AAV capsid for CNS-range gene delivery by interaction with transferrin receptors

Abstract

The present letter invention provides an engineered AAV capsid in which at least one protein on the capsid is modified to include an n-mer motif that promotes transduction of the capsid into the central nervous system (CNS) by interaction with a transferrin receptor. Further embodiments provide a carrier system comprising one or more carriers encoding an AAV capsid and a method of delivering a cargo to a CNS. The methods comprise administering an AAV capsid according to embodiments described herein in vivo or in vitro, and the AVV capsid comprises one or more carrier molecules.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 432,336, filed on December 13, 2022, and U.S. Provisional Application No. 63 / 368,470, filed on July 14, 2022. The entire contents of the above - mentioned applications are incorporated herein by reference in their entirety.

[0003] Statement Regarding Federally Sponsored Research

[0004] 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.

[0005] Reference to Electronic Sequence Listing

[0006] Reference is made to the electronic sequence listing (“BROD - 5515WP_ST26.xml”; size 36,487,898 bytes, created on July 14, 2023), which is incorporated herein by reference in its entirety.

[0007] Reference to Electronic Tables

[0008] 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 July 14, 2022 and designated Serial No. 63 / 368,470. The tables are incorporated herein by reference in their entirety. Field of the Invention

[0009] The subject matter disclosed herein generally relates to enhancing the transduction of engineered AAV capsids into the central nervous system (CNS) through interaction with the transferrin receptor. In certain embodiments described herein, at least one protein on the capsid is modified to include an n - mer motif. Certain embodiments relate to a vector system having one or more vectors encoding an AAV capsid and a method of delivering a cargo to the CNS, wherein the AAV capsid according to the embodiments described herein is administered in vivo or in vitro, and the AAV capsid comprises one or more cargo molecules. Background of the Invention

[0011] The development of gene therapies for neurodevelopmental and neurological disorders has been limited by the inability to effectively deliver genes throughout the CNS. Several studies have reported engineered AAV9 capsids, most notably the AAV-PHP.B family, which are capable of efficient gene transfer throughout the CNS following intravenous administration in adult mice. However, to date, none of the engineered AAV capsids that cross the blood-brain barrier (BBB) and transduce the mouse brain efficiently have been shown to exhibit their enhanced CNS tropism in primates. In this work, the applicant took a mechanism-first approach and designed AAV capsids that interact with the transferrin receptor (TFRC).

[0012] The citation or identification of any document in this application is not an admission that such document is available as prior art for the present invention. Summary of the Invention

[0013] In one aspect, the present disclosure provides a composition comprising a targeting moiety that effectively increases transduction of central nervous system tissue (CNS) by binding to the transferrin receptor (TFRC), optionally further comprising a cargo conjugated or otherwise associated with the targeting moiety. In addition, one aspect provided herein is a vector system comprising one or more vectors that encode a targeting moiety that effectively increases transduction of central nervous system tissue (CNS). Additional embodiments provided herein include polypeptides or particles encoded or produced by the vector systems herein or cells comprising the compositions, vectors, polynucleotides, or particles provided herein. In one aspect, the present disclosure provides a method of delivering one or more cargos to the CNS by administering the compositions provided herein.

[0014] In an exemplary embodiment, the targeting moiety binds to the extracellular domain of TFRC. In an exemplary embodiment, the targeting moiety binds to one or more of the apical, helical, and / or protease-like domains of the extracellular domain. In an exemplary embodiment, the targeting moiety binds to the apical domain.

[0015] In one exemplary embodiment, the targeting moiety comprises an n-mer motif that comprises or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises Y, M, F, and L; X2 comprises S, H, T, and A; X3 comprises K and R; X4 comprises A, G, I, L, M, N, Q, S, T, V, and H; X5 comprises N, G, A, L, M, Q, S, and T; X6 comprises A, T, H, N, F, I, P, L, Y, G, S, V, D, E, M, and Q; and X7 comprises D and N. In one exemplary embodiment, X1 comprises Y, M, and L; X2 comprises S, H, T, and A; X3 comprises K and R; X4 comprises A, G, I, L, M, N, Q, S, T, and V; X5 comprises N; X6 comprises A, T, H, N, F, I, P, L, and Y; and X7 comprises D and N; or X1 comprises Y, M, and L; X2 comprises S, H, T, and A; X3 comprises K and R; X4 comprises A, G, I, L, M, N, Q, S, T, and V; X5 comprises G; X6 comprises A, G, F, H, I, L, N, P, S, T, V, and Y; and X7 comprises D and N; or X1 comprises L and Y; X2 comprises A, H, and S; X3 comprises K and R; X4 comprises A, G, H, I, L, M, N, Q, S, T, and V; X5 comprises G; X6 comprises P; and X7 comprises D; or X1 comprises L and Y; X2 comprises A, H, and S; X3 comprises K and R; X4 comprises A, G, H, I, L, M, N, Q, S, T, and V; X5 comprises G; X6 comprises P; and X7 comprises N; or X1 comprises Y; X2 comprises S; X3 comprises K; X4 comprises A, I, L, M, N, Q, S, T, and V; X5 comprises G; X6 comprises X; and X7 comprises Y, P, T, Q, V, F, L, H, S, A, E, D, I, and M; or X1 comprises L and Y; X2 comprises S; X3 comprises R and K; X4 comprises V, I, T, L, and A; X5 comprises S and A; X6 comprises P, R, Y, F, H, I, K, and W; and X7 comprises D; or X1 comprises F, L, M, and Y; X2 comprises H; X3 comprises K and R; X4 comprises A, L, and M; X5 comprises A, G, L, M, N, Q, S, and T; X6 comprises A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y; and X7 comprises D and N; or X1 comprises L, M, and Y; X2 comprises H; X3 comprises K and R; X4 comprises A, L, and M; X5 comprises A, G, L, M, N, Q, S, and T; X6 comprises A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y; and X7 comprises N;Alternatively, X1 contains L, M, and Y, X2 contains H, X3 contains K and R, X4 contains A, L, and M, X5 contains A, G, L, M, N, Q, S, and T, X6 contains A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y, and X7 contains D; or X1 contains L, M, and Y, X2 contains H, X3 contains K and R, X4 contains L, X5 contains S, Q, G, T, N, and L, X6 contains P, T, V, I, Q, L, and A, and X7 contains D; or X1 contains F, X2 contains S, X3 contains R, X4 contains L, X5 contains G, X6 contains A, H, N, L, V, S, P, and T, and X7 contains N; or X1 contains F, X2 contains A, X3 contains R, X4 contains T, S, and N, X5 contains G, X6 contains Y, F, H, P, and A, and X7 contains N; or X1 contains F, X2 contains H, X3 contains K and R, X4 contains L, X5 contains G, X6 contains I, P, and S, and X7 contains N and D. In one exemplary embodiment, the n-mer 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). In one exemplary embodiment, the n-mer motif is YSRIGPN (SEQ ID NO: 14632).;

[0016] In one exemplary embodiment, the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises Y or L, X2 comprises H, X3 comprises A, X4 comprises K, R, N, and A, X5 comprises G, Q, L, and S, X6 comprises P, L, I, N, D, and T, and X7 comprises N. In one exemplary embodiment, the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7: X1 comprises A, F, H, I, L, N, P, R, S, T, V, and Y, X2 comprises A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, and Y, X3 comprises S, X4 comprises S and T, X5 comprises N, X6 comprises G, and X7 comprises I, R, and V. In one exemplary embodiment, X1 comprises F, L, and Y, X2 comprises D, E, H, N, Q, S, and T, X3 comprises S, X4 comprises S and T, X5 comprises N, X6 comprises G, and X7 comprises I and V; or X1 comprises V, P, I, S, T, H, and A, X2 comprises A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, and Y, X3 comprises S, X4 comprises S and T, X5 comprises N, X6 comprises G, and X7 comprises I and V; or X1 comprises A, F, I, L, P, S, T, V, and Y, X2 comprises D, E, N, Q, S, and T, X3 comprises S, X4 comprises T and S, X5 comprises N, X6 comprises G, and X7 comprises R; or X1 comprises R, X2 comprises E, D, Q, and T, X3 comprises S, X4 comprises S and T, X5 comprises N, X6 comprises G, and X7 comprises I and V. In one exemplary embodiment, the n-mer 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).

[0017] In one exemplary embodiment, the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises R and T, X2 comprises T, L, M, S, G, D, N, E, R, K, Y, and W, X3 comprises G, E, D, I, F, H, S, A, M, P, V, Y, W, Q, and T, X4 comprises D, T, E, H, N, and G, X5 comprises A, V, S, T, and D, X6 comprises Y, F, P, and A, and X7 comprises A and P. In one exemplary embodiment, X1 comprises R, X2 comprises T, M, L, and S, X3 comprises Y, S, A, M, I, F, and P, X4 comprises D, X5 comprises A, V, S, and T, X6 comprises Y and F, and X7 comprises P; or X1 comprises R, X2 comprises T, M, L, and S, X3 comprises Y, S, A, M, I, F, and P, X4 comprises D, X5 comprises A, V, S, and T, X6 comprises Y and F, and X7 comprises A; or X1 comprises R, X2 comprises G, T, D, S, N, and E, X3 comprises E, D, P, S, and G, X4 comprises D, T, E, H, and N, X5 comprises V, A, and T, X6 comprises Y and F, and X7 comprises P; or X1 comprises R, X2 comprises G, L, T, D, and S, X3 comprises D, P, S, and G, X4 comprises D, E, H, and N, X5 comprises V and T, X6 comprises Y and F, and X7 comprises P; or X1 comprises T, X2 comprises R, K, Y, and W, X3 comprises E, W, Y, Q, S, and T, X4 comprises G, X5 comprises D, X6 comprises P and A, and X7 comprises A and P. In one exemplary embodiment, the n-mer 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).

[0018] In one exemplary embodiment, the targeting moiety comprises an n-mer motif that comprises or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises L, X2 comprises C, X3 comprises K and R, X4 comprises P, X5 comprises C, X6 comprises L, S, D, A, N, Q, H, P and V, and X7 comprises E, T, G, A, D, N and S. In one exemplary embodiment, the n-mer motif is LCKPCLD (SEQ ID NO: 36437) or LCKPCPT (SEQ ID NO: 36438). In one exemplary embodiment, the targeting moiety comprises an n-mer motif that comprises or consists of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises Y and F, X2 comprises W, F and Y, X3 comprises S, T, H, A and Q, X4 comprises G, X5 comprises I, T, V, Q, M, H, K and R, X6 comprises I, P, H, L, M, A, Q, T, V, K and R, and X7 comprises A, S, D, E and N. In one exemplary embodiment, X1 comprises Y and F, X2 comprises W, F and Y, X3 comprises T and S, X4 comprises G, X5 comprises I, T, V, Q, M and H, X6 comprises I, P, H, L, M, A, Q, T and V, and X7 comprises A, S, D and E; or X1 comprises Y and F, X2 comprises W, F and Y, X3 comprises T and S, X4 comprises G, X5 comprises I, T, V, Q, M and H, X6 comprises K and R, and X7 comprises A, S, D and E, or X1 comprises Y and F, X2 comprises W, F and Y, X3 comprises T and S, X4 comprises G, X5 comprises K and R, X6 comprises I, P, H, L, M, A, Q, T and V, and X7 comprises A, S, D and E; or X1 comprises Y, X2 comprises F, X3 comprises T, X4 comprises G, X5 comprises K, R, Q, M, H and I, X6 comprises T, R, H, K, V and L, and X7 comprises E; or X1 comprises Y, X2 comprises F, X3 comprises T, S, H and A, X4 comprises G, X5 comprises K, R and T, X6 comprises I, P, H, L, M, A, Q and T, and X7 comprises D and N; or X1 comprises Y, X2 comprises W, X3 comprises T, X4 comprises G, X5 comprises K, M, V and T, X6 comprises P, V, I, H, Q, T, M and L, and X7 comprises E and D; or X1 comprises Y and F, X2 comprises F, X3 comprises S, H, A and Q, X4 comprises G, X5 comprises K, Q and R, X6 comprises I, V, L, K, H, R, Q and M, and X7 comprises E.

[0019] In an exemplary embodiment, the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises K, R, S, G, N, T, M, Q, V, D, I, and E; X2 comprises D, S, N, M, L, G, P, E, and A; X3 comprises E, D, G, S, A, R, Q, T, P, and N; X4 comprises F, Y, T, V, S, N, A, G, and H; X5 comprises T, K, S, R, V, and H; X6 comprises T, S, G, V, A, K, R, N, D, E, and H; and X7 comprises F, W, and Y. In one exemplary embodiment, X1 comprises K and R; X2 comprises D, S, and N; X3 comprises E; X4 comprises F; X5 comprises T, K, S, R, and V; X6 comprises T, S, G, and V; and X7 comprises F, W, and Y; or X1 comprises K and R; X2 comprises D; X3 comprises D; X4 comprises F and Y; X5 comprises T, S, V, and H; X6 comprises T, S, G, V, and A; and X7 comprises F, W, and Y; or X1 comprises S, R, G, N, T, M, and Q; X2 comprises D; X3 comprises G; X4 comprises T, V, S, N, and Y; X5 comprises S; X6 comprises K and R; and X7 comprises W; or X1 comprises R, V, D, I, Q, and K; X2 comprises M, L, and G; X3 comprises S, E, A, R, and Q; X4 comprises D; X5 comprises R; X6 comprises T, A, S, G, K, and N; and X7 comprises W; or X1 comprises D, I, E, Q, V, S, and K; X2 comprises L, M, G, and P; X3 comprises E, A, S, D, Q, and T; X4 comprises S and A; X5 comprises R; X6 comprises D, S, E, T, G, and A; and X7 comprises W; or X1 comprises G; X2 comprises E, S, P, G, and A; X3 comprises D, E, P, and N; X4 comprises G, H, T, S, and N; X5 comprises V; X6 comprises R, K, and S; and X7 comprises W and Y; or X1 comprises R; X2 comprises E; X3 comprises D, E, P, and N; X4 comprises G, H, T, S, and N; X5 comprises V; X6 comprises R, K, and S; and X7 comprises W and Y; or X1 comprises G; X2 comprises G and S; X3 comprises G, E, S, A, P, and D; X4 comprises T, G, and S; X5 comprises S; X6 comprises S, T, H, K, R, A, and N; and X7 comprises W. In one exemplary embodiment, the n-mer motif is selected from the group consisting of: KDEFTTF (SEQ ID NO: 36308), KDDFITY (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).

[0020] In one exemplary embodiment, the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises V, I, R, N, and D, X2 comprises A, G, and S, X3 comprises L, T, S, H, and G, X4 comprises K, R, and E, X5 comprises G, X6 comprises W, R, A, and I, and X7 comprises D and G. In one exemplary embodiment, the n-mer motif is selected from the group consisting of: IALKGWD (SEQ ID NO: 36248), NALEGRD (SEQ ID NO: 36407), VALEGRD (SEQ ID NO: 36604), and ALKGWD (SEQ ID NO: 17701). In one exemplary embodiment, the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises L, M, and W, X2 comprises F, R, W, K, T, and Y, X3 comprises D and S, X4 comprises G, X5 comprises T, X6 comprises P, G, S, N, A, and R, and X7 comprises A, P, S, and Y.

[0021] In one exemplary embodiment, the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises P, N, and K, X2 comprises Y and F, X3 comprises A, X4 comprises R and K, X5 comprises S, X6 comprises P, V, A, R, I, L, S, E, and X7 comprises E, D, M, and L.

[0022] In one exemplary embodiment, a composition comprising a targeting moiety that effectively increases transduction of CNS tissue comprises an n-mer motif that comprises or consists of the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5, wherein 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 optionally comprises A, S, or H, X2 optionally comprises S, T, L, or I, X3 optionally comprises N or G, X4 optionally comprises G, and X5 optionally comprises N, D, I, V, or R. In one exemplary embodiment, the n-mer 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 targeting moiety comprises an n-mer motif that comprises or consists of the amino acid sequence X1-H-X2-L-X3-X4-X5, wherein X1 to X5 are independently selected amino acids. In one exemplary embodiment, the n-mer motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608).

[0023] In one exemplary embodiment, the n-mer 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-mer is selected from any one of the amino acid sequences in Tables 1 to 22, or any combination thereof. In one exemplary embodiment, the n-mer motif is selected from the amino acid sequences of SEQ ID NOs: 10952-20481 and 36241-42428. In one exemplary embodiment, the targeting moiety is a part of the viral capsid protein, including the AAV capsid.

[0024] In one exemplary embodiment, the targeting moiety is inserted or substituted in Loop IV, Loop VIII, or both, of the AAV capsid protein. In one exemplary embodiment, the targeting 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-mer is inserted between two amino acids of one or more capsid proteins such that when the protein containing the n-mer is incorporated into the AAV capsid, the n-mer is on the outside of the AAV capsid. In one exemplary embodiment, the viral capsid protein is an AAV viral capsid protein. In one exemplary embodiment, the n-mer is inserted between amino acids 588 and 589 of the capsid protein of AAV9, or at a similar position in the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh.10. In one exemplary embodiment, the targeting moiety is inserted between two consecutive amino acids within amino acids 451 to 460 of the capsid protein of AAV9, or at a similar position in the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAVrh.10. In one exemplary embodiment, the capsid protein is VP1, VP2, VP3, or a combination thereof.

[0025] 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 comprise an enzyme or an antibody, including a therapeutically useful enzyme or antibody (or an antigen-binding form thereof). In one exemplary embodiment, the polynucleotide encodes a CRISPR-Cas system. In one exemplary embodiment, the cargo is a recombinant AAV genome that is incorporated into a capsid containing an n-mer, and the recombinant AAV genome encodes a therapeutic protein or nucleic acid, including being operably linked to an appropriate regulatory sequence that directs the expression of the protein or nucleic acid in a target tissue. In one exemplary embodiment, the polynucleotide is operably linked to a regulatory sequence that promotes expression in the CNS.

[0026] In one aspect, a viral capsid or viral particle comprises any of the compositions described herein. In one exemplary embodiment, the viral capsid or viral particle further comprises a recombinant viral genome, wherein 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 is operably linked to a regulatory sequence that promotes 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 in the capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh.10.

[0027] In one aspect, the present disclosure provides a composition comprising one or more vectors encoding a targeting moiety that effectively increases transduction of central nervous system tissue (CNS). In an exemplary embodiment, a vector system comprises one or more vectors, wherein at least one of the one or more vectors encodes a targeting moiety that effectively increases transduction of CNS tissue by binding to the transferrin receptor (TFRC), and optionally wherein at least one of the one or more vectors encodes a recombinant AAV genome comprising a transgene encoding a protein or polypeptide. Also provided is a composition comprising a polypeptide or viral capsid that comprises a targeting moiety, such as an n-mer that effectively increases transduction of CNS tissue as described herein, optionally further comprising a payload, such as a recombinant AAV genome encoding a therapeutic protein or nucleic acid, the payload incorporated within, conjugated to, or otherwise associated with the polypeptide comprising the targeting moiety. In an exemplary embodiment, the targeting moiety binds to the extracellular domain of TFRC. In an exemplary embodiment, the targeting moiety binds to one or more of the apical, helical, and / or protease-like domains of the extracellular domain. In an exemplary embodiment, the targeting moiety binds to the apical domain. In an exemplary embodiment, the targeting moiety comprises any of the n-mer motifs described herein. In an exemplary embodiment, the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5, wherein Z1 comprises Y, F, and L, Z2 comprises S, R, and K, and X1 to X5 are independently selected amino acids. In an exemplary embodiment, X1 optionally comprises A, S, or H, X2 optionally comprises S, T, L, or I, X3 optionally comprises N or G, X4 optionally comprises G, and X5 optionally comprises N, D, I, V, or R. In an exemplary embodiment, the n-mer 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).

[0028] In one exemplary embodiment, the targeting moiety encoded by or incorporated into a vector system (including an AAV capsid) comprises an n-mer motif comprising or consisting of the amino acid sequence of X1-H-X2-L-X3-X4-X5, wherein X1 to X5 are independently selected amino acids. In one exemplary embodiment, the n-mer motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608). In one exemplary embodiment, the n-mer motif comprises 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 are vectors or recombinant polypeptides, including engineered AAV capsids, wherein the n-mer is selected from any one of those listed in any one of Tables 1 to 22, or any combination thereof. In one exemplary embodiment, provided herein are vectors wherein the n-mer motif is selected from SEQ ID NOs: 10952-20481 and 36241-42428. In one exemplary embodiment, provided herein are vectors that encode a targeting moiety as part of a viral capsid protein.

[0029] In one exemplary embodiment, the targeting moiety is inserted or substituted in Loop IV and / or Loop VIII of the AAV capsid protein. In one exemplary embodiment, the targeting 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 present disclosure provides a vector wherein the n-mer is inserted between two amino acids of the one or more capsid proteins such that the n-mer is on the exterior of the AAV capsid. In one exemplary embodiment, the present disclosure provides a vector wherein the viral capsid protein is an AAV viral capsid protein. In one exemplary embodiment, the present disclosure provides a vector wherein the n-mer is inserted between amino acids 588 and 589 of the capsid protein of AAV9, or at a similar position in the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74 or AAV rh.10. In one exemplary embodiment, the targeting moiety is inserted between two amino acids within amino acids 451 to 460 of the capsid protein of AAV9, or at a similar position in the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhS, AAV rh.74 or AAVrh.10. In one exemplary embodiment, the present disclosure provides a vector wherein the capsid protein is VP1, VP2, VP3 or a combination thereof.

