exist in a polyploid state (e.g., through specific controls, alterations, inactivations, activations, or modifications of meiosis, cytokinesis, or DNA replication). Polyploid cells may refer to cells in which the entire genome is polyploid, or may refer to cells that are polyploid at a specific genomic locus of interest.

[0210] In some embodiments, the fungal cell is a diploid cell. As used herein, a "diploid" cell can refer to any cell in which the genome is present in two copies. A diploid cell may refer to a cell type that naturally exists in a diploid state, or a cell that has been induced to exist in a diploid state (e.g., through specific control, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the S. cerevisiae strain S228C can be maintained in a haploid or diploid state. A diploid cell may refer to a cell in which the entire genome is diploid, or a cell that is diploid at a specific genomic locus of interest. In some embodiments, the fungal cell is a haploid cell. As used herein, a "haploid" cell can refer to any cell in which the genome is present in one copy. A haploid cell may refer to a cell type that naturally exists in a haploid state, or a cell that has been induced to exist in a haploid state (e.g., through specific control, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the S. cerevisiae strain S228C can be maintained in a haploid or diploid state. A haploid cell may refer to a cell in which the entire genome is haploid, or a cell that is haploid at a specific genomic locus of interest.

[0211] In some embodiments, the engineered cell is a cell obtained from a subject. In some embodiments, the subject is a healthy or non-diseased subject. In some embodiments, the subject is a subject having desired physiological and / or biological characteristics, such that when the engineered AAV capsid particles are generated, they can be related to the desired physiological and / or biological characteristics and / or can modify the desired physiological and / or biological characteristics, and is a subject capable of packaging one or more cargo polynucleotides. Thus, the cargo polynucleotides of the generated engineered AAV capsid particles may be capable of introducing the desired characteristics into recipient cells. In some embodiments, the cargo polynucleotide can modify the polynucleotide of the engineered cell such that the engineered cell has the desired physiological and / or biological characteristics.

[0212] In some embodiments, cells transfected with one or more vectors described herein are used to establish a new cell line containing one or more vector-derived sequences.

[0213] The engineered cell can be used to generate engineered virus (e.g., AAV) capsid polynucleotides, vectors, and / or particles. In some embodiments, the engineered virus (e.g., AAV) capsid polynucleotides, vectors, and / or particles are generated, collected, and / or delivered to a subject in need thereof. In some embodiments, the engineered cell is delivered to a subject. Other uses of the engineered cell are described elsewhere in this specification. In some embodiments, the engineered cell can be included in formulations and / or kits described elsewhere in this specification.

[0214] The processed cells can be stored for short or long terms for later use. Appropriate storage methods are generally known in the art. Further, methods for restoring the stored cells for later use (such as thawing, reconstitution, and other methods that stimulate metabolism in the processed cells) are also generally known in the art.

[0215] Formulation The compositions, polynucleotides, polypeptides, particles, cells, vector systems, and combinations thereof described herein can be included in formulations such as pharmaceutical formulations. In some embodiments, the formulation can be used to generate polypeptides and other particles comprising one or more of the CNS-specific n amino acid lengths described herein. In some embodiments, the formulation can be delivered to a subject in need thereof. In some embodiments, the components of the processed AAV capsid system, processed cells, processed AAV capsid particles, and / or combinations thereof described herein can be included in a formulation that can be delivered to a subject or cells. In some embodiments, the formulation is a pharmaceutical formulation. One or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be supplied to a subject in need thereof, or to cells alone, or as an active ingredient such as in a pharmaceutical formulation. Accordingly, pharmaceutical formulations are further described herein, which comprise one or more of the polypeptides, polynucleotides, vectors, cells, or combinations thereof described herein. In some embodiments, the pharmaceutical formulation can contain one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein in an effective amount. The pharmaceutical formulations described herein can be administered to a subject or cells in need thereof.

[0216] In some embodiments, the amount of one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein contained in a pharmaceutical formulation can range from about 1 pg / kg to about 10 mg / kg, based on the body weight of the subject in need thereof, or the average body weight of a particular patient population to which the pharmaceutical formulation can be administered. The amount of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein contained in a pharmaceutical formulation can range from about 1 pg to about 10 g, about 10 nL to about 10 mL. In embodiments where the pharmaceutical formulation contains one or more cells, the amount of cells can range from about 1 to 1 x 10 2 cells, 1 x 10 3 cells, 1 x 10 4 cells, 1 x 10 5 cells, 1 x 10 6 cells, 1 x 10 7 cells, 1 x 10 8 cells, 1 x 10 9 cells, 1 x 10 10 cells, or up to 1 x 10 2 cells, 1 x 10 3 cells, 1 x 10 4 cells, 1 x 10 5 cells, 1 x 10 6 cells, 1 x 10 7 cells, 1 x 10 8 cells, 1 x 10 9 cells, 1 x 10 10 cells, or up to 1 x 10

[0217] In embodiments where engineered AAV capsid particles are included in the formulation, the formulation contains 1 to 1 x 10 1 、1 x 10 2 、1 x 10 3 、1 x 10 4 、1 x 10 5 、1 x 10 6 、1 x 10 7 、1 x 10 8 、1 x 10 9 、1 x 10 10 、1 x 1011 、 1 x 10 12 、 1 x 10 13 、 1 x 10 14 、 1 x 10 15 、 1 x 10 16 、 1 x 10 17 、 1 x 10 18 、 1 x 10 19 、 or 1 x 10 20 of transducing units (TU) / mL of processed AAV capsid particles. In some embodiments, the formulation can have a volume of 0.1 - 100 mL and 1 - 1 x 10 1 、 1 x 10 2 、 1 x 10 3 、 1 x 10 4 、 1 x 10 5 、 1 x 10 6 、 1 x 10 7 、 1 x 10 8 、 1 x 10 9 、 1 x 10 10 、 1 x 10 11 、 1 x 10 12 、 1 x 10 13 、 1 x 10 14 、 1 x 10 15 、 1 x 10 16 、 1 x 10 17 、 1 x 10 18 、 1 x 10 19 、 or 1 x 10 20 of transducing units (TU) / mL of processed AAV capsid particles.

[0218] pharmaceutically acceptable carriers, excipients, and adjuvants In an embodiment, a pharmaceutical formulation containing one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein in an amount may further contain a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, sugars such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, essential oils, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone that do not react detrimentally with the active composition thereof.

[0219] The pharmaceutical formulation can be sterilized and, if desired, can be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts that affect osmotic pressure, buffering agents, coloring substances, flavoring substances and / or aromatic substances that do not react detrimentally with the active composition.

[0220] In addition to an amount of one or more of the polypeptides, polynucleotides, vectors, cells, engineered AAV capsid particles, nanoparticles, other delivery particles, and combinations thereof described herein, the pharmaceutical formulation can also contain an effective amount of an auxiliary active agent, and examples of the auxiliary active agent include, but are not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatory agents, antihistamines, anti-infective agents, chemotherapeutic agents, and combinations thereof.

[0221] Suitable hormones include, but are not limited to, amino acid-derived hormones (e.g., melatonin and thyroxine), small peptide hormones and protein hormones (e.g., thyrotropin-releasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eicosanoids (e.g., arachidonic acid, lipoxin, and prostaglandin), and steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone, and cortisol). Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12), cytokines (e.g., interferons (e.g., IFN-a, IFN-β, IFN-ε, IFN-K, IFN-ω, and IFN-γ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26, and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotide, glucan, antibody, and aptamer).

[0222] Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammatory agents (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate), paracetamol / acetaminophen, metamizole, nabumetone, phenazone, and quinine.

[0223] Suitable anxiolytics include, but are not limited to, benzodiazepines (e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, chlorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotonin antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), mebicar, fabomotizole, seranek, bromantan, emoxypine, azapirone, barbiturate, hydroxyzine, pregabalin, valdoal, and beta blockers.

[0224] Suitable antipsychotics include, but are not limited to, benperidol, bromperidol, droperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, ciamemazine, dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, periciazine, perphenazine, pipothiazine, prochlorperazine, promazine, promethazine, prothipendyl, thiopropazate, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupenthixol, thiothixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzapine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, diplasidone, zotepine, alstonine, bifeprunox, vitaperzine, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, babicaserin, xanomeline, and dichloranipin.

[0225] Suitable analgesics include, but are not limited to, paracetamol / acetaminophen, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupirtine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate).

[0226] Suitable antispasmodics include, but are not limited to, mebeverine, papaverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, methaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene. Suitable anti-inflammatory agents include, but are not limited to, prednisone, non-steroidal anti-inflammatory drugs (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immunoselective anti-inflammatory derivatives (e.g., submandibular gland peptide-T and its derivatives).

[0227] Suitable antihistamines include H1-receptor antagonists (e.g., acrivastine, azelastine, bilastine, brompheniramine, bucrizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimethindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, meclizine, mirtazapine, olopatadine, orphenadrine, phenindamine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2-receptor antagonists (e.g., cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine), tritoqualine, catechin, cromoglycate, nedocromil, and P2-adrenergic agonists, but are not limited thereto.

[0228] Suitable anti-infective agents include anti-amebic agents (e.g., nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, amphotericin b, and iodoquinol), aminoglycoside antibiotics (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin, praziquantel, albendazole, thiabendazole, oxamniquine), antifungal agents (e.g., azole antifungal agents (e.g., itraconazole, fluconazole, parconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g., caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (e.g., nystatin and amphotericin b), antimalarial agents (e.g., pyrimethamine / sulfadoxine, artemether / lumefantrine, atovaquone / proguanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g., aminosalicylates (e.g., para-aminosalicylic acid), isoniazid / rifampin, isoniazid / pyrazinamide / rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antiviral agents (e.g., amantadine, rimantadine, abacavir / lamivudine, emtricitabine / tenofovir, cobicistat / elvitegravir / emtricitabine / tenofovir, efavirenz / emtricitabine / tenofovir, abacavir / lamivudine / zidovudine, lamivudine / zidovudine, emtricitabine / tenofovir, emtricitabine / ritonavir / lopinavir / tenofovir, interferon α-2V / ribavirin, peginterferon α-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delavirdine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, abacavir,Didobuzin, Stubuzin, Emtricitabine, Zalcitabine, Telbivudine, Simiprevir, Boceprevir, Telaprevir, Ritonavir / Lopinavir, Boceprevir, Darunavir, Ritonavir, Tipranavir, Atazanavir, Nelfinavir, Amprenavir, Indinavir, Saquinavir, Ribavirin, Valacyclovir, Acyclovir, Famciclovir, Ganciclovir, and Valganciclovir), Carbapenem (e.g., Doripenem, Meropenem, Ertapenem, and Silastatin / Imipenem), Cephalosporin (e.g., Cefadroxil, Cefradine, Cefazolin, Cefalexin, Cefepime, Cefazolin, Loracarbef, Cefotetan, Cefuroxime, Cefprozil, Loracarbef, Cefoxitin, Cefaclor, Cefixime, Cefdinir, Cefixime, Cefditoren, Cefotaxime, Cefpodoxime, Ceftazidime, and Cefotaxime), Glycopeptide antibiotics (e.g., Vancomycin, Dalbavancin, Oritavancin, and Telavancin), Glycylcycline (e.g., Tigecycline), Antileprosy agents (e.g., Clofazimine and Thalidomide), Lincomycin and its derivatives (e.g., Clindamycin and Lincomycin), Macrolide and its derivatives (e.g., Telithromycin, Fidaxomicin, Erythromycin, Azithromycin, Clarithromycin, Dirithromycin, and Troleandomycin), Linezolid, Sulfamethoxazole / Trimethoprim, Rifaximin, Chloramphenicol, Fosfomycin, Metronidazole, Aztreonam, Bacitracin, Penicillin (Amoxicillin, Ampicillin, Bacampicillin, Carbenicillin, Piperacillin, Ticarcillin, Amoxicillin / Clavulanate, Ampicillin / Sulbactam, Piperacillin / Tazobactam, Clavulanate / Ticarcillin, Penicillin, Procaine Penicillin, Oxacillin, Dicloxacillin, and Nafcillin), Quinolone (e.g., Romefloxacin, Norfloxacin, Ofloxacin, Gatifloxacin, Moxifloxacin, Ciprofloxacin, Levofloxacin, Gemifloxacin, Moxifloxacin, Cinoxacin, Nalidixic acid, Enoxacin, Grepafloxacin, Gatifloxacin, Trovafloxacin,and sparfloxacin), sulfonamides (e.g., sulfamethoxazole / trimethoprim, sulfasalazine and sulfisoxazole), tetracyclines (e.g., doxycycline, demeclocycline, minocycline, doxycycline / salicylic acid, doxycycline / ω-3 polyunsaturated fatty acids, and tetracycline), and anti-urinary tract infection agents (e.g., nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue), but are not limited thereto.,