[0030] In one exemplary embodiment, the present disclosure provides a vector wherein the cargo is a polynucleotide, one or more polypeptides, a ribonucleoprotein complex. In one exemplary embodiment, the present disclosure provides a vector wherein the polynucleotide encodes one or more polypeptides and / or RNAi oligonucleotides. In one exemplary embodiment, the present disclosure provides a vector wherein the polynucleotide encodes one or more polypeptides. In one exemplary embodiment, the present disclosure provides a vector wherein the one or more polypeptides comprise 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.

[0031] In one aspect, the present disclosure provides a composition comprising a polypeptide encoded or produced by a vector system as described herein. In an exemplary embodiment, the polypeptide is a capsid protein, optionally an AAV capsid polypeptide. In one aspect, the present disclosure provides a composition comprising a particle produced by a vector system as described herein. In an exemplary embodiment, the particle is a viral particle, optionally an AAV particle. In one aspect, the present disclosure provides a composition comprising cells that comprise a composition, vector, polypeptide, or particle as described herein.

[0032] In one aspect, the present disclosure provides a method of delivering one or more payloads to the CNS, comprising: administering in vivo or in vitro an engineered AAV capsid as described herein or a vector as described herein. In an exemplary embodiment, the present disclosure provides a delivery method, wherein the payload is a recombinant AAV genome encoding an RNAi oligonucleotide, a polynucleotide encoding a polypeptide, or a polypeptide, optionally operably linked to a regulatory sequence that promotes expression in a target tissue such as the CNS. In an exemplary embodiment, the present disclosure provides a delivery method, wherein the polypeptide comprises an enzyme or an antibody. In an exemplary embodiment, the present disclosure provides a delivery method, wherein the payload encodes a Cas polypeptide, a guide molecule, or both. In an exemplary embodiment, the present disclosure provides a delivery method, wherein the payload encodes a nuclease or a nucleic acid component of an RNA-guided nuclease. In an exemplary embodiment, the present disclosure provides a delivery method, wherein the payload is one or more polynucleotides encoding a nuclease and a nucleic acid component of an RNA-guided nuclease.

[0033] In one aspect, a method of creating a humanized transgenic non-human animal comprises: delivering one or more cells of a non-human animal to a vector system or recombinant viral particle comprising a recombinant viral genome, wherein the vector system or recombinant viral genome encodes a human transferrin polypeptide, and wherein the encoded human transferrin polypeptide is under the control of a tissue-specific promoter or an miRNA binding element that has selective activity within a desired cell, tissue, or organ.

[0034] In multiple exemplary embodiments, one or more cells are endothelial cells. In one exemplary embodiment, one or more cells are CNS cells. In multiple exemplary embodiments, one or more cells are cells of the CNS vasculature, lung, kidney, liver, or any combination thereof. In multiple exemplary embodiments, the endothelial cells are endothelial cells of the CNS vasculature. In one exemplary embodiment, a recombinant viral particle, optionally an AAV viral particle, comprises a capsid polypeptide, optionally an AAV capsid polypeptide, wherein the capsid polypeptide comprises a CNS-specific n-mer motif. In one exemplary embodiment, the CNS-specific n-mer motif comprises X1-N-X3-X4-X5-X6-X7, wherein X5 is independently selected from K or R, and X1, X3, X4, X6, and X7 are independently selected from any amino acid, optionally wherein the total charge of the n-mer motif at neutral pH is between 0 and +2. In one exemplary embodiment, the CNS-specific n-mer motif comprises NNSTRGG (SEQ ID NO: 42429), GNSARNI (SEQ ID NO: 42430), and GNSVRDF (SEQ ID NO: 42431) or consists of the same. In one exemplary embodiment, the transgenic non-human animal is a rodent, optionally a mouse.

[0035] In one aspect, provided herein is a humanized transgenic non-human animal comprising: one or more cells that express a human transferrin polypeptide, optionally wherein one or more cells are CNS cells. In one exemplary embodiment, the transgenic non-human animal is a rodent, optionally a mouse. A humanized transgenic non-human animal is produced by any of the methods described herein. In one exemplary embodiment, the humanized non-human animal has an inhibited immune system.

[0036] In one aspect, provided herein is a method of screening for an n-mer motif capable of transducing central nervous system (CNS) tissue via binding to the transferrin receptor (TFRC) in a humanized transgenic non-human animal, comprising: introducing one or more compositions comprising a candidate n-mer motif into the humanized non-human transgenic animal described herein; and detecting the binding of the composition to the transferrin receptor (TFRC) and / or detecting the transduction or uptake of one or more CNS cells of the humanized transgenic non-human animal. In one exemplary embodiment, the candidate n-mer motif comprises X1-N-X3-X4-X5-X6-X7 or consists of the same, wherein X5 is independently selected from K or R, and X1, X3, X4, X6, and X7 are independently selected from any amino acid, optionally wherein the total charge of the n-mer 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 of which comprises the candidate n-mer motif.

[0037] In one exemplary embodiment, the viral particle is an AAV viral particle and one or more capsid proteins are AAV capsid proteins, optionally wherein the candidate n-mer motif is inserted between amino acids 588 and 589 of the AAV9 capsid polypeptide or at a similar position in the capsid proteins 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 or encodes a therapeutic nucleic acid or polypeptide, a selectable marker, or a control polypeptide or nucleic acid.

[0038] In one aspect, an in vivo modeling method comprises introducing a second vector system that is capable of targeting a transgenic polypeptide in a transgenic non-human animal as described herein. In a plurality of exemplary embodiments, the non-human animal has an immunosuppressed immune system.

[0039] A method of screening for a vector system capable of transducing central nervous system (CNS) tissue via binding to the transferrin receptor (TFRC) in a humanized transgenic non-human animal comprises: (a) introducing a plurality of vector systems into one or more humanized transgenic non-human animals, and (b) detecting the vector system that binds to the transferrin receptor (TFRC).

[0040] These and other aspects, objects, features, and advantages of the exemplary embodiments will become apparent to those of ordinary skill in the art in view of the implementation of the following exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] A understanding of the features and advantages of the present invention will be obtained by reference to the following embodiments and the accompanying drawings, which illustrate illustrative embodiments in which the principles of the invention may be utilized, in which:

[0042] Figure 1 A structural model of AAV9 highlighting the modified region. The model highlights the loop IV residues (blue) where substitution mutagenesis was performed in some embodiments, and the residues inserted into loop VIII (AA588 - 589) (magenta) in other embodiments. The K449R mutation is shown in green.

[0043] Figures 2A to 2B- Capsids that bind to human TFRC can dramatically enhance the binding and transduction of CHO cells expressing human TFRC. AAVs packaging the indicated AAV-CAG-GFP-2A-luciferase-WPRE-pA genome were applied at 10K vg / cell to control CHO cells or CHO cells expressing human TFRC (hTFRC). (A). The figure shows the indicated AAV binding to the indicated CHO cells. (B) The figure shows the transduction efficiency normalized to AAV for the indicated AAV. Transduction was measured using a luciferase reporter assay 24 hours after virus application.

[0044] Figure 3 - Surface human TFRC protein levels on hCMEC / D3 cells with lentiviruses expressing TFRC. Cells transduced with VSV-G pseudotyped lentiviruses expressing human TFRC at the indicated MOI were incubated with an antibody that binds TFRC at 4°C. Cells were then fixed without membrane permeability and immunostained with anti-TFRC (ThermoFisher, catalog number 14-0719-82) to assess the level of TFRC exposed on the cell surface. Images show immunostaining (green) and nuclei (blue).

[0045] Figure 4 - TFRC-binding capsids transduce hCMEC / D3 cells more efficiently, and lentiviral delivery of TFRC to boost TFRC expression further increases the binding and transduction of hCMEC / D3 cells. The indicated AAVs were applied at 5,000 vg / cell to both control and hCMEC / D3 cells expressing human TFRC. Binding and transduction were assessed as described in Figure 2.

[0046] Figures 5A to 5B - Transduction of hCMEC / D3 cells by AAV-BI19 enhanced relative to AAV9 is blocked by a human TFRC antibody. AAV9 or AAV-BI19 was used to package the AAV-CAG-GFP-2A-luciferase-WPRE-pA genome. The indicated AAVs were applied at 2,000K vg / cell to hCMEC / D3 cells in the presence or absence of the indicated concentration of monoclonal anti-TFRC antibody. Transduction was assessed 24 hours after virus application (A), and binding was assessed at 1 hour (B).

[0047] Figure 6- Increased expression of human TFRC in mouse brain endothelial cells in vivo enhances AAV-BI19 transduction of the CNS. (Left) The schematic shows the experimental design. In a 2-step transduction assay, step one is delivery of the target receptor gene to brain endothelial cells of NSG mice using intravenous administration of AAV-BI30 (Krolak et al. Nature Cardiovascular Research 2022). In this experiment, the target receptor was human TFRC or mouse LY6A, which served as a positive control (LY6A is required for AAV-PHP.eB to efficiently enter the CNS and is absent in NSG mice). Seven days later, the second virus was delivered (AAV-BI19: CAG-mScarlet, which binds hTFRC; or AAV-PHP.eB: CAG-mScarlet, which binds LY6A). AAV-BI19 was delivered to animals that received non-target receptor AAV-BI30:LY6A (upper right), and AAV-PHP.eB was delivered to animals that received hTFRC as a negative control (lower left). In animals that had previously received the AAV-BI19 receptor hTFRC, AAV-BI19 transduction of the CNS was enhanced (lower right). Images show transduction in the mouse brain 3 weeks after delivery of the second virus. All AAVs were delivered at 1e11 vg / animal.

[0048] Figure 7 - Representative SDS-PAGE showing Fc-control (Fc-ctrl), human TFRC-Fc (hTFRC), mouse TFRC-Fc (mTFRC), and marmoset TFRC-Fc (marTFRC). The indicated proteins were produced in HEK293 cells and pulled down with ProA magnetic beads. The proteins were then separated by SDS-PAGE and stained with SyPro Ruby for total protein.

[0049] Figures 8A to 8B - Cluster analysis of 7-mer sequences that bind human TFRC. AAV7-mer inserts that interact with human TFRC were found to form distinct sequence clusters. (A) Sequences were one-hot encoded, projected with UMAP, and clustered with a Gaussian mixture model (k = 30). (B) Cluster sequence logos show the AA frequencies in each cluster, the cluster number (k), and the number of sequences in each cluster (n). A list of the sequences in each cluster is provided in Table 12.

[0050] Figure 9 - Individual binding to CHO cells stably expressing TFRC.

[0051] Figure 10 - BI19-mediated enhanced transduction of hCMEC / D3 cells relative to AAV9 is blocked by anti-human TFRC antibody.

[0052] Figure 11 -Overexpression of TFRC in the murine brain vasculature in vivo increases BI-19 transduction in the CNS.

[0053] Figures 12A to 12B -BI19 actively transports across the human brain vascular layer in vitro. hCMEC cells were cultured until confluent on transwell inserts. The virus pool was added to the top chamber, and the fraction of vector present in the bottom chamber was assessed by qPCR at the indicated times. The virus was added to the cells at 37 °C or at 4 °C. The 4 °C control indicates the amount of viral particles that enter the bottom chamber in the absence of active transport. (B) The figure provides the fraction of AAV genome present in the bottom chamber at 37 °C divided by the fraction of AAV genome present in the bottom chamber at 4 °C.

[0054] Figures 13A to 13C- Increased expression of human TFRC in mouse brain endothelial cells in vivo enhances AAV-BI19 transduction of the CNS. (A) This schematic shows the experimental design. In the two-step transduction assay, step one is to deliver the target receptor gene to the brain endothelial cells of NSG mice using intravenous administration of AAV-BI30 (Krolak et al. Nature Cardiovascular Research 2022). In this experiment, the target receptor is human TFRC or mouse LY6A, which is used as a positive control (LY6A is required for AAV-PHP.eB to efficiently enter the CNS and is absent in NSG mice). (B) Images show sagittal sections of mouse brains transduced with AAV-PHP.eB (left) or AAV-BI19 (right), expressing mouse LY6A or human TFRC by delivering genes encoding these proteins via AAV-BI30 in mice. AAV-BI30: CAG-human TFRC or AAV-BI30: CAG-mouse LY6A vectors were intravenously administered to adult NSG mice at 1e11 vg / animal. After 28 days, the second virus (AAV-BI19, which binds hTFRC; or AAV-PHP.eB, which binds mouse LY6A) was delivered at a dose of 5e11 vg / animal. Both vectors were packaged with the same AAV genome (AAV-CAG-NLS-mScarlet-2A-luciferase-pA). Transduction of the CNS by AAV-BI19 was enhanced in animals that had previously received the AAV-BI19 receptor hTFRC (lower right). AAV-BI19 was delivered to animals that received the non-target receptor AAV-BI30: LY6A (upper right), and AAV-PHP.eB was delivered to animals that received hTFRC as a negative control (lower left). Images show transduction in mouse brains three weeks after delivery of AAV-PHP.eB or AAV-BI19 (assessed by imaging native mScarlet fluorescence).

[0055] Figures 14A to 14D-AAV-BI19 crosses the BBB more efficiently and transduces neurons throughout the mouse brain, in which the extracellular domain of human TFRC is knocked into the mouse Tfrc gene. (A to B) AAV-BI19 or AAV9 packaged with CAG-NLS-mScarlet-2A-luciferase-pA at 8e11 vg / mouse was administered systemically (injected into the retro-orbital sinus) to adult huTFRCKI mice (B-hTFRC, CytoBiogen). Expression was assessed seven days after AAV administration. (A to B) Images show matched exposures of native nuclear mScarlet expression in whole sagittal brain sections from homozygous B-hTFRC mice after transduction with AAV-BI19 (A) or AAV9 (B). (C to D) AAV-BI19 (C) or AAV9 (D) transduction of neurons (NeuN immunostaining) in the huTFRC KI mouse brain. The mScarlet exposures in C and D do not match. The mScarlet exposure in D is longer to show transduction by AAV9.

[0056] Figure 15 -AAV-BI19 transduces human brain endothelial cells in the presence of human holotransferrin. Cells from the hCMEC human brain endothelial cell line were pre-incubated with or without the indicated concentrations of human transferrin and then transduced with AAV-BI19 or AAV9 (5000 vg / cell) packaged with CAG-GFP-2A-luciferase, and transduction was assessed 24 hours later by measuring luciferase activity. The figure shows transduction normalized to AAV9 in the absence of human transferrin.

[0057] Figures 16A to 16E-AAV9 can be reprogrammed to bind human TfR1. (16A) Screened an AAV9-based heptamer NNK capsid library (random heptamer insert between residues 588 and 589 of VP1) for selective binding to huTfR1 as an Fc fusion protein or when transiently overexpressed on HEK293 and CHO cells. (16B) Four huTfR1-binding variants sharing a consensus motif were identified. Each variant was individually generated to carry the CAG-NLS-mScarlet-p2A-Luc-WPRE construct. They showed enhanced species-specific binding (16C) and transduction (16D) of CHO cells stably expressing TFRC from humans but not macaques, marmosets, or mice. For the binding assay, each cell line was seeded at 30,000 cells / well, and after 24 hours, each well was replenished with fresh medium containing each AAV9 variant carrying CAG-NLS-mScarlet-P2A-luciferase-SV40-WPRE at 3,000 vg / cell. The cells were maintained at 4°C for one hour with gentle shaking and washed with PBS. Then, DNA was extracted and qPCR targeting mScarlet was performed. For the transduction assay, each cell line was seeded at 100,000 cells / well, and after 24 hours, each well was replenished with fresh medium containing each AAV9 variant carrying CAG-NLS-mScarlet-P2A-luciferase-SV40-WPRE at 3,000 vg / cell. The cells were maintained at 37°C and 5% CO2 for 24 hours. Then, transduction was measured using the britelite Plus reporter gene assay system. One-way ANOVA tests were performed to assess significant differences; **** indicates P < 0.0001. (16E) Characterization of the binding kinetics between AAV-BI19 and TfR1 in the presence or absence of Holo-Tf. Fitting of the biolayer interferometry (BLI) traces of immobilized AAV-BI19 capsids incubated with 6.25 - 200 nM Peptidisc-reconstituted huTfR1 at 30°C showed strong binding between the variant and the receptor (upper left), while the AAV9 capsid showed no significant association with huTfR1 (upper right). The effect of holotransferrin (Holo-Tf) on AAV-BI19 binding was tested by incubating the AAV-BI19 capsid with 6.25 - 200 nM Peptidisc-reconstituted huTfR1 in the presence of a 200 nM molar excess of Holo-Tf (lower left). In the presence of Holo-Tf, AAV-BI19 bound to huTfR1. AAV9 did not show significant binding to huTfR1 regardless of the presence or absence of Holo-Tf (lower right).

[0058] Figures 17A to 17BTfR1 expression in -CHO cells. By random lentiviral integration of each exogenous construct under the control of the EF-1α promoter, the applicant established stable CHO cell lines expressing human, macaque, marmoset, or mouse TFRC. (17A) RNA was extracted from each cell line, reverse transcribed, and qPCR was performed on the cDNA using species-specific primers (Table 23). (17B) Each cell line was immunostained with the following anti-TfR1 antibodies: abcam ab84036, polyclonal, targeting the N-terminal (mostly intracellular) domain of huTfR1; or OKT9, monoclonal, targeting the apical domain of huTfR1.

[0059] Figures 18A to 18D- The capsid targeting TfR1 transduces human brain endothelial cells via interaction with huTfR1. Variants of the capsid targeting TfR1 exhibit enhanced binding (18A) and transduction (18B) to human brain microvascular endothelial cells (hBMVEC) and hCMEC / D3 cells. For the binding assay, hBMVEC or hCMEC / D3 cells were seeded at 15,000 or 30,000 cells / well, respectively. After 24 hours, the medium in each well was replaced with fresh medium containing each AAV9 variant carrying CAG-NLS-mScarlet-P2A-luciferase-SV40-WPRE at 12,000 or 6,000 vg / cell for hBMVEC or hCMEC / D3 cells, respectively. The cells were maintained at 4 °C for one hour with gentle shaking. Then, DNA was extracted and qPCR targeting mScarlet was performed. For the transduction assay, hBMVEC or hCMEC / D3 cells were seeded at 5,000 or 10,000 cells / well, respectively. After 24 hours, the medium in each well was replaced with fresh medium containing each AAV9 variant carrying CAG-NLS-mScarlet-P2A-luciferase-SV40-WPRE at 12,000 or 6,000 vg / cell for hBMVEC or hCMEC / D3 cells, respectively. Twenty-four hours after addition of AAV, transduction was measured using the britelite Plus reporter gene assay system. A two-tailed t-test was performed to assess significant differences; ****, ***, **, and * indicate P < 0.0001, < 0.001, < 0.01, and < 0.05, respectively. (18C) AAV-BI19 transduction of hCMEC / D3 cells was inhibited by the OKT9 antibody targeting the apical domain of huTfR1, but not by the antibody targeting the transferrin-binding domain of huTfR1. hCMEC / D3 cells were seeded at 7,500 cells / well. Two days later, 100 μL of medium containing the indicated AAV at 3e8 vg / mL and the specified concentration of OKT9 or R&D AF2474 antibody was transferred to each well. Twenty-four hours later, transduction was measured using the britelitePlus reporter gene assay system. Two-way ANOVA was performed to assess significant differences; **** indicates P < 0.0001. (18D) AAV-BI19 actively transports across the endothelial cell barrier. Transwell inserts were seeded with hCMEC cells and grown to confluence. Barcoded AAV-BI19, AAV9, and AAV2 were pooled and applied to the upper chamber of the transwell at 25,000 vg / cell / AAV in medium heated to 37 °C or cooled to 4 °C, as indicated. The amount of virus transported across the transwell at the indicated temperature over three hours was quantified by qPCR and normalized to the corresponding starting titer. Data are represented as quadruplicate ± SEM.Unpaired t-tests were performed to assess significant differences; * indicates P < 0.05, and ns indicates no statistical difference. A dashed line was drawn at 10,000 vg, below which the ability to accurately quantify viral load was reduced.

[0060] Figures 19A to 19D - AAV-BI19 binding to the apical domain of TfR1 depends on the Y247 and T248 residues that are present in the human protein but not in macaque or mouse. (19A) Alignment of the amino acid sequences of the apical domains of human, macaque, and marmoset TfR1 from residues 200 - 380. Differences from the human sequence are highlighted in blue. Amino acid differences between the human and macaque proteins are highlighted in four groups (colored contours, 19A) and on the AlphaFold2 structural model (19B). (19C) CHO cells were transiently transfected to express wild-type human TfR1 (huTfR1) or variants with the indicated substitutions, where human residues were replaced by the corresponding macaque residues or (19D) wild-type macaque TfR1 (macTfR1) or variants with D247Y and S248T substitutions (showing n = 4 replicate transfections / plasmid ± SEM). Cells were maintained at 37 °C and 5% CO2. At 24 h post-transfection, AAV-BI19 or AAV9 encoding ssCAG-NLS-mScarlet-P2A-luciferase-pA was applied at 10,000 vg / cell. After 24 h, viral transduction was measured using the britelitePlus reporter gene assay system. For (19C), one-way ANOVA was performed to determine significant differences in transduction among the groups, using wild-type huTfR1 as a control. To account for multiple comparisons, Dunnett's correction was applied. Only those comparisons with P ≤ 0.05 were reported with an asterisk (* or **** indicate P ≤ 0.05 or ≤ 0.0001, respectively). For (19D), unpaired two-tailed t-tests were performed between the two groups (** indicates P ≤ 0.01).

[0061] Figure 20 - TfR1 expression in B-hTFR1 mice. RNA was extracted from the cortex and spinal cord regions of C57BL / 6J or B-hTFR1 mice that had been treated with AAV9 or AAV-BI19. RNA was reverse transcribed and qPCR was performed on the cDNA using primers spanning exons 1 and 2 of the mouse Tfrc gene (Table 23). Notably, in B-hTFR1 mice, exons 4 to 19 of the mouse Tfrc gene encoding the extracellular region had been replaced by the corresponding human exons. There was no significant difference in TfR1 expression between C57BL / 6J and B-hTFR1 mice (two-way ANOVA).

[0062] Figures 21A to 21I-AAV-BI19 effectively delivers genes to the CNS of knock-in mice expressing the extracellular domain of huTfR1. AAV-BI19 or AAV9 encoding ssCAG-NLS-mScarlet-P2A-luciferase-pA was intravenously injected into adult female C57BL / 6J or C57BL / 6-Tfrltm1TFR1 / Bcgen (B-hTFR1) mice at 5x10 11 vg / mouse. Three weeks later, the biodistribution of vector genome / mouse genome (21A), the mScarlet transcript levels relative to AAV9 in C57BL / 6J mice (21B), and the luciferase activity relative to AAV9 in C57BL / 6J (21C) were measured in different organs. For AAV9 in C57BL / 6J and AAV-BI19 in B-hTFR1, n = 4 mice per group are shown, ±SEM. For AAV9 in B-hTFR1 and AAV-BI19 in C57BL / 6J, n = 3 mice per group are shown, ±SEM. Two-way ANOVA was performed to determine significant differences in organs among the four groups of mice, with AAV-BI19 in B-hTFR1 as the main comparison group and using Bonferroni multiple comparison correction; ****, ***, **, and * indicate P ≤ 0.0001, P ≤ 0.001, P ≤ 0.01, and P ≤ 0.05, respectively, between AAV-BI19 in B-hTFR1 and each of the other groups of mice. Representative (21D) whole brain and (21E) spinal cord images of C57BL / 6J and B-hTFR1 mice treated with AAV-BI19 or AAV9 encoding ssCAG-NLS-mScarlet-P2A-luciferase-pA are shown at a dose of 5e11 vg / mouse. Native mScarlet fluorescence was assessed three weeks after injection. Images show that in the cortex, thalamus, and striatum of B-hTFR1 mice, cells transduced by AAV-BI19 are covered with (21F) NeuN + neurons or (21G) SOX9 + stained cells. The (21H) NeuN + neurons or (21I) SOX9 +Percentage of astrocytes (n = 3 mice for AAV9 in B-hTFR1 and AAV-BI19 in C57BL / 6J mice; n = 4 mice for AAV9 in C57BL / 6J and AAV-BI19 in B-hTFR1 mice; error bars indicate ±SEM). Two-way ANOVA was performed to determine significant differences in transduction among the four groups of mice across the two capsids in specific brain regions, with AAV-BI19 in B-hTFR1 as the main comparison group and using Bonferroni multiple comparison correction. **** indicates P ≤ 0.0001 between AAV-BI19 in B-hTFR1 and each of the other three groups of mice (AAV9 in C57BL / 6J, AAV9 in B-hTFR1, and AAV-BI19 in C57BL / 6J).

[0063] Figures 22A to 22B -AAV-BI19 exhibited similar transduction to AAV9 in the liver and dorsal root ganglia of B-hTFR1 mice. ssAAV-BI19: CAG-NLS-mScarlet-P2A-luciferase-pA or ssAAV9: CAG-NLS-mScarlet-P2A-luciferase-pA was intravenously injected into adult female C57BL / 6J or B-hTFR1 mice at a dose of 5x 10 11 vg / mouse. Three weeks after injection, native fluorescence was assessed in (22A) the liver and (22B) the dorsal root ganglia.

[0064] Figure 23- Increased expression of huTfR1 in mouse brain endothelial cells in vivo enhances AAV-BI19 transduction of the CNS. In a two-step transduction assay, step one was to deliver the target receptor gene to the brain endothelial cells of NSG mice using intravenous administration of AAV-BI30 (Krolak, T., et al. (2022). A high-efficiency AAV for endothelial cell transduction throughout the central nervous system. Nature Cardiovascular Research 1, 389-400.). In this experiment, the target receptor was human TfR1 (huTfR1, encoded by the TFRC gene) or mouse LY6A, which was used as a positive control because LY6A is required for efficient entry of AAV-PHP.eB into the CNS and is absent in NSG mice. AAV-BI30 was systemically administered at 1e11 vg / mouse to deliver AAV-CAG-hTFRC-WPRE-3x-miR122-pA (upper) or AAV-CAG-LY6A-WPRE-3x-miR122-pA (lower). At 28 days post-injection, a second virus was systemically administered (AAV-BI19:CAG-mScarlet at 5e11 vg / mL, which binds huTfR1 (left); or AAV-PHP.eB:CAG-mScarlet, which binds LY6A (right)). This experiment was performed with three mice per condition. Representative images show AAV transduction in sagittal mouse brain sections three weeks after delivery of the second virus. Transduction of the CNS by AAV-PHP.eB and AAV-BI19 was enhanced in animals previously treated with AAV-BI30:LY6A (lower right) or AAV-BI30:hTFRC (upper left). In animals that had received non-target receptor AAV-BI30:LY6A, AAV-BI19 did not show enhanced CNS transduction (lower left). As a negative control, in animals that had received AAV-BI30:hTFRC, AAV-PHP.eB also did not show enhanced CNS transduction (upper right).

[0065] Figure 24 - The vast majority of capsids with heptameric sequences of motif families and genera defined in Table 21 bind human TFR1-Fc fusion protein. For each family / genus, the percentage of sequences contained within the motif measured in the library is provided within the x-axis label.

[0066] Figure 25- Extensive functional characterization of capsids that bind human TFR1. Capsids are selected based on human TFR1-Fc binding and enhanced transduction activity in one or more of the following in vivo assays: transduction of the brains or spinal cords of B-hTFR1 KI mice or a two-step assay with BI30:CAG-human TFRC. Additional functions of capsids with enhanced in vivo tropism in mice expressing human TFRC that are related to selecting the best-performing capsid candidates are then evaluated. Notably, for the following assays, the vast majority of the selected sequences showed elevated enrichment relative to AAV9: B-hTFR1 KI (brain and spinal cord), two-step hTFRC brain, human TFR1-Fc binding, binding to CHO cells expressing human TFRC (CHO-hTFRC), and binding to hCMEC and hCMEC-hTFRC cells. In contrast, these capsids do not positively enrich for transduction of control mice (two-step mice transduced with AAV-BI30-CAG-LY6A). Left, AAVs are encoded into a library with five nucleotide sequences at 1x, 10x, 100x copy numbers relative to other capsid sequences in the library to facilitate accurate comparison in the assays, where 1x AAV9 performs poorly, e.g., in vivo CNS transduction.

[0067] The illustrations in this article are for illustrative purposes only and are not necessarily drawn to scale. Detailed Description

[0068] General Definitions

[0069] Unless otherwise defined, the scientific and technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques used in molecular biology can be found in: Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th Edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (edited by F.M. Ausubel et al.); Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor): Antibodies, A Laboratory Manual (1988) (edited by Harlow and Lane); Antibodies A Laboratory Manual, 2nd Edition 2013 (edited by E.A. Greenfield); Animal Cell Culture (1987) (edited by R.I. Freshney); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Edition, J.Wiley & Sons (New York, N.Y.(1994); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th Edition, John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd Edition (2011).

[0070] As used herein, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include both singular and plural forms.

[0071] The term "optional" or "optionally" means that the subsequent described event, circumstance, or substitution may or may not occur, and the description includes the case where the event or circumstance occurs and the case where it does not occur.

[0072] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the corresponding ranges, as well as the recited endpoints.

[0073] When referring to measurable values such as parameters, amounts, time intervals, etc., the term "about" or "approximately" as used herein means to encompass variations from and variations to the specified value, such as variations of + / - 10% or less, + / - 5% or less, + / - 1% or less, and + / - 0.1% or less, so long as such variations are appropriate for the invention disclosed. It is understood that the value itself to which the modifier "about" or "approximately" refers is also specifically and preferably disclosed.

[0074] As used herein, a "biological sample" can contain whole cells and / or live cells and / or cell debris. A biological sample can contain (or be derived from) "body fluid". The present invention encompasses multiple embodiments, where the body fluid is selected from amniotic fluid, aqueous humor, vitreous humor, bile, serum, milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit, and mixtures of one or more thereof. Biological samples include cell cultures, body fluids, cell cultures from body fluids. Body fluids can be obtained from a mammalian organism, for example, by puncture or other collection or sampling procedures.

[0075] The terms "subject", "individual", and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murine, simian, human, farm animals, sport animals, and pets. Also covered are tissues, cells, and progeny of a biological entity obtained in vivo or cultured in vitro.

[0076] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exclusive description or as a limitation on the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment. Reference throughout this specification to "one embodiment", "an embodiment", "an exemplary embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment", "in an embodiment", or "an exemplary embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may refer to the same embodiment. In addition, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as will be apparent to those skilled in the art from this disclosure. Further, although some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the present invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0077] All publications, published patent documents, and patent applications cited herein are incorporated herein by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0078] Overview

[0079] The embodiments disclosed herein provide targeting moieties that facilitate transduction into the CNS by interacting with the transferrin receptor. These targeting moieties can be incorporated into particles such as viral capsid delivery particles to confer tropism on the delivery particles and facilitate transduction of the CNS. Exemplary CNS tissues include brain and spinal cord tissue. Exemplary CNS cell types include neurons and glial cells. Further embodiments disclosed herein provide a vector system comprising one or more vectors encoding an AAV capsid according to the embodiments described herein. Accordingly, the embodiments disclosed herein provide compositions capable of delivering cargo to the CNS vasculature with enhanced selectivity and efficiency. The embodiments disclosed herein also provide vector systems for generating delivery particles and loading such delivery particles with cargo. Similarly, the embodiments disclosed herein provide methods of using such compositions to target CNS endothelial cells in vitro and in vivo, which are useful for both therapeutic and research purposes.

[0080] Additional features and advantages of the foregoing embodiments are further described below.

[0081] Targeting Moieties and Compositions Thereof

[0082] In multiple exemplary embodiments, the present disclosure provides compositions comprising a targeting moiety having enhanced tropism for endothelial cells of the CNS. Compared to native or wild-type targeting moieties, targeting moieties with enhanced tropism for CNS endothelial cells promote, increase, or otherwise improve binding to the CNS and, in some cases, promote, increase, or otherwise improve transduction of the CNS. This targeting moiety can be directly conjugated to a cargo such as an oligonucleotide or polypeptide to be delivered. Alternatively, the targeting molecule can be incorporated into a delivery particle to provide tropism for endothelial cells of the CNS to the delivery particle. Non-limiting examples of delivery particles are viral capsid particles. In such embodiments, 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 particulate delivery systems are also contemplated and covered herein, where the targeting moiety can be incorporated into or attached to, for example, exosomes or liposomes, as alternative embodiments.

[0083] In a preferred embodiment, the present disclosure provides a composition comprising a targeting moiety that effectively increases transduction of central nervous system (CNS) tissue by binding to the transferrin receptor (TFRC), and optionally further comprising a cargo conjugated or otherwise associated with the targeting moiety. The targeting moiety with increased transduction promotes, enhances, or otherwise improves binding to the CNS compared to a native or wild-type targeting moiety, and in some cases promotes, increases, or otherwise improves transduction of the CNS. In an exemplary embodiment, the targeting moiety binds to the extracellular domain of TFRC. In an exemplary embodiment, the targeting moiety binds to the apical, helical, and / or protease-like domain of the extracellular domain. In an exemplary embodiment, the targeting moiety binds to the apical domain. In an exemplary embodiment, the n-mer is an amino acid sequence of length n. The length of the n-mer can be any length required for transduction of the CNS. In various exemplary embodiments, the n-mer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids in length. In an exemplary embodiment, the n-mer motif has a length of at least 7 amino acids. In an exemplary embodiment, the composition comprising a targeting moiety that effectively increases transduction of CNS tissue comprises an n-mer motif, the n-mer motif comprising 1- the amino acid sequence of or consisting of Z 1- [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 an exemplary embodiment, X1 optionally comprises A, S, or H, X2 optionally comprises S, T, L, or I, X3 optionally comprises N or G, X4 optionally comprises G, and X5 optionally comprises N, D, I, V, or R. In an exemplary embodiment, the targeting moiety comprises an n-mer motif, the n-mer motif comprising the amino acid sequence of or consisting of X1-H-X2-L-X3-X4-X5, where X1 to X5 are independently selected amino acids.

[0084] In multiple exemplary embodiments, the n-mer can be used to increase transduction in target cells, namely CNS cells and tissues. The increase in the transduction efficiency of the n-mer into cells (which can correspond to the tropism efficiency) can be compared with a composition lacking the targeting moiety. For example, inclusion of one or more targeting moieties in the composition can result in an increase in transduction and / or transduction efficiency of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more. In one 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-mer. In one embodiment, transduction and / or transduction efficiency in endothelial cells is increased or enhanced, and in one embodiment is increased in endothelial cells of the vasculature such as the central nervous system vasculature. In multiple embodiments, transduction and / or transduction efficiency in cells of the central nervous system is increased or enhanced. In multiple embodiments, transduction and / or transduction efficiency in neurons and glial cells is increased or enhanced. In one embodiment, a composition comprising an n-mer is selective for target cells when compared to other cell types and / or other viral particles. As used herein, 'selective' and 'cell selective' mean preferentially targeting a cell when compared to other cell types. Preferably, the targeting moiety has at least a 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or greater selectivity or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or greater selectivity for the desired target (e.g., cell, organ, system, such as CNS tissue) or group of targets relative to other targets or cells (e.g., CNS). In one exemplary embodiment, the composition comprising a targeting moiety described herein can have increased uptake, delivery rate, transduction rate, efficiency, amount or a combination thereof in target cells (e.g., across the CNS, such as endothelial cells of the brain endothelium, arteriovenous axis in the brain, retina and spinal cord vasculature) when compared to other cell types (e.g., muscle cells) and / or other viral particles (e.g., AAV lacking the targeting moiety) and other compositions lacking the cell-specific n-mer motif of the present invention.

[0085] In one exemplary embodiment, the n-mer 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-mer motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608). In one exemplary embodiment, the n-mer motif comprises 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, the n-mer is selected from any one of the amino acid sequences in Tables 1-13, or any combination thereof. In one exemplary embodiment, the n-mer motif is selected from peptides having an amino acid sequence of any one of SEQ ID NOs: 10952-20481. In one exemplary embodiment, the targeting moiety is a part of a viral capsid protein (e.g., inserted between contiguous amino acids of a viral capsid protein), including an AAV capsid protein.

[0086] Transferrin receptor (TFRC) binding

[0087] In a preferred embodiment, the targeting moiety binds to the transferrin receptor. TFRC (i.e., the TfR1 protein encoded by the TFRC gene, or CD71) comprises 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 transferrin (TF) with high affinity and is ubiquitously expressed. TFRC1 is a type II transmembrane glycoprotein of approximately 90 kDa and contains approximately 760 amino acids. TFRC1 is typically visible on the cell surface as a disulfide-linked dimer, see Figure 4 .

[0088] The TFRC1 domain contains an extracellular C-terminal domain (approximately 671 amino acids) and contains the TF binding site. The extracellular C-terminal domain contains three subdomains: the apical, helical, and protease-like domains, see Figure 4 . Additionally, the extracellular C-terminal domain contains three N-linked glycosylation sites at asparagine residues 251, 317, and 727 and an O-linked glycosylation site at threonine 104, which contribute to the full function of the receptor. TFRC1 further contains a transmembrane domain (approximately 29 amino acids) and an intracellular N-terminal domain (approximately 61 amino acids). In addition to iron delivery via TF and TFRC1, iron uptake can also occur via apical domain binding by H-ferritin. See also Candelaria, P.V.; et al. Antibodies Targeting the Transferrin Receptor 1 (TfR1) as Direct Anti-Cancer Agents. Frontiers in Immunology, 2021, 12, which is incorporated herein by reference.

[0089] In an exemplary embodiment, the targeting moiety binds to the extracellular domain of TFRC. In an exemplary embodiment, the targeting moiety binds to the apical, helical, and / or protease-like domains. In an exemplary embodiment, the targeting moiety binds to the apical domain.

[0090] Engineered Virus Capsids

[0091] This disclosure describes various embodiments of engineered viral capsids, such as adeno-associated virus (AAV) capsids, which can be engineered to confer cell-selective tropism, such as CNS tissue and cell-specific tropism, on engineered viral particles. The engineered viral capsids can be lentiviral, retroviral, adenoviral, or AAV capsids. The engineered capsids can be included in engineered viral particles (e.g., engineered lentiviral, retroviral, adenoviral, or AAV viral particles) and can confer cell-selective tropism on the engineered viral particles. The engineered viral capsids described herein can include one or more of the engineered viral capsid proteins described herein. The engineered viral capsids described herein can include one or more of the engineered viral capsid proteins described herein, which can contain one or more targeting moieties as described elsewhere herein.

[0092] The engineered viral capsids can be variants of wild-type viral capsids. For example, in some embodiments, the engineered AAV capsids can be variants of wild-type AAV capsids. In some embodiments, the wild-type AAV capsids can be composed of VP1, VP2, VP3 capsid proteins, or combinations thereof. In other words, the engineered AAV capsids can include one or more variants of wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins. In some embodiments, the serotype of the wild-type AAV capsid for reference 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 capsids can have a tropism different from that of the reference wild-type AAV capsids.

[0093] In some embodiments, the targeting moiety is incorporated into a viral protein such as a capsid protein, including but not limited to lentivirus, adenovirus, AAV, bacteriophage, retroviral proteins. In some embodiments, the targeting moiety is positioned between two amino acids of the viral protein such that the targeting moiety is external to the viral capsid (i.e., presented on the surface of the viral capsid). In an exemplary embodiment, the targeting moieties disclosed herein can be inserted between two consecutive amino acids in a wild-type viral protein (VP) (or capsid protein), including regions that are surface-exposed when incorporated into the viral capsid. In some embodiments, the targeting moiety can be inserted between two consecutive amino acids in a variable amino acid region of the viral capsid protein.

[0094] In some embodiments, the targeting moiety may be inserted between two contiguous amino acids in a variable amino acid region of an AAV capsid protein. The core of each wild-type AAV viral protein contains an eight-stranded β-barrel motif (βB to βI) and an α-helix (αA), which are conserved in parvovirus capsids (see, e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structurally variable regions (VRs), also known as "loops," occur in surface loops that connect β-strands, where the β-strands cluster, creating local variations in the capsid surface. AAV has 12 variable regions (also known as 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 targeting moieties may be inserted between two amino acids in one or more of the 12 variable regions of a wild-type AVV capsid protein. In one exemplary embodiment, one or more targeting moieties may each 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 a combination thereof. In one exemplary embodiment, the targeting moiety is inserted or substituted in loop IV and / or loop VIII. In one exemplary embodiment, the targeting 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).

[0095] In one exemplary embodiment, the engineered capsid is a modified AAV1 capsid and can have a targeting moiety motif inserted after or adjacent to amino acid 590 (i.e., between amino acids 590 and 591). In one exemplary embodiment, the engineered capsid is a modified AAV3 capsid and can have a targeting moiety motif inserted after or adjacent to amino acid 586. In one exemplary embodiment, the engineered capsid is a modified AAV4 capsid and can have a targeting moiety motif inserted after or adjacent to amino acid 586. In one exemplary embodiment, the engineered capsid is a modified AAV5 capsid and can have a targeting moiety motif inserted after or adjacent to amino acid 575. In one exemplary embodiment, the engineered capsid is a modified AAV6 capsid and can have a targeting moiety inserted at or adjacent to amino acid 585, and optionally Y705 - 731, T492V, K531E. In one exemplary embodiment, the engineered capsid is a modified AAV8 capsid and can have a targeting moiety inserted after or adjacent to amino acids 585 and 590. In one exemplary embodiment, the engineered capsid is a modified AAV9 capsid and can have a targeting moiety inserted between amino acids 588 and 589. (Büning, 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 heptamer motif inserted between amino acids 588 and 589 of the AAV9 viral protein. SEQ ID NO: 20506 is a reference AAV9 capsid sequence at least with reference to the insertion sites discussed above. In one exemplary embodiment, the engineered capsid can have a heptamer motif inserted between two consecutive amino acids within amino acids 451 to 460 of the capsid protein of the AAV9 viral protein. SEQ ID NO: 20506 is a reference AAV9 capsid sequence at least with reference to the insertion sites discussed above. It should be understood that the targeting moiety can be inserted at similar positions in the AAV viral proteins of other serotypes such as but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh.74, AAV rh.10 capsid polypeptides. In some embodiments as previously discussed, the targeting moiety can be inserted between any two consecutive amino acids within the AAV viral protein, and in some embodiments, the insertion is made in the variable region.