[0229] Suitable chemotherapeutic agents include paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, dacarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, visimodegib, ERwINIaChrysanthemum - derived asparaginase, amifostine, etoposide, flutamide, tamoxifen, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylate, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon α - 2a, gefitinib, romidepsin, ixabepilone, luxolitinib, cabazitaxel, ado - trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium chloride - 89, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octreotide, dasatinib, regorafenib, histrelin, sunitinib, cetuximab, omacetaxine, thioguanine (ThIoguaNINe), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG (Bacillus Calmette - Guérin), temsirolimus, bendamustine hydrochloride, triptorelin, arsenic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafurposide, azathioprine, doxifluridine, vindesine, and all - trans retinoic acid, etc., but not limited thereto.

[0230] In embodiments where there is an auxiliary active agent included in the pharmaceutical formulation in addition to one or more of the polypeptides, polynucleotides, CRISPR-Cas complexes, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein, the amount, such as the effective amount of the auxiliary active agent, will vary depending on the auxiliary active agent. In some embodiments, the amount of the auxiliary active agent ranges from 0.00l micrograms to about 1 milligram. In another embodiment, the amount of the auxiliary active agent ranges from about 0.01 IU to about 1000 IU. In a further embodiment, the amount of the auxiliary active agent ranges from 0.001 mL to about 1 mL. In yet another embodiment, the amount of the auxiliary active agent ranges from about 1% w / w to about 50% w / w of the total pharmaceutical formulation. In additional embodiments, the amount of the auxiliary active agent ranges from about 1% v / v to about 50% v / v of the total pharmaceutical formulation. In yet another embodiment, the amount of the auxiliary active agent ranges from about 1% w / v to about 50% w / v of the total pharmaceutical formulation.

[0231] Dosage form In some embodiments, the pharmaceutical formulations described herein can be in a dosage form. The dosage form can be adapted for administration by any suitable route. Suitable routes include, but are not limited to, rectal, epidural, intracranial, intraocular, inhalation, intranasal, topical (including oral, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intra-articular, intracavernosal, intrathecal, intravitreal, intracerebral, gingival, subgingival, intraventricular, and intradermal. The formulations as described above can be prepared by any method known in the art.

[0232] Dosage forms suitable for parenteral administration and / or suitable for any type of injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intraarticular, intracavernosal, gingival, subgingival, intramedullary, intravitreal, intracerebral, and intraventricular) include aqueous and non-aqueous sterile injection solutions (which may contain antioxidants, buffers, bacteriostats, and solutes to render the composition isotonic with the blood of the subject), as well as aqueous and / or non-aqueous sterile suspensions (which may contain suspending agents and thickening agents). Dosage forms suitable for parenteral administration can be provided in single-unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The dosage can be lyophilized and resuspended in a sterile carrier for reconstitution prior to administration. Immediate injection solutions and suspensions can be prepared from sterile powders, granules, and tablets in some embodiments. See, for example, Glascock, J.J, et al. Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice. J. Vis. Exp. (56), e2968 and Foley CP, et al. Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption. J Control Release. 2014 Dec 28;196:71-78.

[0233] The dosage form can also be prepared to extend or sustain the release of any component. In some embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be the component to be released slowly. In other embodiments, the release of auxiliary components included as necessary is delayed. Suitable methods for delaying the release of components include, but are not limited to, coating or embedding the components with materials such as polymers, waxes, gels, etc. Sustained-release formulations can be prepared as described in standard references. Such references are, for example, "Pharmaceutical dosage form tablets," eds. Liberman et.al. (New York, Marcel Dekker, Inc., 1989), "Remington - The science and practice of pharmacy", 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and "Pharmaceutical dosage forms and drug delivery systems", 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995).

[0234] Dosage forms suitable for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments for treating the eye or other external tissues, such as the mouth or skin, the pharmaceutical formulation is applied as a topical ointment or cream. When formulating as an ointment, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be formulated with a paraffin or water - miscible ointment base. In some embodiments, the active ingredient can be formulated as a cream with an oil - in - water cream base or a water - in - oil base. Dosage forms suitable for topical administration in the mouth include lozenges, troches, and gargles.

[0235] Dosage forms suitable for nasal or inhalation administration include aerosols, solutions, suspension droplets, gels, or dry powders. In some embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein are included in a dosage form suitable for inhalation, and the dosage form is in a particle size-reduced form that can be obtained or is obtainable by micronization. In some embodiments, the particle size of the size-reduced (e.g., micronized) compound, or a salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by appropriate methods known in the art. Dosage forms suitable for administration by inhalation also include powders or mists. For administration as a nasal spray or nasal drops, suitable dosage forms in which the carrier or additive is liquid include aqueous or oily solutions / suspensions of the active ingredient (e.g., one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein, and / or an auxiliary active agent), which can be generated by various types of metered-dose pressurized aerosols, nebulizers, or inhalers.

[0236] In some embodiments, the dosage form can be an aerosol formulation suitable for administration by inhalation. In some of these embodiments, the aerosol formulation can include a solution or fine suspension of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and a pharmaceutically acceptable aqueous or non-aqueous solvent. The aerosol formulation can be supplied in a sterile form, in a single-dose or multi-dose sealed container. In some of these embodiments, the sealed container is a single-dose or multi-dose nasal or aerosol dispenser (e.g., a metered-dose inhaler) equipped with a metering valve, and the container is intended to be discarded when its contents are exhausted.

[0237] When placing an aerosol dosage form in an aerosol dispenser, the dispenser is filled with a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant (including, but not limited to, hydrofluorocarbons). The dosage form of the aerosol formulation in other embodiments is placed in a pump sprayer. The pressurized aerosol formulation can also contain a solution or suspension of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. In further embodiments, the aerosol formulation can also contain cosolvents and / or modifiers incorporated for the purpose of, for example, improving the stability of the formulation and / or the taste and / or the characteristics (amount and / or profile) of the particulate population. Administration of the aerosol formulation can be once a day or several times a day, for example, 2, 3, 4, or 8 times a day, where each time, 1, 2, or 3 doses are delivered.

[0238] For some dosage forms suitable for and / or adapted for inhalation administration, the pharmaceutical formulation is an inhalable dry powder formulation. In addition to one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein, an auxiliary active ingredient, and / or a pharmaceutically acceptable salt thereof, such dosage forms can contain a powder base such as lactose, glucose, trehalose, mannitol, and / or starch. In some of these embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein are in a reduced particle size form. In further embodiments, there are performance modifiers such as L-leucine or another amino acid, cellobiose octaacetate, and / or a metal salt of stearic acid (such as magnesium stearate or calcium stearate).

[0239] In some embodiments, the aerosol dosage form can be adjusted so that each metered dose of the aerosol contains a predetermined amount of the active ingredient (for example, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein).

[0240] Dosage forms suitable for ophthalmic administration may include aqueous and / or non-aqueous sterile solutions that can be made suitable for injection if necessary. Such solutions may optionally include aqueous and non-aqueous sterile suspensions that can include antioxidants, buffers, bacteriostatic agents, solutes that render the composition isotonic with the fluids present in the eye or in the intraocular or periorbital region of interest, and suspending and thickening agents.

[0241] In some embodiments, the dosage form contains a predetermined amount of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein per unit dose. In some embodiments, accordingly, such a predetermined amount of such unit dose can be administered once or more than once a day. Such pharmaceutical formulations can be prepared by any of the methods well known in the art.

[0242] Effective amount In some embodiments, the amount of the primary active agent and / or any adjunct agents can be an effective amount, a minimum effective amount, and / or a therapeutically effective amount. As used herein, "effective amount", "effective concentration", etc. refer to the amount, concentration, etc. of the active agent and / or any adjunct agents contained in a pharmaceutical formulation that achieves one or more therapeutic or desired effects. As used herein, amounts such as "minimum effective", "minimum effective concentration", etc. refer to the lowest amount, concentration, etc. of the active agent and / or any adjunct agents that achieves one or more therapeutic or other desired effects. As used herein, "therapeutically effective amount", "therapeutically effective concentration", etc. refer to the amount, concentration, etc. of the active agent and / or any adjunct agents contained in a pharmaceutical formulation that achieves one or more therapeutic effects. In some embodiments, one or more therapeutic effects include transduction into the CNS.

[0243] In some embodiments, particularly when delivering infectious particles (e.g., virus particles having a main agent or an auxiliary agent as a cargo), the effective amount of the virus particles can be expressed as a titer (plaque-forming units per unit volume) or an MOI (multiplicity of infection). In some embodiments, the effective amount can be from about 1X10 1 particles to 1X10 20 particles or more per pL, nL, μL, mL, or L, for example, about 1x10 1 , 1x10 2 , 1x10 3 , 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 , 1x10 10 , 1x10 11 , 1x10 12 , 1x10 13 , 1x10 14 , 1x10 15 , 1x10 16 , 1x10 17 , 1x10 18 , 1x10 19 , or about 1x10 20 particles (or fewer particles). In some embodiments, the effective titer can be from about 1X10 1 transformation units to 1X10 20 transformation units or more per pL, nL, μL, mL, or L, for example, about 1x10 1 , 1x10 2 , 1x10 3 , 1x10 4 , 1x10 5 , 1x10 6 , 1x10 7 , 1x10 8 , 1x10 9 , 1x10 10 , 1x10 11 , 1x10 12 , 1x013 、 1x10 14 、 1x10 15 、 1x10 16 、 1x10 17 、 1x10 18 、 1x10 19 、 Approximately 1x10 20 transformation units (or less), or any numerical value or partial range within these ranges can be used. In some embodiments, the MOI of the pharmaceutical preparation can be about 0.1 to 10 or more, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 or more, or any numerical value or partial range within these ranges can be used.

[0244] In some embodiments, the effective amount, minimum effective amount, and / or therapeutically effective amount can be an effective concentration, minimum effective concentration, and / or therapeutically effective concentration, respectively, of about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pM, nM, μM, mM, or M, or can be any non-zero amount within these ranges, or any numerical or sub-range within these ranges.

[0245] In some embodiments, the amount of the main active agent and / or any co-active agent present in the pharmaceutical formulation can be a non-zero amount within the range of about 0 to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.9 to 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v, or can be any numerical value or sub-range within these ranges.

[0246] In some embodiments, the amount or effective amount of one or more of the active agents described herein contained in a pharmaceutical formulation can range from about 1 pg / kg to about 10 mg / kg, based on the body weight of the subject in need thereof or the average body weight of a particular patient population to which the pharmaceutical formulation can be administered.

[0247] In embodiments where the pharmaceutical formulation contains an adjuvant, the effective amount of the adjuvant varies depending on the adjuvant, the active agent, the route of administration, the age of the subject, the disease, the stage of the disease, etc., which is within the scope of those skilled in the art.

[0248] An adjuvant active agent can be included in the pharmaceutical formulation as needed, or in a pharmaceutical composition, the adjuvant active agent can be present as a compound, a derivative thereof, or a single compound or pharmaceutical formulation that can be administered simultaneously or sequentially with the pharmaceutical formulation thereof.

[0249] In some embodiments, the effective amount of the co - active agent, if present as required, is any non - zero amount within the range of about 0 to 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v of the total active agent present in the pharmaceutical formulation, or any numerical value or sub - range within these ranges. In some embodiments, the effective amount of the co - active agent is any non - zero amount within the range of about 0 to 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9% w / w, v / v, or w / v of the entire pharmaceutical formulation, or any numerical value or sub - range within these ranges.

[0250] Kit Furthermore, this specification describes a kit. The kit includes one or more of the polypeptides, polynucleotides, vectors, cells, or other components described herein, and combinations thereof, as well as one or more of the pharmaceutical formulations described herein. In embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be provided as a combination kit. As used herein, the term "combination kit" or "parts kit" refers to a combination of the elements included in the kit, or a compound or formulation and additional components that are used for packaging, screening, testing, selling, marketing, delivering, and / or administering a single element (such as an active ingredient). Such additional components include, but are not limited to, packaging, syringes, blister packages, bottles, etc. The combination kit can include one or more of its components (e.g., one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof), or its formulation can be provided as a single formulation (e.g., a solution, a lyophilized powder, etc.) or as separate formulations. The separate components or formulations can be included in a single package or separate packages within the kit. The kit can also include an explanation in a tangible expression medium. The explanation in the tangible expression medium can include information and / or instructions regarding the content of the components and / or formulations included in the kit, can include safety information regarding the content of the component(s) and / or formulation(s) included in the kit, can include information regarding the amount, dosage, use, screening method, recommended component design, and / or recommended treatment regimen(s) of the component(s) and / or formulation(s) included in the kit. As used herein, "tangible expression medium" refers to a medium that is physically tangible or accessible and is not just an abstract thought or an unrecorded spoken word. Examples of "tangible expression medium" include, but are not limited to, words on a cellulose-based material or a plastic material, or data stored in a suitable computer-readable memory format.The data can be stored on a unit device such as a flash memory drive or a CD-ROM, or on a server that can be accessed by the user, for example, via a web interface.

[0251] In one embodiment, the invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises a regulatory element operably linked to one or more engineered polynucleotides (e.g., those comprising a selected n amino acid length) as described elsewhere herein, and optionally a cargo molecule that may be operably linked to the regulatory element as needed. One or more engineered polynucleotides (e.g., those comprising a selected n amino acid length) as described elsewhere herein can be included in the same vector or a different vector as the cargo molecule in embodiments where the kit includes a cargo molecule.

[0252] In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (a) a first regulatory element operably linked to a directory repeat sequence and one or more insertion sites for inserting one or more guide sequences either upstream or downstream (whichever is applicable) of the directory repeat sequence, which, when expressed, causes the guide sequence to induce sequence-specific binding of the Cas9 CRISPR complex to a target sequence in a eukaryotic cell, the Cas9 CRISPR complex comprising a guide sequence hybridized to the target sequence and a Cas9 enzyme complexed therewith, and / or (b) a second regulatory element operably linked to an enzyme coding sequence encoding the Cas9 enzyme comprising a nuclear localization sequence. Optionally, it may comprise a tracr sequence. In some embodiments, the kit comprises components (a) and (b) located on the same vector or different vectors in the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, which, when expressed, each of the two or more guide sequences induces sequence-specific binding of the CRISPR complex to a different target sequence in a eukaryotic cell. In some embodiments, the Cas9 enzyme comprises one or more nuclear localization sequences of sufficient strength to induce accumulation of the CRISPR enzyme in the nucleus of a eukaryotic cell in a detectable amount. In some embodiments, the CRISPR enzyme is a type V or type VI CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme.In some embodiments, the Cas9 enzyme is derived from Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae Cas9 (e.g., modified to have at least one DD or to be associated with at least one DD), may include further variants or mutants of Cas, or may be a chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In some embodiments, the DD-CRISPR enzyme induces a single- or double-strand break at the position of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks or substantially lacks DNA strand cleavage activity (e.g., has a nuclease activity of 5% or less compared to a wild-type enzyme or an enzyme without mutations or modifications that reduce nuclease activity). In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter.In some embodiments, the guide sequence is at least 16 nucleotides in length, 17 nucleotides in length, 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 25 nucleotides in length, or 16 to 30 nucleotides in length, 16 to 25 nucleotides in length, or 16 to 20 nucleotides in length.

[0253] Method of use for delivery of cargo to the CNS Compositions comprising one or more of a cell-selective targeting moiety, an engineered AAV capsid-based polynucleotide, polypeptide, vector(s), engineered cell, engineered AAV capsid particle can generally be used to package and / or deliver one or more cargos to neurons and glial cells of the CNS. In some embodiments, delivery is cell-selectively performed based on the selectivity of the targeting moiety. In some embodiments, this is effected by the tropism of the engineered AAV capsid. This may be at least partially influenced by including one or more n-amino acid long motifs described elsewhere herein. In some embodiments, a composition comprising one or more of a CNS-targeting moiety, including engineered AAV capsid particles (where the capsid incorporates a targeting moiety), is administered to a subject or cell, tissue, and / or organ to facilitate introduction and / or incorporation of the cargo into recipient cells. In other embodiments, engineered cells can be generated that can produce compositions such as polypeptides or other particles (e.g., engineered AAV capsids and viral particles) comprising one or more of the targeting moieties from the polynucleotides, vectors, vector systems, etc. described herein. This includes, but is not limited to, engineered AAV capsid-based molecules (e.g., polynucleotides, vectors, and vector systems, etc.). In some embodiments, the polynucleotides, vectors, vector systems, etc. described herein that can produce compositions such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles) comprising one or more of the targeting moieties can be delivered to cells or tissues in vivo, ex vivo, or in vitro. In some embodiments, upon delivery to a subject, the composition can transform the subject's cells in vivo or ex vivo to generate engineered cells that can produce the compositions described herein comprising one or more of the cell-selective targeting moieties described herein (which includes, but is not limited to, engineered AAV capsid particles).The engineered AAV capsid particles are released from the engineered cells and can be used to deliver cargo molecule(s) to recipient cells in vivo or to generate a personalized engineered composition (e.g., AAV capsid particles) for reintroduction into the subject from which the recipient cells were obtained.

[0254] In some embodiments, the engineered cells are delivered to a subject, where they can release the engineered compositions of the invention (including but not limited to engineered AAV capsid particles), such that the compositions can later deliver cargo (e.g., cargo polynucleotide(s)) to recipient cells. These general processes can be used in a variety of ways in the treatment and / or prevention of diseases or their symptoms in a subject, the generation of model cells, the generation of modified organisms, the provision of cell selection and screening assays, bioproduction, and many other diverse applications.

[0255] In some embodiments, compositions such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles) that contain one or more targeting moieties can be delivered to neurons of the CNS. In another example, a composition containing one or more targeting moieties can be delivered to glial cells of the CNS.

[0256] In some embodiments, an engineered AAV capsid variant library with desired cell selectivity can be generated using engineered AAV capsid polynucleotides, vectors, and their systems to screen for variants with desired cell selectivity. As is clear from the description provided herein, supported by various examples, those with desired cell selectivity in mind can utilize the invention described herein to obtain capsids with desired cell selectivity.

[0257] Treatment methods This specification provides a method for treating a disease or disorder. The method includes administering to a subject in need of treatment a composition disclosed herein to cells of the CNS. In certain embodiments, the composition used in the methods disclosed herein can enhance transduction into neurons and / or glial cells of the CNS, enabling direct delivery of cargo and therapeutic agents to such cell types. Embodiments disclose methods where the cargo is one or more polypeptides.

[0258] disease or disorder In the methods disclosed in embodiments, the disease or disorder is cancer, neuropathy, or an infectious disease.

[0259] In one embodiment, the treatment method includes administering the composition detailed in this specification to a subject in need of treatment. In one exemplary embodiment, the cancer is a neuroepithelial cancer. In one embodiment, the cancer is a neuroepithelial tumor, for example, an astrocytic tumor, for example, diffuse astrocytoma (fibrous, protoplasmic, large cell, mixed), anaplastic (malignant) astrocytoma, glioblastoma (giant cell, variant of gliosarcoma), pilocytic astrocytoma, pleomorphic xanthoastrocytoma, or subependymal giant cell astrocytoma; oligodendroglial tumors, for example, oligodendroglioma, anaplastic (malignant) oligodendroglioma, ependymoma, ependymoma (cellular, papillary, clear cell, tanycytic), anaplastic (malignant) ependymoma, ependymoblastoma, subependymoma; mixed tumors, for example, oligoastrocytoma or anaplastic (malignant) oligoastrocytoma; choroid plexus tumors, for example, choroid plexus papilloma or choroid plexus carcinoma; neuronal and mixed neuronal-glial tumors, for example, gangliocytoma, ganglioglioma, ganglioneuroma, dysembryoplastic neuroepithelial tumor (DNET), cerebellar dysplastic gangliocytoma (Lhermitte-Duclos), desmoplastic infantile astrocytoma / ganglioglioma, central neurocytoma, anaplastic ganglioglioma, cerebellar liponeurocytoma, paraganglioma of the filum terminale; pineal tumors, for example, pineocytoma, pineoblastoma, pineal parenchymal tumor of intermediate differentiation; fetal tumors, for example, medulloblastoma (fibroblastic, large cell, melanotic, medullo-myoblastoma), medulloepithelioma, supratentorial primitive neuroectodermal tumor, neuroblastoma, ganglioneuroblastoma, ependymoblastoma, or PNET such as atypical teratoid / rhabdoid tumor; neuroblastoma tumors, for example, olfactory nerve (sensory neuroblastoma), olfactory neuroepithelioma, neuroblastoma of the adrenal gland and sympathetic nervous system; glioma of unknown origin, for example, astroblastoma, cerebral gliomatosis, chordoid glioma of the third ventricle.