[0096] In one exemplary embodiment, the first, second, third, or fourth amino acid of the target moiety can replace the first, second, third, or fourth amino acid of the polypeptide into which the target moiety is inserted that is before the insertion site. Using AAV as another non-limiting example, one or more of the target moieties can be inserted, for example, between amino acids 588 and 589 of the AAV9 capsid polypeptide, and the insert can replace amino acids 586, 587, and 588 such that the amino acid immediately preceding the target moiety after insertion is residue 585. It will be understood that this principle can be applied to any other insertion situation and is not necessarily limited to insertion between residues 588 and 589 of the AAV9 capsid or equivalent positions in another AAV capsid. It will be further understood that in some embodiments, no amino acids in the polypeptide into which the target moiety is inserted are replaced by the target moiety. In one exemplary embodiment, the AAV capsid protein is selected from SEQ ID NO: 20506.

[0097] In some embodiments, the targeting moiety can also include a polypeptide, polynucleotide, lipid, polymer, sugar, or a combination thereof in addition to including the n-mer motif.

[0098] 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 (such as 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 the SV40 polyadenylation signal.

[0099] In some embodiments, the engineered polynucleotide can be included in a polynucleotide that is configured to express the engineered capsid in a host cell system for producing viral particles. The host cell system can also include a construct that expresses a recombinant viral genome that contains a transgene encoding a polypeptide or nucleic acid that is operably linked to one or more regulatory sequences that facilitate the expression of the transgene in target cells, including a recombinant AAV genome in which the transgene and the regulatory sequences are both flanked by AAV ITR sequences.

[0100] In some embodiments, the polynucleotide encoding the engineered AAV capsid can be included in a polynucleotide that is configured to express the engineered capsid in a host cell system for the production of AAV viral particles. The host cell system can also include a construct that expresses a recombinant AAV viral genome, the recombinant AAV viral genome comprising a transgene encoding a polypeptide or nucleic acid, the polypeptide or nucleic acid operably linked to one or more regulatory sequences that facilitate expression of the transgene in a target cell, including a recombinant AAV genome in which the transgene and the regulatory sequences are both flanked by AAV ITR sequences. In some embodiments, the polynucleotide encoding the engineered AAV capsid can be operably coupled to a polyadenylation tail. In some embodiments, the polyadenylation tail can be the SV40 polyadenylation tail. In some embodiments, the polynucleotide encoding the AAV capsid can be operably coupled to a promoter. In some embodiments, the regulatory sequence that regulates transgene expression is a promoter and can be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is specific for: muscle (e.g., cardiac muscle, skeletal muscle, and / or smooth muscle), neurons and supporting cells (e.g., astrocytes, glial cells, Schwann cells, etc.), fat, 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 discussed elsewhere herein and are generally known in the art and are commercially available. Suitable neuron tissue / cell-specific promoters include, but are not limited to, the GFAP promoter (astrocytes), the SYN1 promoter (neurons), and NSE / RU5′ (mature neurons).

[0101] Further capsid modifications

[0102] In one exemplary embodiment, the viral capsid protein can comprise one or more mutations relative to the wild type. In one exemplary embodiment, the one or more mutations comprise a K449R substitution in the capsid polypeptide of AAV 920507, or a substitution at a similar position in the capsid polypeptide from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh.10. In one exemplary embodiment, the AAV capsid in which K449R is substituted is selected from SEQ ID NO: 20507.

[0103] In multiple exemplary embodiments, the viral capsid protein can include additional targeting motifs in addition to the n-mer motifs of the present disclosure. Without being bound by theory, the additional targeting moiety can be an antibody or a fragment thereof. In some embodiments, the additional targeting moiety can be a molecule or composition capable of recognizing, binding to, attaching to, or otherwise interacting with a binding partner present on the surface of a target cell. Binding partners include, but are not limited to, nucleic acids, proteins, peptides, sugars, fats, or any combination thereof, or any other molecule present on the surface of a target cell. In some embodiments, the binding partner is unique to a cell type or cell state, or is unique 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 a target cell. These additional targeting moieties can be used to target specific cell types or cell states within the group of target cells targeted, for example, by the n-mer motifs. As used herein, "cell state" is used to describe transient elements of cell identity. A cell state can be considered a transient profile or phenotype of a cell. Cell states transiently occur during time-dependent processes, either as a unidirectional progression of time (e.g., during differentiation or after environmental stimuli), or as state fluctuations that are not necessarily unidirectional, and the cell may return to its original state. The fluctuating process can be oscillatory (e.g., the cell cycle or circadian rhythm), or can switch between states without a predetermined order (e.g., due to random or environmentally controlled molecular events). These time-dependent processes can occur transiently in stable cell types (such as in transient environmental responses), or can lead to new distinct types (such as in differentiation). See, e.g., Wagner et al., 2016. Nat Biotechnol. 34(11):1145-1160.

[0104] 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 or fragments thereof are described in more detail elsewhere herein, see, for example, the discussion for exemplary carriers. 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 receptor ligand. 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, for example, Plückthun et al., Annu. Rev. Pharmacol. Toxicol. (2015) 55(1):489-511), an avimer (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, for example, Nord et al., Nat. Biotechnol. (1997) 15(8):772-777). In a number of exemplary embodiments, the additional targeting moiety is a receptor ligand or a binding protein. In some embodiments, the additional targeting moiety is attached to or otherwise conjugated to the capsid surface. In some embodiments, the additional targeting moiety is encoded by the vector that produces the capsids of the present invention as described herein.

[0105] Engineered Vectors and Vector Systems

[0106] The present disclosure also provides vectors and vector systems that can contain one or more of the engineered polynucleotides described herein, which can encode one or more of the target moieties of the present invention, including but not limited to engineered viral polynucleotides (e.g., polynucleotides encoding engineered AAV capsid proteins). In a preferred embodiment, the present disclosure provides a vector system that includes one or more vectors encoding a targeting moiety that effectively increases transduction of central nervous system tissue (CNS), optionally further including a cargo conjugated or otherwise associated with the targeting moiety or further including a construct encoding the cargo, the vector system including a recombinant viral genome containing a transgene. In a preferred embodiment, the targeting moiety encoded in the vector system binds to the transferrin receptor (TFRC). As used in such instances, an engineered viral capsid polynucleotide refers to a polynucleotide described herein that is capable of encoding an engineered viral capsid as described elsewhere herein; and / or any one or more of the polynucleotides capable of encoding one or more engineered viral capsid proteins described elsewhere herein. Additionally, in the case where a vector includes an engineered viral capsid polynucleotide described herein, the vector may also be referred to as or considered an engineered vector or its system, even though not so specifically noted. In various embodiments, a vector can contain a polynucleotide encoding one or more elements of an engineered viral capsid described herein. Vectors and their systems can be used to generate bacteria, fungi, yeast, plant cells, animal cells, and transgenic animals that can express one or more components of an engineered viral capsid, particle, or other composition described herein. Vectors within the scope of the present disclosure are those containing one or more of the polynucleotide sequences described herein. One or more of the polynucleotides that are part of an engineered viral capsid and its system described herein can be included in a vector or vector system.

[0107] In various exemplary embodiments, vectors for generating the rAAV disclosed herein contain the rep gene and the cap gene). The rep gene typically encodes Rep78, Rep68, Rep52, and Rep40 from a single ORF. These replication factors facilitate AAV genome replication and virion assembly. The cap gene also typically encodes three capsid proteins (i.e., virion protein 1 (VP1), VP2, and VP3) from a single ORF. Additionally, these three capsid proteins are regulated by transcription and alternative splicing from the start codon (ACG). The cap gene also encodes the assembly activating protein (AAP) from an in-frame offset ORF. AAP is essential for capsid assembly.

[0108] In some embodiments, the vector may comprise an engineered viral (e.g., AAV) capsid polynucleotide having a 3′ polyadenylation signal. In some embodiments, the 3′ polyadenylation is the SV40 polyadenylation signal. In some embodiments, the vector does not have a splicing regulatory element. In some embodiments, the vector comprises one or more minimal splicing regulatory elements. In some embodiments, the vector may further comprise a modified splicing regulatory element, wherein the modification inactivates the splicing regulatory element. In some embodiments, the modified splicing 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 may be sufficient to induce splicing of a splice acceptor or a splice donor. In some embodiments, the viral (e.g., AAV) capsid polynucleotide is the engineered viral (e.g., AAV) capsid polynucleotide as described elsewhere herein. In some embodiments, the vector does not comprise one or more minimal splicing regulatory elements, modified splicing regulators, splice acceptors, and / or splice donors.

[0109] The vector and / or vector system can be used, for example, to express one or more of an engineered viral (e.g., AAV) capsid and / or other polynucleotides in an expression cell such as a production cell to produce engineered viral (e.g., AAV) particles and / or other compositions (e.g., polypeptides, particles, etc.) comprising the engineered viral (e.g., AAV) capsid or other compositions comprising the n-mer motif of the invention as described elsewhere herein. Other uses of the vectors and vector systems described herein are also within the scope of the present disclosure. Generally and throughout this specification, the term is a tool that allows or facilitates the transfer of an entity from one environment to another. In some instances understood by those of ordinary skill in the art, a "vector" can be a term in the art to refer to a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. A vector can be a replicon such as a plasmid, phage, or cosmid into which another DNA segment can be inserted to replicate the inserted segment. Generally, a vector is capable of replication when associated with appropriate control elements.

[0110] Vectors include, but are not limited to, single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, no free ends (e.g., circular); nucleic acid molecules containing DNA, RNA or both; and variants of polynucleotides known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in a vector for packaging in 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 into host cells. Certain vectors are capable of autonomous replication 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 after introduction into the host cell and thus replicate with the host genome. In addition, certain vectors are capable of directing the expression of genes operably linked thereto. Herein, such vectors are referred to as "expression vectors". Expression vectors commonly used in recombinant DNA technology are usually in the form of plasmids.

[0111] A recombinant expression vector can be constituted by a nucleic acid (e.g., polynucleotide) of the present invention in a form suitable for expressing the nucleic acid in a host cell, meaning that the recombinant expression vector includes one or more regulatory elements, which can be selected based on the host cell to be used for expression, and is operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" and "operatively-linked" are used interchangeably herein and are further defined elsewhere herein. In the context of a vector, the term "operably linked" is intended to mean that a nucleotide sequence of interest is linked to a regulatory element in a manner that permits expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell).

[0112] In some embodiments, the vector can be a bicistronic vector. In some embodiments, the bicistronic vector can be used for one or more elements of the engineered virus (e.g., AAV) capsid system described herein. In some embodiments, the expression of the elements of the engineered virus (e.g., AAV) capsid system described herein can be driven by a suitable constitutive or tissue-specific promoter. In the case where the element of the engineered virus (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, a combination of both is used.

[0113] Cell-based vector amplification and expression

[0114] Vectors can be designed to express one or more elements of the engineered viral (e.g., AAV) capsid systems or other compositions (e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof) containing n-mer motifs described herein in suitable host cells. In one exemplary embodiment, a composition comprising a targeting moiety that effectively increases transduction of CNS tissue comprises an n-mer motif that comprises or consists of the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5, wherein 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 optionally comprises A, S, or H, X2 optionally comprises S, T, L, or I, X3 optionally comprises N or G, X4 optionally comprises G, and X5 optionally comprises N, D, I, V, or R. The n-mer motifs can be 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 targeting moiety comprises an n-mer motif that comprises X 1-The amino acid sequence of H-X2-L-X3-X4-X5 or consisting thereof, wherein X1 to X5 are independently selected amino acids. In one exemplary embodiment, the n-mer motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608). In one exemplary embodiment, the n-mer motif comprises 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).

[0115] In some embodiments, suitable host cells are prokaryotic cells. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. The vector can be virus-based or non-virus-based. In some embodiments, suitable host cells are eukaryotic cells. In some embodiments, suitable host cells are suitable bacterial cells. Suitable bacterial cells include, but are not limited to, bacterial cells from bacteria of the genus Escherichia coli. Many strains of Escherichia coli suitable for expression vectors are known in the art. These include, but are not limited to, Pir1, Stbl2, Stbl3, Stbl4, TOP10, XL1Blue, and XL10 Gold. In some embodiments, the host cell is a suitable insect cell. Suitable insect cells include those from Spodoptera frugiperda. Suitable strains of Spodoptera 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 can be 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, HepGG2, DIKX-X11, J558L, baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEF). Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

[0116] 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, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.). As used herein, a "yeast expression vector" refers to a nucleic acid that contains one or more sequences encoding RNA and / or polypeptide and may further contain any elements required for controlling nucleic acid expression, as well as any elements capable of replicating and maintaining the expression vector inside yeast cells. Many suitable yeast expression vectors and their characteristics are known in the art; for example, various vectors and techniques are described 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 contain, but are not limited to, centromere (CEN) sequences, autonomously replicating sequences (ARS), promoters operably linked to sequences or genes of interest such as RNA polymerase III promoters, terminators such as RNA polymerase III terminators, origins of replication, and marker genes (e.g., auxotrophic, antibiotic, or other selectable markers). Examples of expression vectors for use in yeast can include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integrating plasmids, yeast replicating plasmids, shuttle vectors, and episomal plasmids.

[0117] In some embodiments, the vector is a baculovirus vector or expression vector and is suitable for expressing polynucleotides and / or proteins in insect cells. Baculovirus vectors that can be used for expressing proteins 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 such as Spodoptera frugiperda Sf9 insect cells that are grown in serum-free suspension cultures. Serum-free insect cells can be purchased from suppliers such as Sigma Aldrich (EX-CELL 405).

[0118] In some embodiments, the vector is a mammalian expression vector. In some embodiments, the mammalian expression vector is capable of expressing one or more nucleotides 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). The mammalian expression vector may include one or more suitable regulatory elements capable of controlling the expression of one or more polynucleotides and / or proteins in mammalian cells. For example, commonly used promoters are derived from polyomavirus, adenovirus 2, cytomegalovirus, simian virus 40, and other viruses disclosed herein and known in the art. More details regarding suitable regulatory elements are described elsewhere herein.

[0119] For suitable expression vectors and vector systems 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.

[0120] In some embodiments, the recombinant mammalian expression vector is capable of directing the preferential expression of a nucleic acid in a particular cell type (e.g., expressing a nucleic acid using a tissue-specific regulatory element). 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); lymphoid-specific promoters (Calame and Eaton 1988. Adv. Immunol. 43:235-275), specifically the promoters of the T cell receptor (Winoto and Baltimore, 1989. EMBO J. 8:729-733) and immunoglobulin (Baneiji, et al., 1983. Cell 33:729-740; Queen and Baltimore, 1983. Cell 33:741-748); neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci USA 86:5473-5477); pancreas-specific promoters (Edlund, et al., 1985. Science 230:912-916); and mammary gland-specific promoters (e.g., the whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Also encompassed are developmentally regulated promoters, such as the murine hox promoter (Kessel and Gruss, 1990. Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546). Regarding these prokaryotic and eukaryotic vectors, reference is made to U.S. Patent 6,750,059, the contents of which are incorporated herein by reference in their entirety. Other embodiments may utilize viral vectors, for which reference may be made to U.S. Patent Application 13 / 092,085, the contents of which are incorporated herein by reference in their entirety. Tissue-specific regulatory elements are known in the art, in this regard, reference is made to U.S. Patent 7,776,321, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the regulatory element can be operably linked to a transgene in a recombinant genome packaged by an engineered AAV capsid system, thereby driving the expression of one or more elements of the transgene delivered by the viral vectors described herein in a tissue-specific manner.

[0121] Vectors can be introduced into and propagated in prokaryotic animals or prokaryotic cells. In some embodiments, prokaryotic animals are used to amplify the copy number of a vector to be introduced into eukaryotic cells or as an intermediate vector for generating a vector to be introduced into eukaryotic cells (e.g., amplifying a plasmid as part of a viral vector packaging system). In some embodiments, eukaryotic animals are used to amplify the copy number of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism.

[0122] In some embodiments, the vector can be a fusion vector or a fusion expression vector. In some embodiments, the fusion vector adds a large number of amino acids to the protein encoded therein, such as to the amino terminus, carboxyl terminus, or both of a recombinant protein. Such fusion vectors can be used for one or more purposes, such as: (i) increasing recombinant protein expression; (ii) increasing the solubility of the recombinant protein; and (iii) facilitating 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 prokaryotic animals can be carried out in Escherichia coli using vectors that contain constitutive or inducible promoters that direct the expression of fusion or non-fusion polynucleotides and / or proteins. In some embodiments, the fusion expression vector can include a proteolytic cleavage site that can be introduced at the junction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotide or protein to effect the separation of the recombinant polynucleotide or protein from the fusion vector backbone or other fusion moiety after purification of the fusion polynucleotide or protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin, and enterokinase. Exemplary 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 Escherichia coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0123] In some embodiments, one or more vectors that drive the expression of one or more elements of the engineered viral (e.g., AAV) capsid systems or other compositions containing n-mer motifs described herein are introduced into a host cell such that the expression of the elements of the engineered delivery systems described herein directs the formation of the engineered viral (e.g., AAV) capsid systems or other compositions containing n-mer motifs described herein (including but not limited to engineered gene transfer agent particles, which are described in more detail elsewhere herein). For example, the different elements of the engineered viral (e.g., AAV) capsid systems or other compositions containing n-mer motifs described herein can each be operably linked to separate regulatory elements on separate vectors. RNAs of the different elements of the engineered delivery systems 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 the different elements of the engineered viral (e.g., AAV) capsid systems or other compositions containing n-mer motifs described herein, which incorporate one or more elements of the engineered viral (e.g., AAV) capsid systems or other compositions containing n-mer motifs described herein or one or more cells that incorporate and / or express one or more elements of the engineered viral (e.g., AAV) capsid systems or other compositions containing n-mer motifs described herein.

[0124] In some embodiments, two or more elements expressed by the same or different regulatory elements can be combined in a single vector with one or more additional vectors that 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, such as one element positioned 5′ (“upstream”) or 3′ (“downstream”) relative to a second element. The coding sequence of one element can be positioned on the same or opposite strand of the coding sequence of the second element and in the same or opposite orientation. In some embodiments, a single promoter drives the expression of a transcript encoding one or more of the engineered viral (e.g., AAV) capsid proteins or other compositions containing n-mer motifs described herein, the transcript being embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the engineered polynucleotides of the invention (including but not limited to engineered viral polynucleotides) can be operably linked to and expressed by the same promoter.

[0125] Vector Features

[0126] The vector can include additional features that can confer one or more functionalities to the vector, the polynucleotide to be delivered, the viral particles produced therefrom, or the polypeptides expressed thereby. Such features include, but are not limited to, regulatory elements, selectable markers, molecular identifiers (such as molecular barcodes), stabilizing elements, and the like. Those skilled in the art will understand that the design of the expression vector and the additional features included can depend on factors such as the choice of host cell to be transformed, the desired expression level, and the like.

[0127] Regulatory Elements

[0128] In multiple embodiments, the polynucleotides and / or their vectors described herein (including but not limited to the engineered AAV capsid polynucleotides of the present invention) may include one or more regulatory elements operably linked to the polynucleotide. The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRESs), and other expression control elements (such as transcription termination signals, such as polyadenylation signals and polyuridine (poly-U) 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 direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may direct expression primarily in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, a specific organ (e.g., liver, brain), or a specific cell type (e.g., lymphocytes). Regulatory elements may also direct expression in a time-dependent manner, such as a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue- or cell type-specific. In some embodiments, the vector contains 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 a combination thereof. Examples of pol III promoters include but are not limited to the U6 and H1 promoters. Examples of pol II promoters include but are not limited to the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally together with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally together with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1α promoter. The term "regulatory element" also encompasses enhancer elements, such as the WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0129] 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 Publication WO 2011 / 028929, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the vector can contain a minimal promoter. In some embodiments, the minimal promoter is the Mecp2 promoter, the tRNA promoter, or U6. In yet another embodiment, the minimal promoter is tissue-specific. In some embodiments, the length of the vector polynucleotide minimal promoter and the polynucleotide sequence is less than 4.4 Kb.

[0130] To express a polynucleotide, the vector can include one or more transcriptional and / or translational initiation regulatory sequences, such as a promoter, which directs the transcription of the gene and / or the translation of the encoded protein in a cell. In some embodiments, a constitutive promoter can be employed. Suitable constitutive promoters 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. Suitable constitutive promoters 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 expression in yeast.

[0131] In some embodiments, the regulatory element can be a regulated promoter. A "regulated promoter" refers to a promoter that directs gene expression not constitutively but in a temporally and / or spatially regulated manner and includes tissue-specific, tissue-preferred, and inducible promoters. In some embodiments, the regulated promoter is a tissue-specific promoter as previously discussed elsewhere herein. Regulated promoters include conditional promoters and inducible promoters. In some embodiments, conditional promoters can be used to direct the expression of a polynucleotide in a specific cell type under certain environmental conditions and / or during a specific state of development. Suitable tissue-specific promoters can 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 (αMHC), MYL2 (MLC-2v), TNI3 (cTn1), NPPA (ANF), Slc8a1 (Ncx1)), central nervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, FOXA2 (HNF3 β)), 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, Fer114), endothelial cell-specific promoters (e.g., ENG), pluripotency and embryonic 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 and / or cell-specific promoters are discussed elsewhere herein and can be well known in the art and within the scope of the present disclosure.

[0132] Inducible / conditional promoters can be positive inducible / conditional promoters (e.g., promoters that activate transcription of a polynucleotide upon appropriate interaction with an activated activator, or an inducer (compound, environmental condition, or other stimulus)); or negative / conditional inducible promoters (e.g., promoters that are repressed (e.g., by binding of a repressor) until the repression condition of the promoter is removed (e.g., an inducer binds to the repressor bound to the promoter, stimulates release of the promoter by the repressor, or removes a chemical repressor from the promoter environment). The inducer can be a compound, environmental condition, and other stimuli. Thus, inducible / conditional promoters can respond to any suitable stimulus such as chemical, biological, or other molecular reagents, 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.

[0133] In some embodiments, the vector or its system can include one or more elements capable of translocating an engineered polynucleotide of the invention (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) into a specific cellular component or organelle and / or expressing it in a specific cellular component or organelle. Such organelles can include, but are not limited to, the nucleus, ribosome, endoplasmic reticulum, Golgi apparatus, chloroplast, mitochondrion, vacuole, lysosome, cytoskeleton, plasma membrane, cell wall, peroxisome, centriole, etc.

[0134] Optional Markers and Tags

[0135] 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 otherwise modified to include a polynucleotide encoding or serving as a selectable marker or tag, which can be a polynucleotide or a polypeptide. In some embodiments, a polypeptide encoding a polypeptide selectable marker can be incorporated into the engineered polynucleotide of the invention (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) such that the selectable marker polypeptide is inserted between two amino acids between the N-terminus and the C-terminus of the engineered polypeptide (e.g., an engineered AAV capsid polypeptide) or at the N-terminus and / or C-terminus of the engineered polypeptide (e.g., an engineered AAV capsid polypeptide) upon translation. In some embodiments, the selectable marker or tag is a polynucleotide barcode or a unique molecular identifier (UMI).

[0136] It will be understood that polynucleotides encoding such selectable markers or tags can be incorporated in a suitable manner into polynucleotides encoding one or more components of the engineered AAV capsid systems described herein to permit expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be immediately understood by those of skill in the art in view of the present disclosure. Many such selectable markers and tags are well known in the art and are intended to be within the scope of the present disclosure.

[0137] Suitable selectable markers and tags include, but are not limited to, affinity tags such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP and GST; chromatographic tags such as those composed of polyanionic amino acids such as FLAG tag; epitope tags such as V5 tag, Myc tag, HA tag and NE tag; protein tags that allow specific enzymatic 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 provide resistance to other toxic compounds, including antibiotics such as phleomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT), etc.; DNA and / or RNA segments encoding products that are lacking in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA and / or RNA segments encoding products that can be easily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (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 that were not previously juxtaposed), DNA sequences that are not acted upon or have been acted upon by restriction endonucleases or other DNA modifying enzymes, chemicals, etc.; epitope tags (e.g., GFP, FLAG tag and His tag), and DNA sequences that constitute molecular barcodes or unique molecular identifiers (UMIs), DNA sequences required for specific modifications (e.g., methylation) that allow them to be identified. Those of skill in the art will appreciate other suitable markers.

[0138] Optional tags and labels 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 such as a short linker like GS or GG or a long linker like (GGGGG)3 (SEQ ID NO: 20482) or (GGGG S)3 (SEQ ID NO: 20483), such as a glycine or glycine-serine linker. Other suitable linkers are described elsewhere herein.

[0139] The vector or vector system can include one or more polynucleotides encoding one or more n-mers. In some embodiments, the polynucleotides encoding the n-mers can be included in a vector or vector system such as a viral vector system such that they are expressed within and / or on the resulting viral particles, such that the viral particles can be targeted to specific cells, tissues, organs, etc. In some embodiments, the polynucleotides encoding the n-mers can be included in a vector or vector system such that the engineered polynucleotides of the present invention (e.g., engineered viral (e.g., AAV) capsid polynucleotides) and / or the products expressed therefrom include the n-mer and can be targeted to specific cells, tissues, organs, etc. In some embodiments, such as non-viral carriers, the n-mer can be attached to the carrier (e.g., polymers, lipids, inorganic molecules, etc.) and can target the carrier and any attached or associated engineered polynucleotides of the present invention, engineered polypeptides of the present invention described herein, or other compositions to specific cells, tissues, organs, etc. In some embodiments, the specific cells are CNS cells.

[0140] Cell-free vectors and polynucleotide expression

[0141] In some embodiments, the polynucleotides of the present invention encoding n-mer motifs can be expressed from a vector or suitable polynucleotide in a cell-free in vitro system. In some embodiments, the polynucleotides encoding one or more features of the engineered AAV capsid system can be expressed from a vector or suitable polynucleotide in a cell-free in vitro system. In other words, the polynucleotide can be transcribed and optionally translated in vitro. In vitro transcription / translation systems and suitable vectors are generally known in the art and are commercially available. Generally, in vitro transcription and in vitro translation systems replicate the processes of RNA and protein synthesis, respectively, outside of a cellular environment. Vectors and suitable polynucleotides for in vitro transcription can include T7, SP6, T3, promoter regulatory sequences that can be recognized and acted upon by appropriate polymerases to transcribe the polynucleotide or vector.

[0142] In vitro translation can be independent (e.g., translation of purified polyribonucleotides) 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 the various macromolecular components required for translation of exogenous RNA (e.g., 70S or 80S ribosomes, tRNAs, aminoacyl-tRNAs, synthetases, initiation factors, elongation factors, termination factors, etc.). Other components can be included or added during the translation reaction, including but not limited to amino acids, energy sources (ATP, GTP), energy regeneration systems (phosphocreatine and phosphocreatine kinase (eukaryotic systems)) (phosphoenolpyruvate and pyruvate kinase for bacterial systems), and other cofactors (Mg2+, K+, etc.). As previously mentioned, in vitro translation can be based on RNA or DNA starting materials. Some translation systems can utilize an RNA template as the starting material (e.g., reticulocyte lysates and wheat germ extracts). Some translation systems can utilize a DNA template as the starting material (e.g., E. coli-based systems). In these systems, transcription and translation are coupled, and the DNA is first transcribed into RNA, which is then translated. Suitable standard and coupled cell-free translation systems are generally known in the art and commercially available.

[0143] Codon Optimization of Carrier Polynucleotides

[0144] As described elsewhere herein, the polynucleotides encoding n-mer motifs of the invention described herein and / or other polynucleotides or transgenes contained within a recombinant AAV genome can be codon optimized. In some embodiments, the polynucleotides of the engineered AAV capsid systems described herein can be codon optimized. In some embodiments, one or more polynucleotides contained within the vectors described herein (“vector polynucleotides”), other than the polynucleotides encoding n-mer motifs which are optionally codon optimized, including but not limited to the embodiments of the engineered AAV capsid systems described herein, can be codon optimized. Generally, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. Individual species exhibit particular preferences for certain codons for a given amino acid. Codon bias (differences in codon usage between organisms) is often correlated with the translational efficiency of messenger RNA (mRNA), and it is believed that translational efficiency depends on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The advantages of the selected tRNAs in a cell generally reflect the codons that are most frequently used in peptide synthesis. Thus, genes can be customized for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the “Codon Usage Database” at www.kazusa.or.jp / codon / , and these tables can be adjusted in a number of 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 a particular sequence 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) in the sequence encoding a Cas protein that targets DNA / RNA correspond to the most frequently used codons for a given amino acid.For codon usage in yeast, refer to 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 in plants including seaweeds, refer to 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.

[0145] The carrier polynucleotide can be codon-optimized for expression in a specific cell type, tissue type, organ type, and / or subject type. In some embodiments, the codon-optimized sequence is optimized for expression in eukaryotes, such as humans (i.e., optimized for expression in humans or human cells), or for another eukaryote such as another animal (e.g., a mammal or avian), as described elsewhere herein. Given the description herein, such codon-optimized sequences are within the capabilities of those skilled in the art. In some embodiments, the polynucleotide is codon-optimized for a specific cell type. Such cell types can include, but are not limited to, epidermal cells (including skin cells, gastrointestinal lining cells, other hollow organ lining cells), nerve cells (nerves, brain cells, spinal cord cells, nerve support cells (e.g., astrocytes, glial cells, Schwann cells, etc.)), muscle cells (e.g., cardiac, smooth muscle cells, and skeletal muscle cells), connective tissue cells (adipose and other soft tissue filling cells, bone cells, tendon cells, chondrocytes), blood cells, stem cells and other progenitor cells, immune cells, germ cells, and combinations thereof. Given the description herein, such codon-optimized sequences are within the capabilities of those skilled in the art. In some embodiments, the polynucleotide is codon-optimized for a specific tissue type. Such tissue types can include, but are not limited to, muscle tissue, connective tissue, nerve tissue, and epidermal tissue. Given the description herein, such codon-optimized sequences are within the capabilities of those skilled in the art. 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. Given the description herein, such codon-optimized sequences are within the capabilities of those skilled in the art.

[0146] In some embodiments, the carrier polynucleotide is codon-optimized for expression in a particular cell such as a prokaryotic or eukaryotic cell. The eukaryotic cell can be from or derived from a particular organism such as a plant or mammal, including but not limited to humans, or a non-human eukaryote or an animal or mammal as discussed herein, e.g., a mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.

[0147] Non-viral vectors and carriers

[0148] In some embodiments, the vector is a non-viral vector or vehicle. In some embodiments, compared to viral vectors, non-viral vectors may have the advantages of reduced toxicity and / or immunogenicity and / or increased biosafety. As used herein in this context, the term "non-viral vectors and vehicles" refers to molecules and / or compositions that are not based on one or more components of a virus or viral genome (excluding any nucleotides to be delivered and / or expressed via the non-viral vector), which are capable of attaching to, incorporating, conjugating with the engineered capsid polynucleotides of the present invention described herein (such as engineered AAV capsid polynucleotides) or other compositions and / or otherwise interacting therewith, and are capable of transporting the polynucleotide to a cell and / or expressing the polynucleotide. It should be understood that this does not exclude the inclusion of virus-based polynucleotides to be delivered. For example, if the gRNA to be delivered targets viral components and is inserted or otherwise conjugated to another non-viral vector or vehicle, this will not render the vector a "viral vector". Non-viral vectors and vehicles include naked polynucleotides, chemical-based vehicles, polynucleotide (non-viral)-based vectors, and particle-based vehicles. It should be understood that the term "vector" as used in the context of non-viral vectors and vehicles refers to a polynucleotide vector, and the "vehicle" as used in this context refers to a non-nucleic acid or polynucleotide molecule or composition that attaches to the polynucleotide to be delivered, such as the engineered AAV capsid polynucleotides of the present invention or otherwise interacts therewith.

[0149] Naked Polynucleotides

[0150] In some embodiments, one or more of the engineered AAV capsid polynucleotides or other polynucleotides of the invention described elsewhere herein may be included in a naked polynucleotide. As used herein, the term "naked polynucleotide" in the art refers to a polynucleotide that is not associated with another molecule (such as a protein, lipid, and / or other molecule) that may often help protect it from environmental factors and / or degradation. As used herein, being associated with includes, but is not limited to, being linked to, adhered to, adsorbed to, encapsulated, enclosed therein or therein, mixed with, etc. A naked polynucleotide comprising one or more of the engineered AAV capsid polynucleotides or other polynucleotides of the invention as described herein can be directly delivered to a host cell and optionally expressed therein. The naked polynucleotide can have any suitable two-dimensional and three-dimensional configuration. As non-limiting examples, the naked polynucleotide can be a single-stranded molecule, a double-stranded molecule, a circular molecule (such as a plasmid and an artificial chromosome), a molecule containing single-stranded and double-stranded portions (such as a ribosome), etc. In some embodiments, the naked polynucleotide contains only the engineered AAV capsid polynucleotide or other polynucleotide of the invention. In some embodiments, the naked polynucleotide can contain other nucleic acids and / or polynucleotides in addition to the engineered AAV capsid polynucleotide or other polynucleotide of the invention described elsewhere herein. The naked polynucleotide can include one or more elements of a transposon system. Transposons and their systems are described in more detail elsewhere herein.

[0151] Non-Viral Polynucleotide Vectors

[0152] In some embodiments, one or more of the engineered AAV capsid polynucleotides or other polynucleotides of the invention may be included in a non-viral polynucleotide vector. Suitable non-viral polynucleotide vectors include, but are not limited to, transposon vectors and vector systems, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, plasmids without AR (antibiotic resistance) and minicircles, circular covalently closed vectors (such as microcircles, microvectors, microknots), linear covalently closed vectors ("dumbbell-shaped"), MIDGE (minimalistic immunologically defined gene expression) vectors, MiLV (microlinear vector) vectors, Ministring, mini-intron plasmids, PSK system (post-segregation killing system), ORT (operon repressor titration) plasmids, etc. See, for example, Hardee et al. 2017. Genes. 8(2):65.

[0153] 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-segregation killing system genes. In some embodiments, the non-viral polynucleotide vector is AR-free. In some embodiments, the non-viral polynucleotide vector is a microvector. In some embodiments, the non-viral polynucleotide vector includes a nuclear localization signal. In some embodiments, the non-viral polynucleotide vector can include one or more CpG motifs. In some embodiments, the non-viral polynucleotide vector can include one or more scaffold / matrix attachment regions (S / MARs). See, e.g., Mirkovitch et al. 1984. Cell. 39:223-232; Wong et al. 2015. Adv. Genet. 89:113-152, the techniques and vectors of which are applicable to the present invention. S / MARs are AT-rich sequences that play a role in the spatial organization of chromosomes by attaching DNA loop bases to the nuclear matrix. S / MARs are often found near regulatory elements such as promoters, enhancers, and origins of DNA replication. Including one or more S / MARs can promote replication once per cell cycle to maintain the non-viral polynucleotide vector as an episome in daughter cells. In multiple embodiments, the S / MAR sequence is located downstream of the actively transcribed polynucleotide (e.g., one or more of the engineered AAV capsid polynucleotides or other polynucleotides or molecules of the present invention) included in the non-viral polynucleotide vector. In some embodiments, the S / MAR can be the S / MAR from the β-interferon gene cluster. See, e.g., 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, the techniques and vectors of which are applicable to the present invention.

[0154] 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 capable of moving from one location in a genome to another. There are several classes of transposons. Transposons include retrotransposons and DNA transposons. Retrotransposons require transcription of the polynucleotide, which is then moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. DNA transposons are those that do not require reverse transcription of the polynucleotide, which is 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, meaning that transposition itself does not occur spontaneously. In some of these embodiments, the transposon vector lacks one or more polynucleotide sequences encoding proteins required for transposition. In some embodiments, the non-autonomous transposon vector lacks one or more Ac elements.

[0155] In some embodiments, the non-viral polynucleotide transposon vector system can include a first polynucleotide vector that contains the engineered AAV capsid polynucleotide or other polynucleotide or molecule of the invention described herein, flanked on the 5' and 3' ends by transposon terminal inverted repeats (TIRs); and a second polynucleotide vector that includes a polynucleotide capable of encoding a transposase, the polynucleotide being coupled to a promoter to drive expression of the transposase. When both are expressed in the same cell, the transposase can be expressed by the second vector and can transpose the material between the TIRs on the first vector (e.g., the engineered AAV capsid polynucleotide or other polynucleotide or molecule of the invention) and integrate the material into one or more locations in the host cell genome. In some embodiments, the transposon vector or its system can be configured as a gene trap. In some embodiments, the TIRs can be configured to flank a strong splice acceptor site, followed by a reporter gene and / or other gene (e.g., one or more of the engineered AAV capsid polynucleotide or other polynucleotide or molecule of the invention) and a strong poly A tail. When transposition occurs using the vector or its system, the transposon can insert into an intron of a gene, and the inserted reporter gene or other gene can trigger an incorrect splicing process, thus resulting in activation of the trapped gene.

[0156] Any suitable transposon system can be used. Suitable transposons and their systems can include the Sleeping Beauty transposon system (Tc1 / mariner superfamily) (see, e.g., Ivics et al. 1997. Cell. 91(4):501-510), piggyBac (piggyBac superfamily) (see, e.g., Li et al. 2013 110(25):E2279-E2287 and Yusa et al. 2011. PNAS. 108(4):1531-1536), Tol2 (superfamily hAT), FrogPrince (Tc1 / mariner superfamily) (see, e.g., Miskey et al. 2003 Nucleic Acid Res. 31(23):6873-6881), and variants thereof.

[0157] Chemical Carriers

[0158] In some embodiments, the engineered AAV capsid polynucleotides or other polynucleotides or other molecules of the invention described herein can be conjugated to a chemical carrier. Chemical carriers suitable for delivering polynucleotides can be broadly classified into the following categories: (i) inorganic particles, (ii) lipid-based, (iii) polymer-based, and (iv) peptide-based. They can be classified as (1) those that can form a condensed complex with a polynucleotide (such as the engineered AAV capsid polynucleotide of the invention), (2) those that are capable of targeting specific cells, (3) those that are capable of increasing the delivery of the polynucleotide or other molecule of the invention (such as the engineered AAV capsid polynucleotide) to the nucleus or cytosol of a host cell, (4) those that are capable of dissociating from DNA / RNA in the cytosol of a host cell, and (5) those that are capable of sustained or controlled release. It should be understood that any given chemical carrier can include features from multiple categories. As used herein, the term "particle" refers to any suitable-sized particle for delivering the compositions of the invention described herein (including the particles, polypeptides, polynucleotides, and other compositions described herein). Suitable sizes include large-sized, micron-sized, and nano-sized particles.

[0159] In some embodiments, the non-viral carrier can be an inorganic particle. In some embodiments, the inorganic particle can be a nanoparticle. The inorganic particles can be configured and optimized for different sizes, shapes, and / or porosities. In some embodiments, the inorganic particles are optimized to escape from the reticuloendothelial system. In some embodiments, the inorganic particles can be optimized to protect the trapped molecules from degradation. Suitable inorganic particles that can be used as non-viral carriers in this context can include, but are not limited to, calcium phosphate, silica, metals (such as gold, platinum, silver, palladium, rhodium, osmium, iridium, ruthenium, mercury, copper, rhenium, titanium, niobium, tantalum, and combinations thereof), magnetic compounds, particles, and materials (such as superparamagnetic iron oxide and magnetite), quantum dots, fullerenes (such as carbon nanoparticles, nanotubes, nanobundles, etc.), and combinations thereof. Other suitable inorganic non-viral carriers are discussed elsewhere herein.

[0160] In some embodiments, the non-viral carrier can be lipid-based. Suitable lipid-based carriers can also be described in more detail herein. In some embodiments, the lipid-based carrier includes cationic lipids or zwitterionic lipids that are capable of binding to or otherwise interacting with the negative charges on the polynucleotide to be delivered (such as the engineered AAV capsid polynucleotide of the present invention). In some embodiments, the chemical non-viral carrier system can include a polynucleotide (such as the engineered AAV capsid polynucleotide of the present invention or other compositions or molecules) and a lipid (such as 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 lipid-based non-viral carrier can be a lipid nanoemulsion. The lipid nanoemulsion can be formed by dispersing immiscible liquids in another stabilizing emulsifier and can have particles of about 200 nm composed of lipid, water, and surfactant, and can contain the polynucleotide to be delivered (such as the engineered AAV capsid polynucleotide of the present invention). In some embodiments, the lipid-based non-viral carrier can be a solid lipid particle or nanoparticle.

[0161] In some embodiments, the non-viral vector can be peptide-based. In some embodiments, the peptide-based non-viral vector can include one or more cationic amino acids. In some embodiments, 35% to 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 vector can be used in combination with other types of vectors (such as polymer-based vectors and lipid-based vectors) to functionalize these vectors. In some embodiments, the functionalization is targeted at host cells. Suitable polymers that can be included in the polymer-based non-viral vector can include, but are not limited to, polyethyleneimine (PEI), chitosan, poly(DL-lactide) (PLA), poly(DL-lactide-co-glycolide) (PLGA), dendrimers (see, for example, U.S. Patent Publication 2017 / 0079916, the techniques and compositions of which are applicable to the engineered AAV capsid polynucleotides of the present invention), polymethacrylates, and combinations thereof.

[0162] In some embodiments, the non-viral vector can be configured to release an engineered delivery system polynucleotide associated with or attached to the non-viral vector in response to an external stimulus, such as pH, temperature, osmotic pressure, concentration of a specific molecule or composition (such as calcium, NaCl, etc.), pressure, and the like. In some embodiments, the non-viral vector can be a particle 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 an optional trigger. In some embodiments, the particle can include a polymer selected from the group consisting of polymethacrylates and polyacrylates. In some embodiments, the non-viral particle 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 are applicable to the present invention.

[0163] In some embodiments, the non-viral vector can be a polymer-based vector. In some embodiments, the polymer is cationic or predominantly cationic such that it can interact with a negatively charged polynucleotide to be delivered (such as the engineered AAV capsid polynucleotide of the present invention) in a charge-dependent manner. Polymer-based systems are described in more detail elsewhere herein.