[0260] In one embodiment, the cancer is a primary cancer that has metastasized to the brain or other regions of the central nervous system.

[0261] In one exemplary embodiment, the neuropathy is caused by a neurodegenerative disease or a neurodevelopmental disease. Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease and other memory disorders, amyotrophic lateral sclerosis (ALS), ataxia, Huntington's disease, Parkinson's disease, motor neuron disease, multiple system atrophy, progressive supranuclear palsy, and the like. Examples of neurodevelopmental diseases include, but are not limited to, attention deficit hyperactivity disorder (ADHD), autism, learning disabilities, intellectual disabilities (also referred to as mental retardation), conduct disorders, cerebral palsy, speech disorders, Tourette syndrome, schizophrenia, fragile X syndrome, visual impairments, and hearing impairments.

[0262] Non-human transgenic animal In one aspect, provided is a method of generating a humanized transgenic non-human animal. The method includes delivering a vector system or recombinant viral particles containing a recombinant viral genome to one or more cells of a non-human animal, wherein the vector system or recombinant viral genome encodes a human transferrin polypeptide, and the encoded human transferrin polypeptide is under the control of a tissue-specific promoter or miRNA binding element having selective activity in a desired cell, tissue, or organ. In one exemplary embodiment, the one or more cells are endothelial cells. In one exemplary embodiment, the one or more cells are CNS cells. In one exemplary embodiment, the one or more cells are cells of the CNS vasculature, lung, kidney, liver, or any combination thereof. In one exemplary embodiment, the endothelial cells are cells of the CNS vasculature. In one exemplary embodiment, the recombinant viral particles (optionally AAV viral particles) include a capsid polypeptide (optionally an AAV capsid polypeptide), and the capsid polypeptide includes a CNS-specific n-amino acid long motif. In one exemplary embodiment, the CNS-specific n-amino acid long motif includes X1-N-X3-X4-X5-X6-X7, where X5 is independently selected from K or R, and X1, X3, X4, X6, and X7 are independently selected from any amino acid, and optionally, the overall charge of the n-amino acid long motif at neutral pH is between 0 and +2. In one exemplary embodiment, the CNS-specific n-amino acid long motif includes or consists of NNSTRGG (SEQ ID NO: 42429), GNSARNI (SEQ ID NO: 42430), and GNSVRDF (SEQ ID NO: 42431). In one exemplary embodiment, the transgenic non-human animal is a rodent, optionally a mouse.

[0263] In one aspect, the present specification provides a humanized transgenic non-human animal, which comprises one or more cells that express a human transferrin polypeptide, and optionally, the one or more cells are CNS cells. In one exemplary embodiment, the transgenic non-human animal is a rodent, optionally a mouse. In one aspect, the present specification provides a humanized transgenic non-human animal produced by the method described herein. In one exemplary embodiment, the immune system of the humanized non-human animal is suppressed.

[0264] As is apparent, in the method of the present invention, when the non-human transgenic organism is a multicellular organism, such as an animal or a plant, the modification can occur ex vivo or in vitro, for example, in cell culture, and in some cases, it cannot occur in vivo. In other embodiments, the modification can occur in vivo. In one aspect, the present invention provides a method for modifying an organism or a non-human organism by manipulating a target sequence at a genomic locus of interest, the method comprising delivering a non-natural or engineered composition, for example, via particles (s) or nanoparticles (s) or vectors (s) (e.g., viral vectors, e.g., AAV, adenovirus, lentivirus).

[0265] A single cell or cell population can be preferably modified ex vivo and then reintroduced (e.g., transplanted) to generate a transgenic organism that expresses TFRC in specific cells. In some embodiments, the present invention encompasses a method of modifying a eukaryote, such as a transgenic eukaryote, the method including delivering a non-natural or engineered composition, e.g., via a vector(s) and / or particle(s) and / or nanoparticle(s). The system can include one, two, three, or four different vectors, and the system can include one, two, three, or four different nanoparticle complexes that deliver the component(s) of the system. Thus, components I, II, III, and IV can be arranged on one, two, three, or four different vectors and delivered by one, two, three, or four different particles or nanoparticle complexes or AAV. Alternatively, components I, II, III, and IV can be arranged on the same or different vector(s) / particle(s) / nanoparticle(s), and any combination of arrangements can be envisioned. And complexes that target the CNS or CNS tissue or CNS cells are advantageous.

[0266] In some embodiments, the vector is delivered to a eukaryotic cell of the transgenic eukaryote. In some embodiments, the modification occurs within a eukaryotic cell during cell culture. In one aspect, the present invention provides a method of generating a model eukaryotic cell or a model transgenic eukaryotic organism that includes one or more human proteins.

[0267] In one aspect, the present invention provides a transgenic eukaryote, such as a mouse. In one aspect, the present invention provides a constitutive transgenic eukaryote, such as a mouse strain obtained by mating a transgenic mouse with another mouse strain. In certain embodiments, progeny (or a group of progeny) derived from a transgenic eukaryote, such as a mouse strain, can be successfully propagated for at least five generations without increasing the level of genomic instability or cytotoxicity. In one aspect, the present invention provides a method for simultaneously introducing multiple mutations ex vivo in a tissue, organ, or cell line (of the CNS) or in vivo in a tissue, organ, or cell line (of the CNS). As is apparent, it is envisioned that transgenic non-human eukaryotes (e.g., animal models having multiple mutations at any number of loci) can be generated by using the novel target-directed sub-tools disclosed herein, which are within the scope of the present invention. As is obvious, such transgenic non-human eukaryotes, such as animal models, provide useful tools for research purposes, such as for viral transduction, and open the way for the development and testing of new therapeutic interventions targeting specific tissues that require mutations at multiple loci. Such uses are within the scope of the present invention.

[0268] The eukaryotic cell can comprise a constitutive promoter, or a tissue-specific promoter, or an inducible promoter. The eukaryotic cell can be a non-human transgenic eukaryote, such as a non-human mammal, primate, rodent, mouse, rat, rabbit, canine, dog, dairy cow, cow, sheep, ovine, goat, pig, poultry, avian, chicken, fish, insect, or part of an arthropod, and advantageously can be part of a mouse. The isolated eukaryotic cell or non-human transgenic eukaryote can express additional proteins or enzymes, such as TfR1. The expression of TfR1 can be driven by their coding sequences operably or functionally linked to a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0269] The eukaryotic cell can be a mammalian cell, such as a mouse cell that is part of a transgenic mouse expressing TfR1.

[0270] In one aspect of the practice of the present invention, transgenic non-human eukaryotes, such as animals, are also provided. Preferred examples include animals that contain TfR1 from the perspective of the polynucleotide or protein itself encoding TfR1. In certain aspects, the present invention includes constitutive or conditional or inducible TfR1 non-human eukaryotes, such as animals, such as primates, rodents, such as mice, rats, and rabbits, are preferred, and can include canines or dogs, livestock (dairy cows / cattle, sheep / ovine animals, goats, or pigs), fish, poultry or fowl, such as chickens, and insects or arthropods, and it should be noted that it is advantageous when the animal is a model for human or animal proteins, cells or tissues. This is because it is preferred to use non-human eukaryotes for modeling conditions, for example, through multiple inductions. As illustrated herein, to generate transgenic mice using a construct, pure linear DNA can be injected into the pronuclei of zygotes of pseudopregnant females, such as CB56 females. Subsequently, established lines can be identified, genotypes determined, and backcrossed to CB57 mice. Subsequently, the construct can be cloned and verified, if necessary, for example, by Sanger sequencing. Knock-ins are envisioned (alone or in combination).

[0271] Accordingly, the present invention encompasses non-human eukaryotes, animals, mammals, primates, rodents, etc. that can be used as models, or cells or tissues thereof. For example, the methods of the present invention can be used to generate non-human eukaryotes such as animals, mammals, primates, rodents, or cells, including modification of one or more nucleic acid sequences associated with or related to a cell or tissue (e.g., CNS). In the case of multicellular organisms, the cells can be in vivo or ex vivo. When culturing cells, if appropriate culture conditions are met and preferably the cells are appropriately adapted for this purpose (e.g., stem cells), cell lines can be established. Accordingly, cell lines are also contemplated.

[0272] In one aspect, the present invention can include cells transformed to contain TfR1, such as cells of non-human eukaryotes, such as animals, such as mammals, such as primates, rodents, mice, rats, rabbits, etc., and further human cells. For example, it can include ce...

Claims

1. A composition comprising a target-directed moiety effective in enhancing transduction into central nervous system (CNS) tissue via binding to transferrin receptors (TFRCs), and optionally further comprising cargo bound to or associated with the target-directed moiety.

2. The target-directing portion binds to the extracellular domain of the TFRC, Depending on the circumstances, the target-directing portion may bind to one or more of the apical domain of the extracellular domain, the helical domain of the extracellular domain, and / or the protease-like domain of the extracellular domain. Depending on the circumstances, the target-directing portion may be coupled to the apical domain. The composition according to claim 1.

3. The target-directing portion includes an n-amino acid length motif, and the n-amino acid length motif is X １ -X ２ -X ３ -X ４ -X ５ -X ６ -X ７ The amino acid sequence includes or consists of the above amino acid sequence, where X １ It contains amino acids selected from Y, M, F, and L. X ２ contains an amino acid selected from S, H, T, and A, X ３ It contains amino acids selected from K and R, X ４ It contains amino acids selected from A, G, I, L, M, N, Q, S, T, V, and H. X ５ It contains amino acids selected from N, G, A, L, M, Q, S, and T. X ６ It contains amino acids selected from A, T, H, N, F, I, P, L, Y, G, S, V, D, E, M, and Q. X ７ It contains amino acids selected from D and N. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains an amino acid selected from Y or L, X 2 contains amino acid H, X 3 contains amino acid A, X4 contains amino acids selected from K, R, N, and A. X 5 contains amino acids selected from G, Q, L, and S. X 6 comprises amino acids selected from P, L, I, N, D, and T. X7 contains an amino acid selected from N. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from A, F, H, I, L, N, P, R, S, T, V, and Y. X2 comprises amino acids selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, and Y. X 3 contains amino acid S, X4 contains amino acids selected from S and T, X 5 contains amino acid N, X 6 contains amino acid G, X7 contains amino acids selected from I, R, and V. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from R and T, X2 comprises amino acids selected from T, L, M, S, G, D, N, E, R, K, Y, and W. X3 contains amino acids selected from G, E, D, I, F, H, S, A, M, P, V, Y, W, Q, and T. X4 contains amino acids selected from D, T, E, H, N, and G. X 5 contains amino acids selected from A, V, S, T, and D. X 6 contains amino acids selected from Y, F, P, and A. X7 contains amino acids selected from A and P. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X 1 contains amino acid L, X2 contains amino acid C, X3 contains amino acids selected from K and R, X4 contains amino acid P, X 5 contains amino acid C, X 6 contains amino acids selected from L, S, D, A, N, Q, H, P, and V. X7 contains amino acids selected from E, T, G, A, D, N, and S. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from Y and F, X2 contains amino acids selected from W, F, and Y. X3 contains amino acids selected from S, T, H, A, and Q. X 4 contains amino acid G, X5 contains amino acids selected from I, T, V, Q, M, H, K, and R. X 6 contains amino acids selected from I, P, H, L, M, A, Q, T, V, K, and R. X7 contains amino acids selected from A, S, D, E, and N. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from K, R, S, G, N, T, M, Q, V, D, I, and E. X2 comprises amino acids selected from D, S, N, M, L, G, P, E, and A. X3 contains amino acids selected from E, D, G, S, A, R, Q, T, P, and N. X4 contains amino acids selected from F, Y, T, V, S, N, A, G, and H. X 5 contains amino acids selected from T, K, S, R, V, and H. X 6 comprises amino acids selected from T, S, G, V, A, K, R, N, D, E, and H. X7 contains amino acids selected from F, W, and Y. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from V, I, R, N, and D. X2 contains amino acids selected from A, G, and S. X3 contains amino acids selected from L, T, S, H, and G. X4 contains amino acids selected from K, R, and E. X 5 contains amino acid G, X 6 contains amino acids selected from W, R, A, and I. X7 contains amino acids selected from D and G. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from L, M, and W. X2 contains amino acids selected from F, R, W, K, T, and Y. X3 contains amino acids selected from D and S, X 4 contains amino acid G, X 5 contains amino acid T, X 6 contains amino acids selected from P, G, S, N, A, and R. X7 contains amino acids selected from A, P, S, and Y. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from P, N, and K. X2 contains amino acids selected from Y and F, X 3 contains amino acid A, X4 contains amino acids selected from R and K, X 5 contains amino acid S, X 6 contains amino acids selected from P, V, A, R, I, L, S, and E. X7 contains amino acids selected from E, D, M, and L. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5, where Z1 is selected from the group consisting of Y, F, and L. Z2 is selected from the group consisting of S, R, and K. X1 to X5 are amino acids that are selected independently. or The target-directed portion includes an n-amino acid length motif, which includes or consists of the amino acid sequence X1-H-X2-L-X3-X4-X5, where X1 to X5 are amino acids that are selected independently. or The aforementioned target-directed n-amino acid long motif is a 7-mer peptide having an amino acid sequence selected from any one of the sequence numbers 10952-20481 and 36241-42428. The composition according to claim 1.

4. The n-amino acid length motif includes or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X １ It contains amino acids selected from Y, M, and L. X ２ It contains amino acids selected from S, H, T, and A. X ３ It contains amino acids selected from K and R, X ４ It contains amino acids selected from A, G, I, L, M, N, Q, S, T, and V. X ５ It contains amino acid N, X ６ It contains amino acids selected from A, T, H, N, F, I, P, L, and Y. X ７ It contains amino acids selected from D and N. or X １ It contains amino acids selected from Y, M, and L. X ２ It contains amino acids selected from S, H, T, and A. X ３ It contains amino acids selected from K and R, X ４ It contains amino acids selected from A, G, I, L, M, N, Q, S, T, and V. X ５ It contains amino acid G, X ６ It contains amino acids selected from A, G, F, H, I, L, N, P, S, T, V, and Y. X ７ It contains amino acids selected from D and N, or X １ It contains amino acids selected from L and Y, X ２ It contains amino acids selected from A, H, and S, X ３ It contains amino acids selected from K and R, X ４ It contains amino acids selected from A, G, H, I, L, M, N, Q, S, T, and V. X ５ It contains amino acid G, X ６ It contains amino acid P, X ７ It contains amino acid D, or X １ It contains amino acids selected from L and Y, X ２ It contains amino acids selected from A, H, and S, X ３ It contains amino acids selected from K and R, X ４ It contains amino acids selected from A, G, H, I, L, M, N, Q, S, T, and V. X ５ It contains amino acid G, X ６ It contains amino acid P, X ７ It contains amino acid N, or X １ It contains amino acid Y, X ２ It contains amino acid S, X ３ It contains amino acid K, X ４ It contains amino acids selected from A, I, L, M, N, Q, S, T, and V. X ５ It contains amino acid G, X ６ It contains amino acid X, X ７ It contains amino acids selected from Y, P, T, Q, V, F, L, H, S, A, E, D, I, and M. or X １ It contains amino acids selected from L and Y, X ２ It contains amino acid S, X ３ It contains amino acids selected from R and K, X ４ It contains amino acids selected from V, I, T, L, and A. X ５ It contains amino acids selected from S and A, X ６ It contains amino acids selected from P, R, Y, F, H, I, K, and W. X ７ It contains amino acid D, or X １ It contains amino acids selected from F, L, M, and Y. X ２ It contains amino acid H, X ３ It contains amino acids selected from K and R, X ４ It contains amino acids selected from A, L, and M. X ５ It contains amino acids selected from A, G, L, M, N, Q, S, and T. X ６ It contains amino acids selected from A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y. X ７ It contains amino acids selected from D and N. or X １ It contains amino acids selected from L, M, and Y. X ２ It contains amino acid H, X ３ It contains amino acids selected from K and R, X ４ It contains amino acids selected from A, L, and M. X ５ It contains amino acids selected from A, G, L, M, N, Q, S, and T. X ６ It contains amino acids selected from A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y. X ７ It contains amino acid N, or X １ It contains amino acids selected from L, M, and Y. X ２ It contains amino acid H, X ３ It contains amino acids selected from K and R, X ４ It contains amino acids selected from A, L, and M. X ５ It contains amino acids selected from A, G, L, M, N, Q, S, and T. X ６ It contains amino acids selected from A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y. X ７ It contains amino acid D, or X １ It contains amino acids selected from L, M, and Y. X ２ It contains amino acid H, X ３ It contains amino acids selected from K and R, X ４ It contains amino acid L, X ５ It contains amino acids selected from S, Q, G, T, N, and L. X ６ It contains amino acids selected from P, T, V, I, Q, L, and A. X ７ It contains amino acid D, or X １ It contains amino acid F, X ２ It contains amino acid S, X ３ It contains amino acid R, X ４ It contains amino acid L, X ５ It contains amino acid G, X ６ comprises an amino acid selected from A, H, N, L, V, S, P, and T, X ７ It contains amino acid N, or X １ It contains amino acid F, X ２ It contains amino acid A, X ３ It contains amino acid R, X ４ It contains amino acids selected from T, S, and N, X ５ It contains amino acid G, X ６ contains an amino acid selected from Y, F, H, P, and A, X ７ It contains amino acid N, or X １ It contains amino acid F, X ２ It contains amino acid H, X ３ It contains amino acids selected from K and R, X ４ It contains amino acid L, X ５ It contains amino acid G, X ６ It contains amino acids selected from I, P, and S, X ７ It contains amino acids selected from N and D. or X1 contains amino acids selected from F, L, and Y. X2 comprises amino acids selected from D, E, H, N, Q, S, and T. X 3 contains amino acid S, X4 contains amino acids selected from S and T, X 5 contains amino acid N, X 6 contains amino acid G, X7 contains amino acids selected from I and V. or X1 contains amino acids selected from V, P, I, S, T, H, and A. X2 contains amino acids selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, and Y. X 3 contains amino acid S, X4 contains amino acids selected from S and T, X 5 contains amino acid N, X 6 contains amino acid G, X7 contains amino acids selected from I and V. or X1 comprises amino acids selected from A, F, I, L, P, S, T, V, and Y. X2 comprises amino acids selected from D, E, N, Q, S, and T. X 3 contains amino acid S, X4 contains amino acids selected from T and S, X 5 contains amino acid N, X 6 contains amino acid G, X7 contains amino acid R. or X 1 contains amino acid R, X2 comprises amino acids selected from E, D, Q, and T. X 3 contains amino acid S, X4 contains amino acids selected from S and T, X 5 contains amino acid N, X 6 contains amino acid G. X7 contains amino acids selected from I and V. or X 1 contains amino acid R, X2 contains amino acids selected from T, M, L, and S. X3 contains amino acids selected from Y, S, A, M, I, F, and P. X4 contains amino acid D, X 5 contains amino acids selected from A, V, S, and T. X 6 contains amino acids selected from Y and F. X7 contains amino acid P. or X 1 contains amino acid R, X2 contains amino acids selected from T, M, L, and S. X3 contains amino acids selected from Y, S, A, M, I, F, and P. X4 contains amino acid D, X 5 contains amino acids selected from A, V, S, and T. X 6 contains amino acids selected from Y and F. X 7 contains amino acid A. or X 1 contains amino acid R, X2 contains amino acids selected from G, T, D, S, N, and E. X3 contains amino acids selected from E, D, P, S, and G. X4 contains amino acids selected from D, T, E, H, and N. X 5 contains amino acids selected from V, A, and T. X 6 contains amino acids selected from Y and F. X7 contains amino acid P. or X 1 contains amino acid R, X2 contains amino acids selected from G, L, T, D, and S. X3 contains amino acids selected from D, P, S, and G. X4 contains amino acids selected from D, E, H, and N. X 5 contains amino acids selected from V and T, X 6 contains amino acids selected from Y and F. X7 contains amino acid P. or X1 contains amino acid T, X2 contains amino acids selected from R, K, Y, and W. X3 contains amino acids selected from E, W, Y, Q, S, and T. X 4 contains amino acid G, X 5 contains amino acid D, X 6 contains amino acids selected from P and A, X7 contains amino acids selected from A and P. or X1 contains amino acids selected from Y and F, X2 contains amino acids selected from W, F, and Y. X3 contains amino acids selected from T and S, X 4 contains amino acid G, X 5 contains amino acids selected from I, T, V, Q, M, and H. X 6 contains amino acids selected from I, P, H, L, M, A, Q, T, and V. X7 contains amino acids selected from A, S, D, and E. or X1 contains amino acids selected from Y and F, X2 contains amino acids selected from W, F, and Y. X3 contains amino acids selected from T and S, X 4 contains amino acid G, X 5 contains amino acids selected from I, T, V, Q, M, and H. X 6 contains amino acids selected from K and R, X7 contains amino acids selected from A, S, D, and E. or X1 contains amino acids selected from Y and F, X2 contains amino acids selected from W, F, and Y. X3 contains amino acids selected from T and S, X 4 contains amino acid G, X 5 contains amino acids selected from K and R, X 6 contains amino acids selected from I, P, H, L, M, A, Q, T, and V. X7 contains amino acids selected from A, S, D, and E. or X1 contains amino acid Y, X 2 contains amino acid F, X 3 contains amino acid T, X 4 contains amino acid G, X 5 contains amino acids selected from K, R, Q, M, H, and I. X 6 contains amino acids selected from T, R, H, K, V, and L. X 7 contains amino acid E. or X1 contains amino acid Y, X 2 contains amino acid F, X3 contains amino acids selected from T, S, H, and A. X 4 contains amino acid G, X 5 contains amino acids selected from K, R, and T. X 6 contains amino acids selected from I, P, H, L, M, A, Q, and T. X7 contains amino acids selected from D and N. or X1 contains amino acid Y, X 2 contains amino acid W, X 3 contains amino acid T, X 4 contains amino acid G, X5 contains amino acids selected from K, M, V, and T. X 6 contains amino acids selected from P, V, I, H, Q, T, M, and L. X7 contains amino acids selected from E and D. or X1 contains amino acids selected from Y and F, X 2 contains amino acid F, X3 contains amino acids selected from S, H, A, and Q. X 4 contains amino acid G, X 5 contains amino acids selected from K, Q, and R. X 6 contains amino acids selected from I, V, L, K, H, R, Q, and M. X 7 contains amino acid E. or X1 contains amino acids selected from K and R, X2 contains amino acids selected from D, S, and N. X 3 contains amino acid E, X 4 contains amino acid F, X 5 contains amino acids selected from T, K, S, R, and V. X 6 contains amino acids selected from T, S, G, and V. X7 contains amino acids selected from F, W, and Y. or X1 contains amino acids selected from K and R. X 2 contains amino acid D, X 3 contains amino acid D, X4 contains amino acids selected from F and Y, X 5 contains amino acids selected from T, S, V, and H. X 6 contains amino acids selected from T, S, G, V, and A. X7 contains amino acids selected from F, W, and Y. or X1 contains amino acids selected from S, R, G, N, T, M, and Q. X 2 contains amino acid D, X 3 contains amino acid G, X4 contains amino acids selected from T, V, S, N, and Y. X 5 contains amino acid S, X 6 contains amino acids selected from K and R, X 7 contains amino acid W. or X1 comprises amino acids selected from R, V, D, I, Q, and K. X2 contains amino acids selected from M, L, and G. X3 contains amino acids selected from S, E, A, R, and Q. X4 contains amino acid D, X 5 contains amino acid R, X 6 contains amino acids selected from T, A, S, G, K, and N. X 7 contains amino acid W. or X1 comprises amino acids selected from D, I, E, Q, V, S, and K. X2 contains amino acids selected from L, M, G, and P. X3 contains amino acids selected from E, A, S, D, Q, and T. X 4 contains amino acids selected from S and A, X 5 contains amino acid R, X 6 contains amino acids selected from D, S, E, T, G, and A. X 7 contains amino acid W. or X1 contains amino acid G, X2 contains amino acids selected from E, S, P, G, and A. X3 contains amino acids selected from D, E, P, and N. X4 contains amino acids selected from G, H, T, S, and N. X 5 contains amino acid V, X 6 contains amino acids selected from R, K, and S. X7 contains amino acids selected from W and Y. or X 1 contains amino acid R, X 2 contains amino acid E, X3 contains amino acids selected from D, E, P, and N. X4 contains amino acids selected from G, H, T, S, and N. X 5 contains amino acid V, X 6 contains amino acids selected from R, K, and S. X7 contains amino acids selected from W and Y. or X1 contains amino acid G, X2 contains amino acids selected from G and S, X3 contains amino acids selected from G, E, S, A, P, and D. X4 contains amino acids selected from T, G, and S. X 5 includes S, X 6 contains amino acids selected from S, T, H, K, R, A, and N. X 7 contains amino acid W. or The aforementioned n-amino acid length motif includes or consists of the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5. X1 contains an amino acid selected from A, S, or H, X 2 contains an amino acid selected from S, T, L, or I, X 3 contains an amino acid selected from N or G, X 4 contains amino acid G, X 5 contains an amino acid selected from N, D, I, V, or R: or any combination thereof The composition according to claim 1.