[0164] Viral Vectors

[0165] In some embodiments, the vector is a viral vector. As used herein in this context, the term "viral vector" in the art refers to a polynucleotide-based vector that contains one or more elements derived from or based on a virus, and that, when used alone or in combination with one or more viral vectors (such as in a viral vector system), is capable of expressing a polynucleotide such as an engineered AAV capsid polynucleotide of the invention, a cargo, or other composition or molecule in a viral particle, packaging it into the viral particle, and producing the viral particle. Viral vectors and their systems can be used to produce viral particles for delivery and / or expression and / or generation of one or more compositions of the invention described herein (including but not limited to any viral particle and associated cargo). A viral vector can be part of a viral vector system involving multiple vectors. In some embodiments, systems incorporating multiple viral vectors can increase the safety of these systems. Suitable viral vectors can include adenovirus-based vectors, adeno-associated vectors, helper-dependent adenovirus (HdAd) vectors, hybrid adenovirus vectors, and the like. Other embodiments of viral vectors and viral particles produced therefrom are described elsewhere herein. In some embodiments, the viral vector is configured to produce non-replicating viral particles to enhance the safety of these systems.

[0166] Adenoviral Vectors, Helper-Dependent Adenoviral Vectors, and Hybrid Adenoviral Vectors

[0167] In some embodiments, the vector can be an adenovirus vector. In some embodiments, the adenovirus vector can include elements such that the viral particles produced using the vector or its system can be serotype 2, 5, or 9. In some embodiments, the polynucleotide to be delivered via the adenovirus particle can be up to about 8 kb. Thus, in some embodiments, the adenovirus vector can include a DNA polynucleotide to be delivered, the size of which can range from about 0.001 kb to about 8 kb. Adenovirus vectors have been successfully used in several instances (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 an engineered AAV capsid that forms the adenovirus particle.

[0168] In some embodiments, the vector can be a helper-dependent adenoviral vector or a system thereof. Also known in the art as "gutless" or "empty shell" vectors and are a modified first generation of adenoviral vectors (see, e.g., Thrasher et al. 2006. Nature. 443: E5-7). In an embodiment of a helper-dependent adenoviral vector system, one vector (helper) can contain all the viral genes required for replication but contain a conditional genetic defect in the packaging domain. The second vector of the system can contain only the termini of the viral genome, one or more engineered AAV capsid polynucleotides, and the native packaging recognition signals, which can allow for selective packaging and release from cells (see, e.g., Cideciyan et al. 2009. N Engl J Med. 361: 725-727). Helper-dependent adenoviral vector systems have been successfully used for gene delivery in several instances (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 disclosures can be applicable to include and deliver the engineered AAV capsid polynucleotides described herein. In some embodiments, the polynucleotide to be delivered via viral particles generated from a helper-dependent adenoviral vector or a system thereof can be up to about 38 kb. Thus, in some embodiments, the adenoviral vector can include a DNA polynucleotide to be delivered that ranges 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).

[0169] In some embodiments, the vector is a hybrid adenoviral vector or a system thereof. The hybrid adenoviral vector consists of the high transduction efficiency of a gene-deleted adenoviral vector and the long-term genomic integration potential of gene transfer based on adeno-associated virus, retrovirus, lentivirus, and transposons. In some embodiments, such hybrid vector systems can result in stable transduction and restricted integration sites. See, e.g., 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), where the techniques and vectors described can be modified and adapted to the engineered AAV capsid systems of the present invention. In some embodiments, the hybrid adenoviral vector can include one or more characteristics of a retrovirus and / or an adeno-associated virus. In some embodiments, the hybrid adenoviral vector can include one or more characteristics of a spuma retrovirus or foamy virus (FV). See, e.g., Ehrhardt et al. 2007. Mol. Ther. 15:146-156 and Liu et al. 2007. Mol. Ther. 15:1834-1841, where the techniques and vectors described can be modified and adapted to the engineered AAV capsid systems of the present invention. Advantages of using one or more characteristics of FV in a hybrid adenoviral vector or a system thereof can include the ability of the resulting viral particles to infect a wide variety of cells, the large packaging capacity compared to other retroviruses, and the ability to persist in quiescent (non-dividing) cells. See also, e.g., Ehrhardt et al. 2007. Mol. Ther. 156:146-156 and Shuji et al. 2011. Mol. Ther. 19:76-82, where the techniques and vectors described can be modified and adapted to the engineered AAV capsid systems of the present invention.

[0170] Adeno-Associated Vectors

[0171] In one embodiment, the engineered vector or its system can be an adeno-associated vector (AAV). See, for example, 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). Although AAV is similar to adenoviral vectors in some of its characteristics, AAV has some deficiencies in its replication and / or pathogenicity and may therefore be safer than adenoviral vectors. In some embodiments, AAV can integrate into specific sites on chromosome 19 of human cells without observable side effects. In some embodiments, the capacity of the AAV vector, its system, and / or AAV particles can be up to about 4.7 kb. The AAV vector or its system can include one or more of the engineered capsid polynucleotides described herein.

[0172] The AAV vector or its system can be operably linked to a regulatory sequence that encodes one or more regulatory molecules. In some embodiments, the regulatory molecule can be a promoter, enhancer, repressor, etc., which are described in more detail elsewhere herein. In some embodiments, the AAV vector or its system can include one or more polynucleotides that can encode one or more regulatory proteins. In some embodiments, the promoter can be the tissue-specific promoter discussed previously. In some embodiments, the tissue-specific promoter can drive the expression of the engineered capsid AAV capsid polynucleotide described herein.

[0173] The AAV vector or its system can include one or more polynucleotides that can encode one or more capsid proteins, such as the engineered AAV capsid proteins described elsewhere herein. The engineered capsid proteins are capable of assembling into the protein shell (engineered capsid) of the AAV virus particle. The engineered capsid can have cell, tissue, and / or organ-specific tropism.

[0174] In some embodiments, the AAV vector or its system can include one or more adenovirus helper factors or polynucleotides that can encode one or more adenovirus helper factors. Such adenovirus helper factors can include, but are not limited to, E1A, E1B, E2A, E4ORF6, and VARNA. In some embodiments, a host cell line is generated that expresses one or more of the adenovirus helper factors.

[0175] An AAV vector or its system can be configured to produce 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. The AAV can be selected with respect to the cells to be targeted; for example, AAV serotypes 1, 2, 5, 9, or hybrid capsids AAV-1, AAV-2, AAV-5, AAV-9, or any combination thereof can be selected for targeting the brain and / or neuronal cells; and AAV-4 can be selected for targeting cardiac tissue; and AAV-8 can be selected for delivery to the liver. Thus, in some embodiments, an AAV vector or its system capable of producing AAV particles that can target the brain and / or neuronal cells can be configured to generate AAV particles having serotype 1, 2, 5, or hybrid capsids AAV-1, AAV-2, AAV-5, or any combination thereof. In some embodiments, an AAV vector or its system capable of producing AAV particles that can target cardiac tissue can be configured to generate AAV particles having the AAV-4 serotype. In some embodiments, an AAV vector or its system capable of producing AAV particles that can target the liver can be configured to generate AAV having the AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21:75-80.

[0176] It should be understood that although different serotypes can provide a certain level of cell, tissue, and / or organ specificity, each serotype is still pleiotropic. Thus, if a serotype is used to target a tissue with low transduction efficiency for that serotype, it may lead to tissue toxicity. Therefore, in addition to achieving some tissue targeting ability by selecting a specific serotype of AAV, it should be understood that the tropism of AAV serotypes can be altered by the engineered AAV capsids described herein. As described elsewhere herein, wild-type AAV variants of any serotype can be generated by the methods described herein and determined to have a specific cell-specific tropism, which can be the same as or different from the tropism of the reference wild-type AAV serotype. In some embodiments, the cell, tissue, and / or specificity of a wild-type serotype can be enhanced (e.g., making it more selective or specific for a particular cell type to which the serotype is already biased). For example, in humans, wild-type AAV-9 is biased towards 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, the preference for, e.g., the brain can be reduced or eliminated and / or the specificity (septicity) can be increased such that, in comparison, the brain specificity appears reduced and thus the specificity for muscle is enhanced compared to wild-type AAV-9. As previously mentioned, engineered capsids and / or capsid protein variants comprising wild-type AAV serotypes may have a tropism different from that of the wild-type reference AAV serotype. For example, engineered AAV capsids and / or capsid protein variants of AAV-9 can be specific for tissues in humans other than muscle or brain.

[0177] In some embodiments, the AAV vector is a hybrid AAV vector or system. A hybrid AAV is an AAV that includes a genome having elements from one serotype and is packaged in a capsid derived from at least one different serotype. For example, if the AAV vector is rAAV2 / 5 to be produced and if the production method is based on the helper-free, transient transfection method discussed below, the first plasmid and the third plasmid (adeno-associated plasmid) will be the same as those discussed for rAAV2 production. However, the second plasmid, pRepCap, will be different. In the plasmid herein called pRep2 / Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5. The production protocol is the same as the method mentioned above for AAV2 production. The resulting rAAV is called rAAV2 / 5, where the genome is based on recombinant AAV2 and the capsid is based on AAV5. It is assumed that the cell or tissue tropism exhibited by the AAV2 / 5 hybrid virus should be the same as that of AAV5. It should be understood that wild-type hybrid AAV particles suffer from the same specificity problems as the non-hybrid wild-type serotypes discussed previously.

[0178] By generating hybrid AAVs that can include engineered AAV capsids described elsewhere herein, the advantages achieved by wild-type-based hybrid AAV systems can be combined with the increased and customizable cell specificity achievable with available engineered AAV capsids. It should be understood that a hybrid AAV can contain an engineered AAV capsid that contains a genome having elements of a serotype different from a reference wild-type serotype, and the engineered AAV capsid is a variant thereof. For example, a hybrid AAV can be produced that includes an engineered AAV capsid that is a variant of the AAV-9 serotype, and this serotype can be used to package a genome containing components (such as AAV2 ITRs) from the AAV-2 serotype. As with the wild-type-based hybrid AAVs discussed previously, the tropism of the resulting AAV particles will be that of the engineered AAV capsid.

[0179] A list of certain wild-type AAV serotypes with respect to these cells can be found in Grimm, D. et al., J. Virol. 82:5887 - 5911 (2008), reproduced below as Table A. Further tropism details can be found in Srivastava. 2017. Curr. Opin. Virol. 21:75 - 80, as previously discussed.

[0180]

[0181]

[0182] In multiple exemplary embodiments, the AAV vector or its system is AAV rh.74 or AAV rh.10.

[0183] In multiple exemplary embodiments, the AAV vector or its system is configured as a "gutless" vector, similar to that described for retroviral vectors. In some embodiments, the "gutless" AAV vector or its system can have cis-acting viral DNA elements that are involved in genome amplification and packaging associated with a heterologous sequence of interest (such as a transgene encoding a therapeutic protein or nucleic acid of interest).

[0184] Vectors Encoding Transgenes

[0185] In one exemplary embodiment, a vector encoding a transgene (also referred to as an "artificial genome") contains a transgene flanked on both sides by AAV ITRs for delivery. Recombinant AAV (rAAV) propagation only requires approximately 145 bp of AAV ITRs as they are involved in vector production, induce transgene expression, and ensure continuous cell transduction. Thus, approximately 96% of the AAV genome can be removed for gene therapy. For example, the rep and cap genes can be replaced with an expression cassette containing a promoter (such as those described herein), a therapeutic transgene (e.g., IDS), and a poly(A) tail, forming the essence of all AAV vectors.

[0186] In multiple exemplary embodiments, additional modifications can be implemented to further enhance the efficacy of AAV. For example, AAV ITRs can be modified to increase the expression of the rAAV vector after transduction, which can allow the transgene to be expressed without second-strand DNA synthesis; promoters can be modified to increase transcription; and codons in the transgene can be engineered to alter mRNA production and / or translation.

[0187] In multiple exemplary embodiments, the ITRs are modified to overcome second-strand synthesis after infection. AAV transduction efficiency 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 a suitable substrate for the Rep68 and Rep78 proteins. As a result, terminal resolution of replication is eliminated, and a specific self-complementary AAV (scAAV) replication intermediate is produced. The scAAV intermediate contains positive and negative DNA strands fused by the modified ITRs encapsulated in the viral particle capsid. Wild-type AAV packages a single positive or negative-strand DNA. The modified scAAV intermediate is delivered to the nucleus where these positive and negative strands instantaneously anneal to form dsDNA.

[0188] In multiple exemplary embodiments, cis-elements are optimized for targeted delivery. The cis-elements are optimized because the packaging capacity of AAV is limited. In one exemplary embodiment, small cis-elements replace long promoter sequences for the delivery of large therapeutic transgenes (e.g., 4.4 - 4.5 kbs).

[0189] In multiple exemplary embodiments, several strategies can be used to deliver transgenes using AAV vectors. Exemplary method 1 utilizes AAV genomes tandemly integrated via homologous recombination through the ITR sequences. In this method, the transgene cassette can be divided into two or more vectors and then delivered to the same cell. After the virus is uncoated, a complete transgene is formed through homologous recombination between the two or more fragments.

[0190] In exemplary method 2, truncated transgenic fragments of different lengths are packaged into different AAV viral particles at undefined positions on the vector genome. Homologous recombination of overlapping regions of different AAV vector genomes or annealing of different AAV vector genomes in complementary regions via single-stranded templates gives rise to transgenic cassettes. In multiple exemplary embodiments, overlapping fragments can be added to the ends of individual AAV vectors to facilitate homologous recombination.

[0191] In exemplary method 3, a hybrid dual vector incorporates overlapping regions with intron splicing sites in a split vector transgene. Method 3 uses the concatemerization activity of the AAV genome to bring together separate AAV vector genomes. Recombination (e.g., the starting vector is split in half, with each half carrying 5′ and 3′ splicing elements respectively), and splicing provides a suitable transgenic protein. This strategy may increase the expression of a fully functional protein.

[0192] In exemplary method 4, the AAV genome is cross-packaged into the capsid of other parvoviruses, thereby forming chimeric vectors. In exemplary method 5, intein-mediated protein trans-splicing is used. Intein catalyzes protein splicing, thereby causing the ligation of two polypeptides via trans-splicing (this method is analogous to intron-mediated RNA splicing). Multiple AAV vectors are delivered to the same cell. Each AAV vector encodes a fragment of the target protein, which is flanked by short split inteins. The full-length protein is formed after protein trans-splicing. 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.

[0193] Vector construction

[0194] The vectors described herein can be constructed using any suitable process or technique. In some embodiments, one or more suitable recombinant and / or cloning methods or techniques can be used for the vectors described herein. Suitable recombinant and / or cloning techniques and / or methods can include, but are not limited to, those described in U.S. Application Publication No. 2004-0171156A1. Other suitable methods and techniques are described elsewhere herein.

[0195] The construction of recombinant AAV vectors has been described in numerous publications, including 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 discussed elsewhere herein.

[0196] In some embodiments, the vector can have one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites (e.g., about or more than 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.

[0197] Delivery vehicles, vectors, particles, nanoparticles, formulations, and components thereof for expressing one or more elements of the engineered AAV capsid systems described herein can be used as in the foregoing documents such as International Patent Application Publication WO 2014 / 093622 (PCT / US2013 / 074667) and are discussed in more detail herein.

[0198] Generation of viral particles from viral vectors

[0199] AAV Particle Production

[0200] There are two strategies for generating AAV particles from AAV vectors and their systems such as those described herein, depending on the manner of providing adenovirus helper factors (helper versus no helper). In some embodiments, a method for generating AAV particles from an AAV vector and its system can include transfecting an adenovirus together with the AAV vector into a cell line stably carrying AAV replication and capsid-encoding polynucleotides, the AAV vector containing a polynucleotide to be packaged and delivered by the resulting AAV particles (e.g., an engineered AAV capsid polynucleotide). In some embodiments, a method for generating AAV particles from an AAV vector and its system can be a "no helper" method, which includes co-transfecting a suitable producer cell line with three vectors (e.g., plasmid vectors): (1) an AAV vector containing a polynucleotide of interest (e.g., a transgene encoding a therapeutic protein or nucleic acid, operably linked to a regulatory element that promotes expression in a target tissue) between 2 ITRs; (2) a vector carrying a polynucleotide encoding AAV Rep-Cap, including the engineered capsid proteins described herein; and a helper polynucleotide. Those skilled in the art will understand the various methods and their variants as helper and no helper, as well as the different advantages of each system.

[0201] The engineered AAV vectors and their systems described herein can be generated by any of these methods.

[0202] Vector and Viral Particle Delivery

[0203] The vectors (including non-viral carriers) described herein can be introduced into host cells to produce transcripts, proteins, or peptides, including fusion proteins or peptides encoded by the nucleic acids described herein (e.g., engineered AAV capsid system transcripts, proteins, enzymes, their mutant forms, their fusion proteins, etc.) and viral particles (such as from viral vectors and their systems).

[0204] AAV capsids prepared from one or more engineered AAV capsid polynucleotides can be used to deliver recombinant AAV genomes encoding a therapeutic protein or nucleic acid of interest. Alternatively, delivery can be using an adenovirus or other plasmid or viral vector type as previously described, specifically, using formulations and doses from, for example, U.S. Patent Nos. 8,454,972 (regarding adenovirus formulations, doses), 8,404,658 (regarding AAV formulations, doses), and 5,846,946 (regarding DNA plasmid formulations, doses) as well as clinical trials involving lentiviruses, AAVs, and adenoviruses and publications regarding such clinical trials. For example, for AAV, the route of administration, formulation, and dose can be as in U.S. Patent No. 8,454,972 as well as in clinical trials involving AAV. For adenovirus, the route of administration, formulation, and dose can be as in U.S. Patent No. 8,404,658 as well as in clinical trials involving adenovirus.

[0205] For plasmid delivery, the route of administration, formulation, and dosage can be as described in U.S. Patent No. 5,846,946 and in clinical studies involving plasmids. In some embodiments, the dosage can be based on or extrapolated to an average 70 kg individual (e.g., adult male), and can be adjusted for patients, subjects, mammals of different weights and species. The frequency of administration is within the capabilities of a medical or veterinary practitioner (e.g., physician, veterinarian), depending on common factors including the age, sex, general health, other medical conditions of the patient or subject, and the particular medical condition or symptom being addressed. The viral vector can be injected or otherwise delivered to the tissue or cell of interest.

[0206] For in vivo delivery, AAV is superior to other viral vectors for two reasons such as: low toxicity (which may be due to the purification method not requiring ultracentrifugation of cellular particles that may activate the immune response), and low likelihood of insertional mutagenesis due to not integrating into the host genome.

[0207] The vectors and viral particles described herein can be delivered to host cells in vitro, in vivo, and / or ex vivo. Delivery can occur by any suitable method including but not limited to physical methods, chemical methods, and biological methods. Physical delivery methods are those that use physical forces to counteract the cell's membrane barrier to facilitate intracellular delivery of the vector. Suitable physical methods include but are not limited to needles (e.g., injection), ballistic polynucleotides (e.g., particle bombardment, microprojectile gene transfer, and gene gun), electroporation, sonoporation, photoporation, magnetofection, hydroporation, and mechanical massage. Chemical methods are those that use chemicals to induce changes in cell membrane permeability or other characteristics to facilitate entry of the vector into the cell. For example, the environmental pH can be altered, which can induce a change in cell membrane permeability. Biological methods are those that rely on and utilize the biological processes or biological characteristics of the host cell to facilitate the transport of the vector (with or without a carrier) into the cell. For example, the vector and / or its carrier can stimulate endocytosis or a similar process in the cell to facilitate uptake of the vector into the cell.

[0208] Delivery of engineered AAV capsid system components (such as polynucleotides encoding engineered AAV capsids and / or capsid proteins) to cells via particles. As used herein, the term "particle" refers to any appropriately sized particle for delivering the engineered AAV capsid system components described herein. Appropriate sizes include large-sized, micron-sized, and nano-sized particles. In some embodiments, any one of the engineered AAV capsid system components (such as the polypeptides, polynucleotides, vectors, and combinations thereof described herein) can be attached, conjugated, integrated, or otherwise associated with one or more of the particles or components described herein. Then, the particles described herein can be administered to cells or organisms by any appropriate route and / or technique. In some embodiments, particle delivery can be selected, which can be advantageous for delivering polynucleotide or vector components. It should be understood that in multiple embodiments, particle delivery can also be advantageous for other engineered capsid system molecules and formulations described elsewhere herein.

[0209] Engineered Virus Particles Comprising Engineered Viruses (e.g., AAV) Capsids

[0210] This document also describes engineered virus particles (also referred to herein and elsewhere as "engineered viral particles"), which can contain an engineered virus capsid (such as an AAV capsid, referred to as an "engineered AAV particle") as described in detail elsewhere herein. It should be understood that the 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 as previously described. An engineered AAV capsid is a capsid that contains one or more engineered AAV capsid proteins as described elsewhere herein. In some embodiments, the engineered AAV particles can include from 1 to 60 of the engineered AAV capsid proteins described herein. In some embodiments, the 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, the engineered AAV particles can contain from 0 to 59 wild-type AAV capsid proteins. In some embodiments, the 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, the engineered AAV particles can include one or more of the n-mer motifs described previously.

[0211] Engineered AAV particles can include one or more cargo polynucleotides. The cargo polynucleotides are discussed in more detail elsewhere herein. Methods for making engineered AAV particles from viral and non-viral vectors are described elsewhere herein. Formulations containing engineered virus particles are described elsewhere herein.

[0212] Exemplary cargo

[0213] The n-mer can be conjugated to or otherwise associated with a cargo. The cargo can include any molecule capable of being conjugated to or associated with the n-mer described herein. The cargo can include, but is not limited to, nucleotides, oligonucleotides, polynucleotides, amino acids, peptides, polypeptides, riboproteins, lipids, sugars, pharmaceutically active agents (such as drugs, imaging agents, and other diagnostic agents, etc.), compounds, and combinations thereof. In some embodiments, the cargo is DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences of ribozymes for inhibiting the translation or transcription of tumor-essential proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatory agents, antihistamines, anti-infective agents, radiosensitizers, chemotherapeutic agents, radioactively active compounds, imaging agents, and combinations thereof. In various embodiments, the cargo is a recombinant AAV genome containing a transgene, such as a therapeutic protein or nucleic acid encoding a therapeutic protein or nucleic acid operably linked to a regulatory sequence directing the expression of the therapeutic protein or nucleic acid in a target tissue, the transgene flanked by AAV ITR sequences.

[0214] In some embodiments, the cargo is capable of treating or preventing neurological diseases or disorders, details of which are described herein.

[0215] In some embodiments, the cargo is N-morpholino, peptide-linked N-morpholino, antisense oligonucleotide, PMO, therapeutic transgene, polynucleotide encoding a therapeutic polypeptide or peptide, PPMO, one or more peptides, one or more polynucleotides encoding a CRISPR-Cas protein, guide RNA, or both, ribonucleoprotein, wherein the ribonucleoprotein comprises a CRISPR-Cas system molecule, therapeutic transgene RNA, or other gene-modifying or therapeutic RNA and / or protein, or any combination thereof.

[0216] In some embodiments, one or more of the n-mers described herein are directly attached to the cargo. In some embodiments, one or more of the n-mers described herein are indirectly conjugated to the cargo, such as via a linker molecule. In some embodiments, one or more of the n-mers described herein are conjugated to or associated with a polypeptide or other particle that is conjugated to, attached to, packages, and / or contains the cargo.

[0217] Exemplary particles include, but are not limited to, viral particles (e.g., viral capsids, including bacteriophage capsids), polysomes, liposomes, nanoparticles, microparticles, exosomes, micelles, etc. As used herein, the term "nanoparticle" includes nanoscale deposits of homogeneous or heterogeneous materials. Nanoparticles can be regular or irregular in shape and can be formed from multiple co-deposited particles that form a composite nanoscale particle. Nanoparticles can generally be spherical in shape or have 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, columnar, discoidal, etc. In some embodiments, the nanoparticles have a substantially spherical shape.

[0218] Carrier polynucleotide

[0219] Carriers are also described elsewhere herein. In some embodiments, the carrier is a carrier polynucleotide, which can be packaged in an engineered viral particle and subsequently delivered to a cell. In some embodiments, the delivery is cell-selective, such as neurons and glial cells of the central nervous system. In some embodiments, one or more carrier polynucleotides are part of the engineered viral (e.g., AAV) genome of a viral (e.g., AAV) system and are packaged within an engineered capsid containing the targeting moiety of the present invention. The carrier polynucleotide can be packaged in an engineered viral (e.g., AAV) particle, which can be delivered to, for example, a cell. In some embodiments, the carrier polynucleotide is capable of modifying 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 the genetic instructions for a trait or characteristic in an organism. The term gene can refer to the translated and / or untranslated regions of a genome. "Gene" can refer to a specific sequence of DNA that is transcribed into an RNA transcript, which can be translated into a polypeptide or be a catalytic RNA molecule, including but not limited to tRNA, siRNA, piRNA, miRNA, long non-coding RNA, and shRNA. Modifications of polynucleotides, genes, transcripts, etc. include all genetic engineering techniques, including but not limited to gene editing; and conventional recombinant gene modification techniques (e.g., techniques of whole or partial gene insertion, deletion, and mutagenesis (e.g., insertional and deletional mutagenesis).

[0220] In a number of exemplary embodiments, the carrier molecule is a polynucleotide that is or can encode a vaccine. In a number of exemplary embodiments, the carrier molecule is a polynucleotide encoding an antibody.

[0221] Interfering RNA

[0222] In certain exemplary embodiments, one or more polynucleotides may encode one or more interfering RNAs. Interfering RNAs are RNA molecules capable of inhibiting gene expression. Exemplary types of interfering RNAs include small interfering RNAs (siRNAs), microRNAs (miRNAs), and short hairpin RNAs (shRNAs).

[0223] In certain exemplary embodiments, the interfering RNA may be an siRNA. A small interfering RNA (siRNA) molecule is capable of inhibiting target gene expression through interfering RNA. siRNAs can be chemically synthesized, or can be obtained by in vitro transcription, or can be synthesized in vivo in target cells. siRNAs may comprise double-stranded RNAs that are 15 to 40 nucleotides in length and may contain overhang regions 3′ and / or 5′ 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 function by post-transcriptionally degrading or silencing target messengers. In some cases, exogenous polynucleotides encode shRNAs. In shRNAs, the antiparallel strands that form the siRNA are joined by a loop or hairpin region.

[0224] Interfering RNAs (e.g., siRNAs) can inhibit gene expression to promote long-term survival and functionality of cells after transplantation into a subject. In some instances, the interfering RNAs inhibit genes in the TGFβ pathway, such as TGFβ, TGFβ receptors, and SMAD proteins. In some instances, the interfering RNAs inhibit genes in the colony stimulating factor 1 (CSF1) pathway, such as CSF1 and CSF1 receptor. In certain embodiments, one or more interfering RNAs inhibit genes in both the CSF1 pathway and the TGFβ pathway. TGFβ pathway genes can include one or more of the following: ACVR1, ACVR1C, ACVR2A, ACVR2B, ACVRL1, AMH, AMHR2, BMP2, BMP4, BMP5, BMP6, BMP7, BMP8A, BMP8B, BMPR1A, BMPR1B, BMPR2, CDKN2B, CHRD, COMP, CREBBP, CUL 1, 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.

[0225] In some embodiments, the carrier polynucleotide is an RNAi molecule, an antisense molecule, and / or a gene silencing oligonucleotide, or a polynucleotide encoding an RNAi molecule, an antisense molecule, and / or a gene silencing oligonucleotide.

[0226] As used herein, "gene silencing oligonucleotide" refers to any oligonucleotide that, alone or together with other gene silencing oligonucleotides, can utilize the endogenous mechanisms, molecules, proteins, enzymes, and / or other cellular machinery of the cell or exogenous molecules, agents, proteins, enzymes, and / or polynucleotides to cause a global or specific decrease or elimination in gene expression, RNA levels, RNA translation, or RNA transcription, and can cause a decrease or effective loss of protein expression and / or function of non-coding RNA compared to wild-type or a suitable control. This is synonymous with the phrase "gene knockdown." The decrease in gene expression, RNA levels, RNA translation, RNA transcription, and / or protein expression can be in the range of 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 decrease. "Gene silencing oligonucleotides" include, but are not limited to, any antisense oligonucleotides, ribozymes, any oligonucleotides (single-stranded or double-stranded) used to stimulate the RNA interference (RNAi) pathway in cells (collectively referred to as RNAi oligonucleotides), small interfering RNA (siRNA), microRNA, and short hairpin RNA (shRNA). Commercially available programs and tools are available that can design the nucleotide sequences of gene silencing oligonucleotides for the desired gene based on the gene sequence and other information available to those of skill in the art.

[0227] In some embodiments, a cargo polynucleotide such as an encoded polynucleotide is flanked at least by a reverse transcriptase element polypeptide encoding the 3′UTR of the polynucleotide or a portion thereof (such as the proximal region of about 500 base pairs of the 3′UTR). In some embodiments, a cargo polynucleotide such as an encoded polynucleotide is flanked (e.g., endogenously or engineered) by a reverse transcriptase element polypeptide (such as a retroviral gag protein or a gag homolog) at the 5′UTR. In some embodiments, a cargo polynucleotide such as an encoded polynucleotide is flanked by (e.g., endogenously or engineered) reverse transcriptase element polypeptides encoding the 5′ and 3′UTRs of the polynucleotide. In some embodiments, the flanking reverse transcriptase element polypeptides of the polynucleotide UTRs are 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 they flank. These and other packaging elements are described in more detail elsewhere herein.

[0228] Genetically Modified Carrier Polynucleotides

[0229] In some embodiments, a cargo molecule can be a polynucleotide or a polypeptide or a polynucleotide encoding a polypeptide that, when delivered alone or as part of a system (whether delivered with other components of the system or not), operates to modify the genome, epigenome, and / or transcriptome of the cell to which it is delivered. Such systems include, but are not limited to, CRISPR-Cas systems. Other gene modification systems, such as TALENs, zinc finger nucleases, Cre-Lox, N-morpholino, etc., are other non-limiting examples of gene modification systems, one or more components of which can be delivered by engineered viral (e.g., AAV) particles described herein.

[0230] 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 a CRISPR-Cas system). In some embodiments, the cargo molecule is or encodes a gRNA. As used herein, CRISPR-Cas systems are intended to be encompassed by class 1 and class 2 CRISPR-Cas systems and derivatives of CRISPR-Cas systems such as base editors, prime editors, and CRISPR-associated transposase (CAST) systems.

[0231] In some embodiments, the cargo molecule can be a polynucleotide or a polypeptide or a polynucleotide encoding a polypeptide that, when delivered alone or as part of a system (whether delivered together with other components of the system or not), operates to modify the genome, epigenome, and / or transcriptome of the cells to which it is delivered such that it treats or prevents a disease, disorder, or a symptom thereof of a neurological disease or disorder, and / or a virus (such as a single-stranded RNA virus). In some embodiments, the cargo molecule operates to modify the genome, epigenome, and / or transcriptome of the cells to which it is delivered, whether or not delivered together with other components of the system, such that it treats or prevents a neurological disease or disorder further described herein.

[0232] In some embodiments, the cargo molecule operates to modify the genome, epigenome, and / or transcriptome of the cells to which it is delivered, whether or not delivered together with other components of the system, such that it can modify the GAA gene, such as any of those described in U.S. Patent Application Publication 2019 / 0284555, the disclosure of which is incorporated herein by reference in its entirety and is applicable to the present invention.

[0233] In some embodiments, the cargo molecule is or encodes an antisense oligomer or an RNA molecule, such as those described in U.S. Patent Application Publications US2016 / 0251398, US2015 / 0267202, and US2018 / 0216111, the disclosures of which are incorporated herein by reference in their entireties and are applicable to the present invention.

[0234] In some embodiments, the cargo molecule can be a peptide-oligomer, conjugate, such as those described in International Patent Application Publication WO2017 / 106304A1, the disclosure of which is incorporated herein by reference in its entirety and is applicable to the present invention.

[0235] One embodiment of the present invention encompasses a method of modifying a locus of interest to alter gene expression in a cell by introducing any of the compositions described herein into the cell.

[0236] One embodiment of the present invention is to include the above elements in a single composition or in individual compositions. These compositions can be advantageously administered to a host to elicit a functional effect at the gene level.

[0237] Polypeptide

[0238] In certain exemplary embodiments, the cargo molecule can be one or more polypeptides or can be a nucleic acid encoding a polypeptide. The polypeptide can be a full-length polypeptide or a functional fragment or domain thereof, i.e., a fragment or domain that maintains the functions required of the full-length polypeptide. As used in this section, "protein" means a full-length protein as well as its functional fragments and domains. A variety of polypeptides can be delivered using the engineered delivery vehicles described herein, including but not limited to secreted proteins, immunomodulatory proteins, antifibrotic proteins, proteins that promote tissue regeneration and / or transplant survival functions, hormones, antimicrobial proteins, antifibrotic polypeptides, and antibodies. One or more polypeptides can also comprise a combination of the foregoing exemplary classes of polypeptides. It should be understood that the polypeptides described herein can also be delivered via the engineered delivery vehicles and systems described herein by delivering the corresponding encoding polynucleotides.

[0239] Antibodies

[0240] In certain embodiments, one or more polypeptides can comprise one or more antibodies. The term "antibody" is used interchangeably herein with the term "immunoglobulin" and includes intact antibodies, fragments of antibodies such as Fab, F(ab')2 fragments, scFv, and intact antibodies and fragments that have been mutated in their constant and / or variable regions (e.g., mutations that result in chimeric, partially humanized, or fully humanized antibodies, and mutations that result in antibodies having desired properties such as enhanced binding and / or reduced FcR binding). The term "fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or full antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of an intact or full antibody or antibody chain. Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, V HH and scF and / or Fv fragments. As used herein, an antibody protein preparation having less than about 50% non-antibody protein (also referred to herein as "contaminating protein") or chemical precursors is considered to be "substantially free of". 40%, 30%, 20%, 10%, and more preferably 5% (by dry weight) of non-antibody protein or chemical precursors is considered substantially free of. When an antibody protein or a bioactive portion thereof is produced recombinantly, it is also preferred to be substantially free of culture medium, i.e., the culture medium accounts for 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.

[0241] In multiple exemplary embodiments, 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 "binding portion" (or "antibody portion") of the antibody includes one or more complete domains, such as a pair of complete domains, and antibody fragments that retain the ability to specifically bind to a target molecule. It has been shown that the binding function of an antibody can be carried out by fragments of a full-length antibody. Binding fragments are produced by recombinant DNA techniques 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.

[0242] In some embodiments, the vehicle or antibody is an antibody fragment or portion. In some embodiments, the vehicle is an epitope-binding protein. Examples of portions of an antibody or epitope-binding protein are encompassed by the definitions of the present invention that include the following: (i) a Fab fragment that has V L , C L , V H and C H domains; (ii) a Fab' fragment that is a Fab fragment having one or more cysteine residues at the C-terminus of the C H domain; (iii) an Fd fragment that has V H and C H domains; (iv) an Fd′ fragment that has V H and C H domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) an Fv fragment that has V L and V H domains of a single arm of an antibody; (vi) a dAb fragment (Ward et al., 341 Nature 544 (1989)) that consists of a V H domain or a V L domain that binds an antigen; (vii) an isolated CDR region or regions that are present in a functional framework; (viii) an F(ab')2 fragment that is a bivalent fragment that includes two Fab' fragments linked at the hinge region by a disulfide bridge; (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 "bispecific antibody" having two antigen-binding sites that includes a heavy-chain variable domain (V L ) linked to a light-chain variable domain (V H in the same polypeptide chain)(See, e.g., EP 404,097; WO 93 / 11161; Hollinger et al., 90 PNAS 6444 (1993)); (xi) "linear antibody", which comprises a pair of tandem Fd segments (V H -C h 1-V H -C h 1), which together with a complementary light chain oligopeptide form a pair of "antigen binding regions" (Zapata et al., Protein Eng. 8(10): 1057-62 (1995); and U.S. Patent No. 5,641,870).

[0243] The term "antigen-binding fragment" refers to an immunoglobulin or polypeptide fragment of an antibody that binds an antigen or competes with a full antibody (i.e., the full antibody from which they are derived) for antigen binding (i.e., specifically binds). Thus, such antibodies or fragments thereof are included within the scope of the present invention provided that the antibody or fragment specifically binds to a target molecule.

[0244] 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 that contains the heavy chain variable region (V H ) and the light chain variable region (V L ) of an immunoglobulin linked by 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 or threonine residues to increase or alter solubility. V H and light (V L ) can be linked via the linker in any order. In some embodiments, the N-terminus of V H is coupled via the linker to the C-terminus of (V L ). In some embodiments, the C-terminus of V H is coupled via the linker to the (V L) N-terminal conjugation. In some embodiments, the scFv is a bivalent or trivalent scFv. In some embodiments, the bivalent or trivalent scFv is bispecific or trispecific, meaning that they can target 2 or 3 different epitopes, respectively. See also, 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:10.100 6 / 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.; 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;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; et al., J Mol Biol.2001 Feb 2;305(5):989-1010.doi:10.1006 / jmbi.2000.4265。

[0245] As used herein, "heavy chain antibody", "VHH" or "single domain antibody" (sdAb) refers to an antibody that consists of only two heavy chains and lacks the two light chains commonly found in antibodies (see, e.g., Henry and MacKenzie, Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs. 2018 Aug-Sep;10(6):815-826). A VHH can refer to an antibody or a VHH domain. A single domain antibody (sdAb), also known as a "nanobody", is defined herein as an antibody fragment consisting of a single monomeric variable antibody domain. As used herein, "VHH" and "nanobody" are used interchangeably. The approximately 12-15 kDa variable domains of these antibodies (VHH and VNAR) can be recombinantly produced and can recognize antigens in the absence of the remainder of the antibody heavy chain. In a normal antibody, the antigen-binding region consists of the variable domains of the heavy and light chains (VH and VL). Heavy chain antibodies can bind antigens but have only a VH domain. In certain embodiments, the heavy chain antibody is an antibody derived from cartilaginous fish (immunoglobulin new antigen receptor (IgNAR)) or a camelid ungulate. Non-limiting examples of camelids include dromedary camels, camels, llamas, and alpacas.

[0246] The term "antibody" is intended to cover any Ig class or any Ig subclass (e.g., the IgG1, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any source (e.g., humans and non-human primates, and rodents, lagomorphs, goats, cows, horses, sheep, etc.).

[0247] As used herein, the term "Ig class" or "immunoglobulin class" refers to the five classes of immunoglobulins that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE. The term "Ig subclass" refers to the two subclasses (H and L) of IgM, the three subclasses of IgA (IgA1, IgA2, and secretory IgA), and the four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4) that have been identified in humans and higher mammals. Antibodies can exist in monomeric or polymeric forms; for example, IgM antibodies exist as pentamers, while IgA antibodies exist as monomers, dimers, or multimers.

[0248] The term "IgG subclass" refers to the four subclasses of the immunoglobulin class IgG - IgG1, IgG2, IgG3, and IgG4, which have been identified in humans and higher mammals by the heavy chains of immunoglobulins V1 to γ4, respectively. The term "single-chain immunoglobulin" or "single-chain antibody" (used interchangeably herein) refers to a protein having a double-polypeptide chain structure composed 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 an antigen. The term "domain" refers to a globular region of a heavy or light chain polypeptide that contains, for example, peptide loops (such as those containing 3 or 4 peptide loops) stabilized by β-sheets and / or intrachain disulfide bonds. Based on the relative lack of sequence variation within the domain for various class members in the case of "constant" domains, or the significant variation within the domain for various class members in the case of "variable" domains, the domains are further referred to herein as "constant" or "variable". An antibody or polypeptide "domain" is often referred to interchangeably as an antibody or polypeptide "region". The "constant" domain of an antibody light chain may be referred to interchangeably as the "light chain constant region", "light chain constant domain", "CL" region, or "CL" domain. The "constant" domain of an antibody heavy chain may be referred to interchangeably as the "heavy chain constant region", "heavy chain constant domain", "CH" region, or "CH" domain. The "variable" domain of an antibody light chain may be referred to interchangeably as the "light chain variable region", "light chain variable domain", "VL" region, or "VL" domain. The "variable" domain of an antibody heavy chain may be referred to interchangeably as the "heavy chain constant region", "heavy chain constant domain", "VH" region, or "VH" domain.

[0249] The term "region" may also refer to a part or portion of an antibody chain or antibody chain domain (such as a part or portion of a heavy or light chain, or a part or portion of a constant or variable domain, as defined herein), as well as a more discrete part or portion of said chain or domain. For example, the light and heavy chains or the light and heavy chain variable domains include "complementary determining regions" or "CDRs" that are interspersed between "framework regions" or "FRs", as defined herein.

[0250] The term "conformation" refers to the tertiary structure of a protein or polypeptide (such as an antibody, antibody chain, its domain or region). For example, the phrase "light (or heavy) chain conformation" refers to the tertiary structure of the light (or heavy) chain variable region, and the phrase "antibody conformation" or "antibody fragment conformation" refers to the tertiary structure of an antibody or its fragment.

[0251] The term "antibody-like protein scaffold" or "engineered protein scaffold" broadly encompasses protein non-immunoglobulin specific binding agents, typically obtained by combinatorial engineering (such as the combination of site-directed random mutagenesis with phage display or other molecular selection techniques). Generally, such scaffolds are derived from robust small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from the stably folded extracellular domains of cell surface receptors (such as protein A, fibronectin, or ankyrin repeats).

[0252] Such scaffolds have been extensively reviewed in 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), and include but are not limited to affibodies, the Z-domain based on staphylococcal protein A, which is a 58-residue three-helix bundle that provides interfaces on both of its α-helices (Nygren, Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 2008, 275: 2668-2676); engineered Kunitz domains, small polypeptides based on 58 residues and robust, disulfide-crosslinked serine protease inhibitors, typically of human origin (e.g., LACI-D1), which can be engineered for different protease specificities (Nixon and Wood, Engineered protein inhibitors of proteases.Curr Opin DrugDiscov Dev 2006, 9: 261-268); monomeric antibodies or adnectins based on the tenth fibronectin type III extracellular domain (10Fn3), which adopt an Ig-like β-sandwich fold (94 residues), have 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 lipocalins, a diverse family of eight-stranded β-barrel proteins (approximately 180 residues) that naturally form binding sites for small ligands through four structurally variable loops at the open ends, and 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) that provide a rigid interface typically generated by three repeated β-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 cysteine-rich knottin peptides (Kolmar Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins. FEBS J 2008, 275: 2684-2690).