5. The composition according to claim 4, wherein the n-amino acid length motif is selected from the group consisting of LHRLGPN (SEQ ID NO: 36834), YSRIGPN (SEQ ID NO: 14632), LHRLGPN (SEQ ID NO: 36834), LHRLGPD (SEQ ID NO: 36413), LHRAGPD (SEQ ID NO: 36894), YSRIGPD (SEQ ID NO: 38223), LSRIGPD (SEQ ID NO: 36274), LARSGPD (SEQ ID NO: 18035), YSRNSDN (SEQ ID NO: 16626), LHKAGPN (SEQ ID NO: 36305), LSRIGPN (SEQ ID NO: 36347), LAKSGPN (SEQ ID NO: 36287), YARNGPN (SEQ ID NO: 14048), and YSRNSDN (SEQ ID NO: 16626).

6. The composition according to claim 4, wherein the n-amino acid long motif is selected from the group consisting of FRSTNGV (SEQ ID NO: 16070), VESTNGR (SEQ ID NO: 36431), VDSTNGV (SEQ ID NO: 12206), VQSTNGV (SEQ ID NO: 36423), VSSTNGV (SEQ ID NO: 12333), TESTNGR (SEQ ID NO: 17558), VQSTNGI (SEQ ID NO: 11292), and FVSTNGV (SEQ ID NO: 11162).

7. The composition according to claim 4, wherein the n-amino acid length motif is selected from the group consisting of RGEDVYP (SEQ ID NO: 36864), RLEDVFP (SEQ ID NO: 36264), RTYDSYP (SEQ ID NO: 37938), RTYDAYP (SEQ ID NO: 38571), RTYDSFP (SEQ ID NO: 37806), RTETVYP (SEQ ID NO: 36486), RTETVFP (SEQ ID NO: 36389), and RTEHVFP (SEQ ID NO: 36603).

8. The composition according to claim 3, wherein the n-amino acid long motif is composed of LCKPCLD (SEQ ID NO: 36437) or LCKPCPT (SEQ ID NO: 36438).

9. The composition according to claim 4, wherein the n-amino acid length motif is selected from the group consisting of KDEFTTF (SEQ ID NO: 36308), KDDFTTY (SEQ ID NO: 36336), RDEFTTY (SEQ ID NO: 36615), KDEFSTY (SEQ ID NO: 36390), RDEFTSF (SEQ ID NO: 36701), and REDHVSW (SEQ ID NO: 37067).

10. The composition according to claim 3, wherein the n-amino acid length motif is selected from the group consisting of IALKGWD (SEQ ID NO: 36248), NALEGRD (SEQ ID NO: 36407), VALEGRD (SEQ ID NO: 36604), and VALKGWD (SEQ ID NO: 17701).

11. The composition according to claim 3, wherein the n-amino acid length motif is selected from the group consisting of YSRIGPN (SEQ ID NO: 14632), YSRLNMN (SEQ ID NO: 14301), YSRLNKD (SEQ ID NO: 16577), and YHRLSNN (SEQ ID NO: 16636).

12. The composition according to claim 3, wherein the n-amino acid length motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608).

13. The aforementioned n-amino acid length motifs are PSATNGV (SEQ ID NO: 20486), QVSTNGI (SEQ ID NO: 16021), SYSSNGV (SEQ ID NO: 16234), HQSSNGV (SEQ ID NO: 15978), VGSINGI (SEQ ID NO: 16199), AMSTNGR (SEQ ID NO: 16000), SASTNGV (SEQ ID NO: 16127), YMSTNGV (SEQ ID NO: 16042), YYSSNGV (SEQ ID NO: 1 The composition according to claim 3, comprising 6206), VHSTNGI (SEQ ID NO: 16134), PLSTNGV (SEQ ID NO: 16233), VYSTNGV (SEQ ID NO: 16059), IISTNGV (SEQ ID NO: 16054), RSVSSNGV (SEQ ID NO: 20502), YKSSSNGV (SEQ ID NO: 16123), FRSTNGV (SEQ ID NO: 16070), and / or FVSTNGV (SEQ ID NO: 11162).

14. The composition according to claim 3, wherein the n amino acid length is selected from any one of the amino acid sequences listed in Tables 1 to 22, SEQ ID NOs: 10952 to 20481 and 36241 to 42428, or any combination thereof.

15. The aforementioned target-directing portion is part of the viral capsid protein, Depending on the circumstances, the target-directing portion may be inserted into or replaced in loop IV of the AAV capsid protein, loop VIII of the AAV capsid protein, or both loop IV and loop VIII of the AAV capsid protein. Depending on the case, the target-directing portion may be IPFSRRVNPDT (SEQ ID NO: 20285), LGFARTGAAD (SEQ ID NO: 20274), LGFTKSSGSD (SEQ ID NO: 20270), LRYSKTQGES (SEQ ID NO: 20266), SPYARSSAGV (SEQ ID NO: 20271), or VGWSRLDLTT (SEQ ID NO: 20262). The composition according to claim 1.

16. The target-directing portion is inserted into or substituted in loop IV of the AAV capsid protein, loop VIII of the AAV capsid protein, or both loop IV and loop VIII of the AAV capsid protein. Depending on the case, the target-directing portion may be IPFSRRVNPDT (SEQ ID NO: 20285), LGFARTGAAD (SEQ ID NO: 20274), LGFTKSSGSD (SEQ ID NO: 20270), LRYSKTQGES (SEQ ID NO: 20266), SPYARSSAGV (SEQ ID NO: 20271), or VGWSRLDLTT (SEQ ID NO: 20262). The composition according to claim 3.

17. The composition according to claim 15, wherein the target-directing portion is inserted between two amino acids of one or more capsid proteins, so that the target-directing portion is located outside the AAV capsid.

18. The aforementioned viral capsid protein is the AAV viral capsid protein. Depending on the circumstances, the capsid protein may be VP1, VP2, VP3, or a combination thereof. The composition according to claim 15.

19. The target-directed portion is inserted between amino acids 588 and 589 of the AAV9 capsid protein, or is inserted at a similar position in the capsid proteins derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh. 74, or AAVrh.

10. or The target-directed portion is inserted between two consecutive amino acids within amino acids 451-460 of the AAV9 capsid protein, or is inserted at a similar position in the capsid proteins derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh. 74, or AAVrh.

10. The composition according to claim 1.

20. The cargo consists of polynucleotides, a recombinant AAV genome containing a transgene, one or more polypeptides, and a ribonucleoprotein complex. In some cases, the polynucleotide is operably linked to a regulatory sequence that promotes expression in the CNS. Depending on the circumstances, the polynucleotide may encode one or more polypeptides and / or RNAi oligonucleotides. Depending on the circumstances, the one or more polypeptides may include an enzyme or an antibody, and In some cases, the polynucleotide encodes the CRISPR-Cas system. The composition according to claim 1.

21. The composition according to claim 20, wherein the polynucleotide is operably linked to a regulatory sequence that promotes expression in the CNS.

22. The composition comprises the composition described in claim 1, In some cases, the virus capsid or virus particle is an AAV virus capsid or an AAV virus particle. In some cases, the target-directing portion is inserted between amino acids 588 and 589 of the AAV9 capsid protein, or at a similar position in the capsid proteins derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh. 74, or AAVrh.

10. Virus capsid or virus particle.

23. Further comprising a recombinant viral genome, the recombinant viral genome encoding a therapeutic protein or nucleic acid, a regulatory polypeptide or nucleic acid, and / or a selectable marker polypeptide or nucleic acid, In some cases, the therapeutic protein or nucleic acid, the regulatory polypeptide or nucleic acid, and / or the selectable marker polypeptide or nucleic acid are operably linked to a regulatory sequence that promotes expression in the CNS, and In some cases, the recombinant viral genome is a recombinant AAV viral genome. The viral capsid or viral particle according to claim 22.

24. A vector system comprising one or more vectors, wherein at least one of the one or more vectors encodes a target-directed moiety effective in enhancing transduction into central nervous system tissue (CNS) via binding to a transferrin receptor (TFRC), optionally at least one of the one or more vectors encodes a recombinant AAV genome comprising a transgene encoding a protein or polypeptide, and optionally further comprising a cargo.

25. The target-directing portion binds to the extracellular domain of the TFRC, Depending on the circumstances, the target-directing portion may bind to one or more of the apical domain of the extracellular domain, the helical domain of the extracellular domain, and / or the protease-like domain of the extracellular domain. The vector system according to claim 24.

26. The target-directing portion includes an n-amino acid length motif, The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from Y, M, F, and L. X2 contains amino acids selected from S, H, T, and A. X3 contains amino acids selected from K and R, X4 contains amino acids selected from A, G, I, L, M, N, Q, S, T, V, and H. X5 contains amino acids selected from N, G, A, L, M, Q, S, and T. X 6 contains amino acids selected from A, T, H, N, F, I, P, L, Y, G, S, V, D, E, M, and Q. X7 contains amino acids selected from D and N. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains an amino acid selected from Y or L, X 2 contains amino acid H, X 3 contains amino acid A, X4 contains amino acids selected from K, R, N, and A. X 5 contains amino acids selected from G, Q, L, and S. X 6 comprises amino acids selected from P, L, I, N, D, and T. X 7 contains amino acid N. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from A, F, H, I, L, N, P, R, S, T, V, and Y. X2 comprises amino acids selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, and Y. X 3 contains amino acid S, X4 contains amino acids selected from S and T, X 5 contains amino acid N, X 6 contains amino acid G, X7 contains amino acids selected from I, R, and V. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from R and T, X2 comprises amino acids selected from T, L, M, S, G, D, N, E, R, K, Y, and W. X3 contains amino acids selected from G, E, D, I, F, H, S, A, M, P, V, Y, W, Q, and T. X4 contains amino acids selected from D, T, E, H, N, and G. X 5 contains amino acids selected from A, V, S, T, and D. X 6 contains amino acids selected from Y, F, P, and A. X7 contains amino acids selected from A and P. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X 1 contains amino acid L, X2 contains amino acid C, X3 contains amino acids selected from K and R, X4 contains amino acid P, X 5 contains amino acid C, X 6 contains amino acids selected from L, S, D, A, N, Q, H, P, and V. X7 contains amino acids selected from E, T, G, A, D, N, and S. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from Y and F, X2 contains amino acids selected from W, F, and Y. X3 contains amino acids selected from S, T, H, A, and Q. X 4 contains amino acid G, X5 contains amino acids selected from I, T, V, Q, M, H, K, and R. X 6 contains amino acids selected from I, P, H, L, M, A, Q, T, V, K, and R. X7 contains amino acids selected from A, S, D, E, and N. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from K, R, S, G, N, T, M, Q, V, D, I, and E. X2 comprises amino acids selected from D, S, N, M, L, G, P, E, and A. X3 contains amino acids selected from E, D, G, S, A, R, Q, T, P, and N. X4 contains amino acids selected from F, Y, T, V, S, N, A, G, and H. X 5 contains amino acids selected from T, K, S, R, V, and H. X 6 comprises amino acids selected from T, S, G, V, A, K, R, N, D, E, and H. X7 contains amino acids selected from F, W, and Y. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from V, I, R, N, and D. X2 contains amino acids selected from A, G, and S. X3 contains amino acids selected from L, T, S, H, and G. X4 contains amino acids selected from K, R, and E. X 5 contains amino acid G, X 6 contains amino acids selected from W, R, A, and I. X7 contains amino acids selected from D and G. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from L, M, and W. X2 contains amino acids selected from F, R, W, K, T, and Y. X3 contains amino acids selected from D and S, X 4 contains amino acid G, X 5 contains amino acid T, X 6 contains amino acids selected from P, G, S, N, A, and R. X7 contains amino acids selected from A, P, S, and Y. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from P, N, and K. X2 contains amino acids selected from Y and F, X 3 contains amino acid A, X4 contains amino acids selected from R and K, X 5 contains amino acid S, X 6 contains amino acids selected from P, V, A, R, I, L, S, and E. X7 contains amino acids selected from E, D, M, and L. or The aforementioned target-directed n-amino acid long motif peptide is a 7-mer peptide having the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5, where Z1 is selected from the group consisting of Y, F, and L. Z2 is selected from the group consisting of S, R, and K. X1 to X5 are amino acids that are selected independently. or The aforementioned target-directed n-amino acid long motif is a 7-mer peptide having the amino acid sequence X1-H-X2-L-X3-X4-X5, where X1 to X5 are independently selected amino acids. or The aforementioned target-directed n-amino acid long motif is a 7-mer peptide having one of the amino acid sequences from SEQ ID NOs: 10952-20481 and 36241-42428. The vector system according to claim 24.

27. The vector system according to claim 26, wherein the n amino acid length is selected from any one of those listed in Tables 1 to 22, or any combination thereof.

28. The aforementioned target-directing portion is part of the viral capsid protein, In some cases, the viral capsid protein is the AAV viral capsid protein. Depending on the case, the capsid protein may be VP1, VP2, VP3, or a combination thereof. Depending on the circumstances, the target-directing portion is inserted into or replaced in loop IV and / or loop VIII. In some cases, the target-directing portion is inserted between two amino acids of one or more capsid proteins, thereby placing the target-directing portion outside the AAV capsid. In some cases, the target-directing portion is inserted between amino acids 588 and 589 of the AAV9 capsid protein, or at a similar position in the capsid proteins derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh. 74, or AAVrh.

10. In some cases, the target-directing portion is inserted between amino acids 451-460 of the AAV9 capsid protein, or inserted at a similar position in the capsid proteins derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh. 74, or AAVrh.

10. The vector system according to claim 24.

29. The vector system according to claim 24, wherein the target-directing portion is IPFSRRVNPDT (SEQ ID NO: 20285), LGFARTGAAD (SEQ ID NO: 20274), LGFTKSSGSD (SEQ ID NO: 20270), LRYSKTQGES (SEQ ID NO: 20266), SPYARSSAGV (SEQ ID NO: 20271), and VGWSRLDLTT (SEQ ID NO: 20262).

30. The cargo consists of polynucleotides, a recombinant AAV genome containing a transgene, one or more polypeptides, and a ribonucleoprotein complex. In some cases, the polynucleotide is operably linked to a regulatory sequence that promotes expression in the CNS. Depending on the circumstances, the polynucleotide may encode one or more polypeptides and / or RNAi oligonucleotides. Depending on the circumstances, the one or more polypeptides may include an enzyme or an antibody. In some cases, the polynucleotide encodes the CRISPR-Cas system. The vector system according to claim 24.

31. A polypeptide encoded or generated by the vector system described in claim 24, wherein the polypeptide is optionally a capsid protein and optionally an AAV capsid polypeptide.

32. Particles generated by the vector system described in claim 24, wherein the particles are optionally virus particles and optionally AAV particles.

33. A cell comprising the composition according to claim 1, a vector system comprising one or more vectors wherein at least one of the one or more vectors encodes the composition, a polypeptide encoded or produced by the vector system, or particles produced by the vector system.

34. A method for delivering one or more cargoes to the CNS, The procedure includes administering the capsid and / or viral particles described in claim 22 in vivo or in vitro. Depending on the circumstances, the cargo may be a recombinant AAV genome containing a transgene, a polynucleotide encoding an RNAi oligonucleotide, a polynucleotide encoding a polypeptide, or a polypeptide, and depending on the circumstances, the polypeptide may include an enzyme or an antibody. Depending on the circumstances, the cargo may be a Cas polypeptide, a guide molecule, or both. Depending on the circumstances, the cargo may encode a nucleic acid component of a nuclease or RNA-induced nuclease. In some cases, the cargo is one or more polynucleotides encoding nucleic acid components of the nuclease and the RNA-inducing nuclease. The aforementioned method.

35. A method for creating humanized transgenic non-human animals, This involves delivering a vector system or recombinant viral particles containing a recombinant viral genome to one or more cells of a non-human animal. The vector system or the recombinant viral genome encodes a human transferrin polypeptide, the encoded human transferrin polypeptide being under the control of a tissue-specific promoter or miRNA-binding element having selective activity in a desired cell, tissue, or organ. In some cases, the recombinant virus particles, and in some cases the AAV virus particles, include a capsid polypeptide, and in some cases the AAV capsid polypeptide, and the capsid polypeptide includes a CNS-specific n-amino acid long motif. In some cases, the CNS-specific n-amino acid length motif comprises X1-N-X3-X4-X5-X6-X7, where X5 is independently selected from K or R, and X1, X3, X4, X6, and X7 are independently selected from any amino acid, and in some cases, the total charge of the n-amino acid length motif at neutral pH is 0 to +2. Depending on the case, the CNS-specific n-amino acid long motif may include NNSTRGG (SEQ ID NO: 42429), GNSARNI (SEQ ID NO: 42430), and GNSVRDF (SEQ ID NO: 42431), or consist of NNSTRGG (SEQ ID NO: 42429), GNSARNI (SEQ ID NO: 42430), and GNSVRDF (SEQ ID NO: 42431). In some cases, the transgenic non-human animal is a rodent, and in some cases, a mouse. The aforementioned method.

36. The one or more cells are endothelial cells, CNS cells, or the endothelial cells are cells of the CNS vascular structure. In some cases, one or more of the cells are cells from the CNS vascular structure, lungs, kidneys, liver, or any combination thereof. The method according to claim 35.

37. A humanized transgenic non-human animal comprising one or more cells expressing a human transferrin polypeptide, wherein the one or more cells are CNS cells, and wherein the transgenic non-human animal is a rodent, and optionally a mouse.

38. A humanized transgenic non-human animal prepared by the method of claim 35, wherein the humanized non-human animal has a suppressed immune system.

39. A method for screening n-amino acid length motifs that can induce transduction into central nervous system (CNS) tissue via binding to transferrin receptors (TFRCs) in humanized transgenic non-human animals, Introducing one or more compositions containing candidate n-amino acid length motifs into the humanized non-human transgenic animal described in claim 35, This includes detecting the binding of the composition to a transferrin receptor (TFRC) and / or detecting transduction or uptake by one or more CNS cells of the humanized transgenic non-human animal, Depending on the case, the candidate n-amino acid length motif may include X1-N-X3-X4-X5-X6-X7 or consist of X1-N-X3-X4-X5-X6-X7, where, X5 is selected independently from K or R. X1, X3, X4, X6, and X7 are independently selected from any amino acid. Depending on the circumstances, the total charge of the n-amino acid length motif at a neutral pH is between 0 and +2. The aforementioned method.

40. Each of the compositions is a viral particle containing one or more capsid proteins that include the candidate n-amino acid length motif. Depending on the circumstances, the virus particle may be an AAV virus particle, and the one or more capsid proteins may be AAV capsid proteins. In some cases, the candidate n-amino acid length motif is inserted between amino acids 588 and 589 of the AAV9 capsid polypeptide, or inserted at a similar position in a capsid protein derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh. 74, or AAVrh.

10. Depending on the circumstances, at least one of the one or more compositions may further include cargo. Depending on the circumstances, the cargo may be a therapeutic nucleic acid or polypeptide, a selectable marker, or a regulatory polypeptide or nucleic acid, or may encode a therapeutic nucleic acid or polypeptide, a selectable marker, or a regulatory polypeptide or nucleic acid. The method according to claim 39.

41. (1) A recombinant capsid protein having an n-amino acid long motif peptide targeting TFR1, wherein the peptide is inserted into the capsid protein so that when the peptide is incorporated into an AAV particle, the peptide is presented on the surface; and (2) a recombinant AAV genome containing a transgene encoding a therapeutic polypeptide or polynucleotide, comprising a recombinant AAV particle. The transgene is operably linked to one or more regulatory sequences that promote the expression of the transgene in CNS cells and is sandwiched between AAV ITR sequences, and the AAV particles exhibit enhanced binding, transduction, or transgene expression in CNS cells compared to AAV particles having a target-directed reference capsid that does not contain the n-amino acid length motif. Depending on the circumstances, the recombinant capsid protein may be the AAV9 capsid protein (SEQ ID NO: 20506), the AAV9 K449R capsid protein (SEQ ID NO: 20507), or a capsid protein having at least 90%, 90%, or 99% sequence identity with the AAV9 capsid protein, and which forms a capsid that transduces CNS cells. In some cases, the peptide is inserted between amino acids 588 and 589 of the AAV9 capsid protein, or at a corresponding position in another AAV capsid protein, or the peptide is inserted between two consecutive amino acids within amino acids 451-460 of the AAV9 capsid protein, or at a corresponding position in another AAV capsid protein. Recombinant AAV particles.

42. The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from Y, M, F, and L. X2 contains amino acids selected from S, H, T, and A. X3 contains amino acids selected from K and R, X4 contains amino acids selected from A, G, I, L, M, N, Q, S, T, V, and H. X5 contains amino acids selected from N, G, A, L, M, Q, S, and T. X 6 contains amino acids selected from A, T, H, N, F, I, P, L, Y, G, S, V, D, E, M, and Q. X7 contains amino acids selected from D and N. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains an amino acid selected from Y or L, X 2 contains amino acid H, X 3 contains amino acid A, X4 contains amino acids selected from K, R, N, and A. X 5 contains amino acids selected from G, Q, L, and S. X 6 comprises amino acids selected from P, L, I, N, D, and T. X 7 contains amino acid N. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from A, F, H, I, L, N, P, R, S, T, V, and Y. X2 comprises amino acids selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, and Y. X 3 contains amino acid S, X4 contains amino acids selected from S and T, X 5 contains amino acid N, X 6 contains amino acid G, X7 contains amino acids selected from I, R, and V. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from R and T, X2 comprises amino acids selected from T, L, M, S, G, D, N, E, R, K, Y, and W. X3 contains amino acids selected from G, E, D, I, F, H, S, A, M, P, V, Y, W, Q, and T. X4 contains amino acids selected from D, T, E, H, N, and G. X 5 contains amino acids selected from A, V, S, T, and D. X 6 contains amino acids selected from Y, F, P, and A. X7 contains amino acids selected from A and P. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X 1 contains amino acid L, X2 contains amino acid C, X3 contains amino acids selected from K and R, X4 contains amino acid P, X 5 contains amino acid C, X 6 contains amino acids selected from L, S, D, A, N, Q, H, P, and V. X7 contains amino acids selected from E, T, G, A, D, N, and S. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from Y and F, X2 contains amino acids selected from W, F, and Y. X3 contains amino acids selected from S, T, H, A, and Q. X 4 contains amino acid G, X5 contains amino acids selected from I, T, V, Q, M, H, K, and R. X 6 contains amino acids selected from I, P, H, L, M, A, Q, T, V, K, and R. X7 contains amino acids selected from A, S, D, E, and N. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from K, R, S, G, N, T, M, Q, V, D, I, and E. X2 comprises amino acids selected from D, S, N, M, L, G, P, E, and A. X3 contains amino acids selected from E, D, G, S, A, R, Q, T, P, and N. X4 contains amino acids selected from F, Y, T, V, S, N, A, G, and H. X 5 contains amino acids selected from T, K, S, R, V, and H. X 6 comprises amino acids selected from T, S, G, V, A, K, R, N, D, E, and H. X7 contains amino acids selected from F, W, and Y. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from V, I, R, N, and D. X2 contains amino acids selected from A, G, and S. X3 contains amino acids selected from L, T, S, H, and G. X4 contains amino acids selected from K, R, and E. X 5 contains amino acid G, X 6 contains amino acids selected from W, R, A, and I. X7 contains amino acids selected from D and G. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from L, M, and W. X2 contains amino acids selected from F, R, W, K, T, and Y. X3 contains amino acids selected from D and S, X 4 contains amino acid G, X 5 contains amino acid T, X 6 contains amino acids selected from P, G, S, N, A, and R. X7 contains amino acids selected from A, P, S, and Y. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from P, N, and K. X2 contains amino acids selected from Y and F, X 3 contains amino acid A, X4 contains amino acids selected from R and K, X 5 contains amino acid S, X 6 contains amino acids selected from P, V, A, R, I, L, S, and E. X7 contains amino acids selected from E, D, M, and L. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5, where Z1 is selected from the group consisting of Y, F, and L. Z2 is selected from the group consisting of S, R, and K. X1 to X5 are amino acids that are selected independently. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-H-X2-L-X3-X4-X5, where X1 to X5 are independently selected amino acids. or The n-amino acid motif peptide targeting TFR1 is a 7-mer peptide having one of the amino acid sequences from SEQ ID NOs: 10952-20481 and 36241-42428. Recombinant AAV particles according to claim 41.

43. A method for delivering a therapeutic polypeptide or polynucleotide to a CNS of a subject in need thereof, comprising administering recombinant AAV particles according to claim 41 to the subject.

44. A recombinant AAV capsid protein having an n-amino acid long motif peptide targeting TFR1, wherein the peptide is inserted into the capsid protein, and thereby, when incorporated into an AAV particle, the peptide is presented on the surface. Depending on the circumstances, the recombinant capsid protein may be the AAV9 capsid protein (SEQ ID NO: 20506), the AAV9 K449R capsid protein (SEQ ID NO: 20507), or a capsid protein having at least 90%, 90%, or 99% sequence identity with the AAV9 capsid protein, and which forms a capsid that transduces CNS cells. In some cases, the peptide is inserted between amino acids 588 and 589 of the AAV9 capsid protein, or at a corresponding position in another AAV capsid protein. The recombinant AAV capsid protein mentioned above.

45. The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from Y, M, F, and L. X2 contains amino acids selected from S, H, T, and A. X3 contains amino acids selected from K and R, X4 contains amino acids selected from A, G, I, L, M, N, Q, S, T, V, and H. X5 contains amino acids selected from N, G, A, L, M, Q, S, and T. X 6 contains amino acids selected from A, T, H, N, F, I, P, L, Y, G, S, V, D, E, M, and Q. X7 contains amino acids selected from D and N. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains an amino acid selected from Y or L, X 2 contains amino acid H, X 3 contains amino acid A, X4 contains amino acids selected from K, R, N, and A. X 5 contains amino acids selected from G, Q, L, and S. X 6 comprises amino acids selected from P, L, I, N, D, and T. X 7 contains amino acid N. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from A, F, H, I, L, N, P, R, S, T, V, and Y. X2 comprises amino acids selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, and Y. X 3 contains amino acid S, X4 contains amino acids selected from S and T, X 5 contains amino acid N, X 6 contains amino acid G, X7 contains amino acids selected from I, R, and V. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from R and T, X2 comprises amino acids selected from T, L, M, S, G, D, N, E, R, K, Y, and W. X3 contains amino acids selected from G, E, D, I, F, H, S, A, M, P, V, Y, W, Q, and T. X4 contains amino acids selected from D, T, E, H, N, and G. X 5 contains amino acids selected from A, V, S, T, and D. X 6 contains amino acids selected from Y, F, P, and A. X7 contains amino acids selected from A and P. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X 1 contains amino acid L, X2 contains amino acid C, X3 contains amino acids selected from K and R, X4 contains amino acid P, X 5 contains amino acid C, X 6 contains amino acids selected from L, S, D, A, N, Q, H, P, and V. X7 contains amino acids selected from E, T, G, A, D, N, and S. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from Y and F, X2 contains amino acids selected from W, F, and Y. X3 contains amino acids selected from S, T, H, A, and Q. X 4 contains amino acid G, X5 contains amino acids selected from I, T, V, Q, M, H, K, and R. X 6 contains amino acids selected from I, P, H, L, M, A, Q, T, V, K, and R. X7 contains amino acids selected from A, S, D, E, and N. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from K, R, S, G, N, T, M, Q, V, D, I, and E. X2 comprises amino acids selected from D, S, N, M, L, G, P, E, and A. X3 contains amino acids selected from E, D, G, S, A, R, Q, T, P, and N. X4 contains amino acids selected from F, Y, T, V, S, N, A, G, and H. X 5 contains amino acids selected from T, K, S, R, V, and H. X 6 comprises amino acids selected from T, S, G, V, A, K, R, N, D, E, and H. X7 contains amino acids selected from F, W, and Y. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 comprises amino acids selected from V, I, R, N, and D. X2 contains amino acids selected from A, G, and S. X3 contains amino acids selected from L, T, S, H, and G. X4 contains amino acids selected from K, R, and E. X 5 contains amino acid G, X 6 contains amino acids selected from W, R, A, and I. X7 contains amino acids selected from D and G. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from L, M, and W. X2 contains amino acids selected from F, R, W, K, T, and Y. X3 contains amino acids selected from D and S, X 4 contains amino acid G, X 5 contains amino acid T, X 6 contains amino acids selected from P, G, S, N, A, and R. X7 contains amino acids selected from A, P, S, and Y. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 contains amino acids selected from P, N, and K. X2 contains amino acids selected from Y and F, X 3 contains amino acid A, X4 contains amino acids selected from R and K, X 5 contains amino acid S, X 6 contains amino acids selected from P, V, A, R, I, L, S, and E. X7 contains amino acids selected from E, D, M, and L. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5, where Z1 is selected from the group consisting of Y, F, and L. Z2 is selected from the group consisting of S, R, and K. X1 to X5 are amino acids that are selected independently. or The n-amino acid long motif peptide targeting TFR1 is a 7-mer peptide having the amino acid sequence X1-H-X2-L-X3-X4-X5, where X1 to X5 are independently selected amino acids. or The n-amino acid motif peptide targeting TFR1 is a 7-mer peptide having one of the amino acid sequences from SEQ ID NOs: 10952-20481 and 36241-42428. The recombinant AAV capsid protein according to claim 44.

46. A host cell for generating recombinant AAV particles according to claim 41, comprising: a first construct comprising nucleic acid encoding the recombinant AAV capsid protein; and a second construct comprising nucleic acid encoding the recombinant AAV genome.

47. A method for producing recombinant AAV particles according to claim 41, comprising: culturing host cells under conditions sufficient for the production of recombinant AAV particles; and collecting the recombinant AAV particles.