[0253] "Specific binding" of an antibody means that the antibody exhibits a distinct affinity for a particular antigen or epitope and generally does not exhibit significant cross-reactivity. "Distinct" binding includes binding with an affinity of at least 25 μM. Antibodies having an affinity greater than 1 x 10 7 M -1 (or a dissociation coefficient of 1 μM or less or a dissociation coefficient of 1 nM or less) typically bind with correspondingly greater specificity. Intermediate values of those detailed herein are also intended to be within the scope of the present invention, and the antibodies of the present invention bind with an affinity in the range of 100 nM or less, 75 nM or less, 50 nM or less, 25 nM or less, such as 10 nM or less, 5 nM or less, 1 nM or less, or in some embodiments, 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 will not significantly bind to entities other than its target (e.g., different epitopes or different molecules). For example, an antibody that specifically binds to a target molecule will significantly bind that target molecule but will not significantly react with non-target molecules or peptides. An antibody specific for a particular epitope will, for example, not significantly cross-react with distal epitopes on the same protein or peptide. Specific binding can be determined according to any art-recognized means for determining such binding. Preferably, specific binding is determined according to Scatchard analysis and / or competitive binding assays.

[0254] As used herein, the term "affinity" refers to the strength of binding between a single antigen-combining site and an antigenic determinant. Affinity depends on the tightness of the stereochemical fit between the antibody-combining site and the antigenic determinant, on the size of the area of contact between them, on the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium dialysis or by the dynamic BIACORE TM method. The dissociation constant Kd and the association constant Ka are quantitative measures of affinity.

[0255] As used herein, the term "monoclonal antibody" refers to an antibody derived from a clone population of antibody-producing cells (e.g., B lymphocytes or B cells) that is homogeneous in structure and antigen specificity. The term "polyclonal antibody" refers to multiple antibodies originating from different clone populations of antibody-producing cells that are heterogeneous in their structure and epitope specificity but recognize a common antigen. Monoclonal and polyclonal antibodies can exist as crude preparations in body fluids; or can be purified as described herein. A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains a minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which the residues from the recipient hypervariable regions are replaced by residues from the hypervariable regions of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, the FR residues of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, humanized antibodies can contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody will contain substantially all of at least one and typically two variable domains, wherein 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 those of the human immunoglobulin sequence. A humanized antibody optionally will also contain at least a portion of the immunoglobulin constant region (Fc), which typically is the constant region of a human immunoglobulin.

[0256] As used herein, a "blocking" antibody or antibody "antagonist" is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In certain embodiments, the blocking antibody or antagonist antibody or a portion thereof described herein completely inhibits the biological activity of the antigen.

[0257] Antibodies can act as agonists or antagonists of the polypeptides they recognize. For example, the present invention includes antibodies that partially or completely disrupt receptor / ligand interactions. The present invention provides both receptor-specific antibodies and ligand-specific antibodies. The present invention also provides receptor-specific antibodies that do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signal transduction) can be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting phosphorylation (e.g., tyrosine or serine / threonine) of the receptor or one of its downstream substrates by immunoprecipitation followed by immunoblot analysis. In specific embodiments, antibodies are provided that inhibit 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% relative to the activity in the absence of the antibody.

[0258] The present invention also provides 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 the ligand and prevent ligand binding to the receptor, as well as antibodies that bind the ligand and prevent receptor activation but do not prevent ligand binding to the receptor. The present invention further includes antibodies that activate the receptor. Such antibodies can act as receptor agonists, i.e., enhance or activate all or part of the biological activity of ligand-mediated receptor activation, such as by inducing dimerization of the receptor. Antibodies can be defined as agonists, antagonists, or inverse agonists of biological activity, encompassing the specific biological activity of the peptides disclosed herein. Antibody agonists and antagonists can be prepared using methods known in the art. See, for example, PCT Publication WO 96 / 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. 111(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).

[0259] Antibodies as defined by the present invention include derivatives that are modified, i.e., covalently attached to the antibody by any type of molecule such that the covalent attachment does not prevent the antibody from generating an anti-idiotypic response. By way of example, and not limitation, antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cell ligand or other protein, etc. Any of a variety of chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis with tunicamycin, etc. In addition, the derivative can contain one or more non-classical amino acids.

[0260] Engineered Cells and Organisms Expressing the Engineered AAV Capsid

[0261] Engineered cells are described herein that 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 are expressed in the engineered cells. In some embodiments, the engineered cells are capable of producing an engineered AAV capsid protein and / or an engineered AAV capsid particle as described elsewhere herein. Modified or engineered organisms are also described herein that can include one or more of the engineered cells described herein. As described elsewhere herein, the engineered cells can be engineered to express a cargo molecule (e.g., a cargo polynucleotide) dependent on or independent of the engineered AAV capsid polynucleotide, e.g., packaged within an engineered AAV capsid as described herein.

[0262] A wide variety of animals, plants, seaweeds, fungi, yeasts, etc., and animal, plant, seaweed, fungal, yeast cell or tissue systems can be engineered to express one or more nucleic acid constructs of the engineered AAV capsid system described herein using the various transformation methods mentioned elsewhere herein. This can produce organisms that can produce engineered AAV capsid particles, such as for production purposes, engineered AAV capsid design and / or generation, and / or model organisms. In some embodiments, polynucleotides encoding one or more components of the engineered AAV capsid system described herein can be stably or transiently incorporated into one or more cells of a plant, animal, seaweed, fungus, and / or yeast or tissue system. In some embodiments, one or more of the engineered AAV capsid system polynucleotides are genomically incorporated into one or more cells of a plant, animal, seaweed, fungus, and / or yeast or tissue system. Further embodiments of modified organisms and systems are described elsewhere herein. In some embodiments, one or more components of the engineered AAV capsid system described herein are expressed in one or more cells of a plant, animal, seaweed, fungus, yeast, or tissue system.

[0263] Engineered Cells

[0264] This disclosure describes various embodiments of engineered cells that can include one or more of the engineered AAV capsid system polynucleotides, polypeptides, vectors, and / or vector systems described elsewhere herein. In some embodiments, the cells can express one or more of the engineered AAV capsid polynucleotides and can produce one or more engineered AAV capsid particles, which are described in more detail herein. Such cells are also referred to herein as "producer cells." It should be understood that these engineered cells are different from the "modified cells" described elsewhere herein in that modified cells are not necessarily producer cells unless they include one or more of the engineered AAV capsid polynucleotides, engineered AAV capsid vectors, or other vectors described herein that enable the cells to produce engineered AAV capsid particles. Modified cells can be recipient cells of engineered AAV capsid particles and, in some embodiments, can be modified by engineered AAV capsid particles and / or cargo polynucleotides delivered to the recipient cells. Modified cells are discussed in more detail elsewhere herein. The term modification can be used in connection with cell modification that is independent of the cell being a recipient cell. For example, isolated cells can be modified prior to receiving engineered AAV capsid molecules.

[0265] In one embodiment, the invention provides a non-human eukaryotic organism; e.g., a multicellular eukaryotic organism, comprising a eukaryotic host cell containing one or more components of the engineered delivery system described herein according to any of the embodiments described. In other embodiments, the invention provides a eukaryotic organism; preferably, a multicellular eukaryotic organism, comprising a eukaryotic host cell containing one or more components of the engineered delivery system described herein according to any of the embodiments described. In some embodiments, the organism is a host for AAV.

[0266] In certain embodiments, the obtained cells or parts of plants, algae, fungi, yeast, etc. are transgenic plants, comprising an exogenous DNA sequence incorporated into all or part of the genome of the cells.

[0267] The engineered cell can be a prokaryotic cell. The prokaryotic cell can be a bacterial cell. The prokaryotic cell can be an archaeal cell. The bacterial cell can be any suitable bacterial cell. Suitable bacterial cells can be from the genera Escherichia, Bacillus, Lactobacillus, Rhodococcus, Rhodobacter, Synechococcus, Synechoystis, Pseudomonas, Psedoaltermonas, Stenotrophamonas, and Streptomyces. Suitable bacterial cells include, but are not limited to, Escherichia coli cells, Caulobacter crescentus cells, Rhodobacter sphaeroides cells, Psedoaltermonas haloplanktis cells. Suitable bacterial strains include, but are not limited to, BL21(DE3), DL21(DE3)-pLysS, BL21 Star-pLysS, BL21-SI, BL21-AI, Tuner, Tuner pLysS, Origami, Origami B pLysS, Rosetta, RosettapLysS, Rosetta-gami-pLysS, BL21 CodonPlus, AD494, BL2trxB, HMS174, NovaBlue(DE3), BLR, C41(DE3), C43(DE3), Lemo21(DE3), Shuffle T7, ArcticExpress, and ArticExpress(DE3).

[0268] The engineered cells can be eukaryotic cells. The eukaryotic cells can belong to or be derived from a particular organism such as a plant or a mammal, including but not limited to humans, or non-human eukaryotes or animals or mammals as discussed herein, such as mice, rats, rabbits, dogs, livestock or non-human mammals or primates. In some embodiments, the engineered cells can be a cell line. Examples of cell lines include but are not limited to 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, HepG2, HeLaB, HeLaT4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epidermis, BALB / 3T3 mouse embryonic 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, CHODhfr- / - , COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalc1c7, 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 line, Peer, PNT-1A / PNT2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR and their transgenic variants. The cell lines can be obtained from a variety of sources known to those skilled in the art (see, for example, American Type Culture Collection (ATCC) (Manassas, Va.)).

[0269] 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, neuron-supporting cells, intestinal cells, epidermal cells, endothelial cells, stem cells and other progenitor cells, adrenal cells, chondrocytes, and combinations thereof.

[0270] In some embodiments, the engineered cell can be a fungal cell. As used herein, "fungal cell" refers to any type of eukaryotic cell within the Fungi kingdom. The phyla within the Fungi kingdom include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cells can include yeasts, molds, and filamentous fungi. In some embodiments, the fungal cell is a yeast cell.

[0271] As used herein, the term "yeast cell" refers to any fungal cell within the phyla Ascomycota and Basidiomycota. Yeast cells can include budding yeast cells, fission yeast cells, and mold cells. Without being limited to these organisms, many types of yeast used in laboratory and industrial settings are part of the phylum Ascomycota. In some embodiments, the yeast cell is a Saccharomyces cerevisiae, Kluyveromyces marxianus, or Issatchenkia orientalis cell. Other yeast cells can include, but are not limited to, Candida spp. (e.g., Candida albicans), Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichia pastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis and Kluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa), Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g., Issatchenkia orientalis, also known as 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 as a filament, i.e., a hypha or mycelium. Examples of filamentous fungal cells can include, but are not limited to, Aspergillus spp. (e.g., Aspergillus niger), Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g., Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

[0272] In some embodiments, the fungal cell is an industrial strain. As used herein, an "industrial strain" refers to any strain of fungal cells used in or isolated from an industrial process, such as the production of a product on a commercial or industrial scale. An industrial strain can refer to a fungal species typically used in an industrial process, or it can 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 can include fermentation (such as in the production of food or beverage products), distillation, biofuel production, production of compounds, and production of polypeptides. Examples of industrial strains can include, but are not limited to, JAY270 and ATCC4124.

[0273] In some embodiments, the fungal cell is a polyploid cell. As used herein, a "polyploid" cell can refer to any cell in which its genome exists in more than one copy. A polyploid cell can refer to a type of cell that is naturally visible in a polyploid state, or it can refer to a cell that has been induced to exist in a polyploid state (such as by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). A polyploid cell can refer to a cell in which the entire genome is polyploid; or it can refer to a cell that is polyploid at a specific locus of interest.

[0274] In some embodiments, the fungal cell is a diploid cell. As used herein, a "diploid" cell can refer to any cell in which its genome exists in two copies. A diploid cell can refer to a type of cell that is naturally visible in a diploid state, or it can refer to a cell that has been induced to exist in a diploid state (such as by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the Saccharomyces cerevisiae strain S228C can be maintained in a haploid or diploid state. A diploid cell can refer to a cell in which the entire genome is diploid; or it can refer to a cell that is diploid at a specific 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 its genome exists in one copy. A haploid cell can refer to a type of cell that is naturally visible in a haploid state, or it can refer to a cell that has been induced to exist in a haploid state (such as by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the Saccharomyces cerevisiae strain S228C can be maintained in a haploid or diploid state. A haploid cell can refer to a cell in which the entire genome is haploid; or it can refer to a cell that is haploid at a specific locus of interest.

[0275] In some embodiments, the engineered cells are cells 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 engineered AAV capsid particles are produced, they can package one or more cargo polynucleotides that may be related to the desired physiological and / or biological characteristics and / or capable of modifying the desired physiological and / or biological characteristics. Thus, the cargo polynucleotides of the engineered AAV capsid particles produced can transfer the desired characteristics to recipient cells. In some embodiments, the cargo polynucleotide is capable of modifying the polynucleotide of the engineered cell such that the engineered cell has the desired physiological and / or biological characteristics.

[0276] In some embodiments, cells transfected with one or more of the vectors described herein are used to establish a new cell line comprising one or more vector-derived sequences.

[0277] Engineered cells can be used to produce engineered viral (e.g., AAV) capsid polynucleotides, vectors, and / or particles. In some embodiments, the engineered viral (e.g., AAV) capsid polynucleotides, vectors, and / or particles are produced, harvested, and / or delivered to a subject in need thereof. In some embodiments, the engineered cells are delivered to a subject. Other uses of the engineered cells are described elsewhere herein. In some embodiments, the engineered cells can be included in formulations and / or kits described elsewhere herein.

[0278] Engineered cells can be stored short-term or long-term for later use. Suitable storage methods are generally known in the art. In addition, methods for recovering the stored cells for later use, such as thawing, reconstitution, and otherwise stimulating metabolism in the engineered cells after storage, are also generally known in the art.

[0279] Formulations

[0280] 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 formulations can be used to generate polypeptides and other particles including one or more CNS-specific n-mers described herein. In some embodiments, the formulations can be delivered to a subject in need thereof. In some embodiments, the components of the engineered AAV capsid systems, engineered cells, engineered AAV capsid particles, and / or combinations thereof described herein can be included in formulations that can be delivered to a subject or cell. In some embodiments, the formulations are pharmaceutical formulations. One or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be provided, either alone or as an active ingredient such as in a pharmaceutical formulation, to a subject or cell in need thereof. Accordingly, pharmaceutical formulations are also described herein that contain an amount of one or more of the polypeptides, polynucleotides, vectors, cells, or combinations thereof described herein. In some embodiments, the pharmaceutical formulations can contain an effective amount of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The pharmaceutical formulations described herein can be administered to a subject or cell in need thereof.

[0281] In some embodiments, based on the body weight of the subject in need thereof or the average body weight of a specific patient population to which the pharmaceutical formulation can be administered, the amount of one or more of the polypeptides, polynucleotides, vectors, cells, viral particles, nanoparticles, other delivery particles, and combinations thereof described herein contained in the pharmaceutical formulation can range from about 1 pg / kg to about 10 mg / kg. The amount of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein contained in the 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 can be in the range of about 1 cell to 1x10 2 、1x10 3 、1x10 4 、1x10 5 、1x10 6 、1x10 7 、1x10 8 、1x10 9 、1x10 10 or more cells. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can be from about 1 cell to 1x10 2 、1x10 3 、1x10 4 、1x10 5 、1x10 6 、1x10 7 、1x10 8 、1x10 9, 1 x 10 10 or more cells.

[0282] In many embodiments where engineered AAV capsid particles are included in a formulation, the formulation may contain from 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 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 transducing units (TU) / mL of engineered AAV capsid particles. In some embodiments, the volume of the formulation can be from 0.1 to 100 mL and can contain from 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 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 transducing units (TU) / mL of engineered AAV capsid particles.

[0283] Pharmaceutically acceptable carriers, excipients, and agents

[0284] In multiple embodiments, a pharmaceutical formulation containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, viral particles, nanoparticles, other delivery particles, and combinations thereof described herein may further include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohols, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose, or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oils, fatty acid esters, hydroxypropylmethyl cellulose, and polyvinylpyrrolidone, which do not react detrimentally with the active composition.

[0285] The pharmaceutical formulation may be sterilized and, if desired, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts affecting osmotic pressure, buffers, coloring agents, flavoring agents, and / or aromatic substances, etc., which do not react detrimentally with the active composition.

[0286] 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 may also include an effective amount of co-active agents, including but 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.

[0287] 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, antidiuretic hormone, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eicosanoids (e.g., arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g., estradiol, testosterone, tetrahydrotestosterone, 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-α, IFN-β, IFN-ε, IFN-κ, IFN-ω, and IFN-γ), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26, and CXCL7), phosphorothioate cytosine-guanosine, oligodeoxynucleotides, dextran, antibodies, and aptamers).

[0288] Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammatory drugs (such as ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (such as choline salicylate, magnesium salicylate, and sodium salicylate), paracetamol / acetaminophen, metamizole, nabumetone, phenazone, and quinine.

[0289] Suitable anxiolytics include, but are not limited to, benzodiazepines (such as alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotonergic antidepressants (such as selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), mebicar, fabomotizole, selank, bromantane, emoxypine, azapirones, barbiturates, hydroxyzine, pregabalin, validol, and beta blockers.

[0290] Suitable antipsychotics include but are not limited to benperidol, bromoperidol, droperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thiopropazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, 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, ziprasidone, zotepine, alstonie, bifeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, and zicronapine.

[0291] Suitable painkillers include, but are not limited to, paracetamol / acetaminophen, non-steroidal anti-inflammatory drugs (such as ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (such as rofecoxib, celecoxib, and etoricoxib), opioid drugs (such as morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, noradrenaline, flupirtine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (such as choline salicylate, magnesium salicylate, and sodium salicylate).

[0292] Suitable antispasmodics include, but are not limited to, mebeverine, papaverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine and dantrolene. Suitable anti-inflammatory agents include, but are not limited to, prednisone, non-steroidal anti-inflammatory agents (such as ibuprofen, naproxen, ketoprofen and nimesulide), COX-2 inhibitors (such as rofecoxib, celecoxib and etoricoxib) and immunoselective anti-inflammatory derivatives (such as submandibular gland peptide-T and its derivatives).

[0293] Suitable antihistamines include, but are not limited to, H1 receptor antagonists (such as acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, dextrochlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, meclizine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2 receptor antagonists (such as cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine), tritoqualine, catechins, cromoglicate, nedocromil, and p2-adrenergic agonists.

[0294] Suitable anti-infective drugs include, but are not limited to, anti-amoebic drugs (such as nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, amphotericin b, and iodoquinol), aminoglycosides (such as paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (such as pyrantel, mebendazole, ivermectin, praziquantel, albendazole, thiabendazole, oxamniquine), antifungal drugs (such as azole antifungal drugs (such as itraconazole, fluconazole, parconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (such as caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine, flucytosine, and polyenes (such as nystatin and amphotericin b), antimalarials (such as pyrimethamine / sulfadoxine), artemether / lumefantrine, atovaquone / proguanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), anti-tuberculosis drugs (such as aminosalicylates (such as aminosalicylic acid), isoniazid / rifampin, isoniazid / pyrazinamide / rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antiviral drugs (such as amantadine, rimantadine,Abacavir / lamivudine, emtricitabine / tenofovir, cobicistat / elvitegravir / emtricitabine / tenofovir, efavirenz / emtricitabine / tenofovir, abacavir / lamivudine / zidovudine, lamivudine / zidovudine, emtricitabine / tenofovir, emtricitabine / lopinavir / ritonavir / 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, zidovudine, stavudine, emtricitabine, zalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir / ritonavir, boceprevir, darunavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, saquinavir, ribavirin, valacyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g., doripenem, meropenem,Ertapenem and cilastatin / imipenem, cephalosporins (such as ceftobiprole, cephradine, cefazolin, cephalexin, cefepime, cefazoline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, ceftizoxime, and ceftazidime), glycopeptide antibiotics (such as vancomycin, dalbavancin, oritavancin, and telavancin), glycylcyclines (such as tigecycline), antileprosy drugs (such as clofazimine and thalidomide), lincomycin and its derivatives (such as clindamycin and lincomycin), macrolides and their derivatives (such as telithromycin, fidaxomicin, erythromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole / trimethoprim, rifaximin, chloramphenicol, fosfomycin, metronidazole, aztreonam, bacitracin, penicillins (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin / clavulanate, amoxicillin / sulbactam, piperacillin / tazobactam, clavulanate / ticarcillin, penicillin, procaine penicillin, oxacillin,Dicloxacillin and nafcillin, quinolones (such as lomefloxacin, norfloxacin, ofloxacin, gatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafloxacin, and sparfloxacin), sulfonamides (such as sulfamethoxazole / trimetoprim, sulfasalazine, and sulfisoxazole), tetracyclines (such as doxycycline, demeclocycline, minocycline, doxycycline / salicylic acid, doxycycline / ω-3 polyunsaturated fatty acids, and tetracycline), and urinary tract anti-infectives (such as nitrofurantoin, methenamine, fosfomycin, cinoxacin, nalidixic acid, trimetoprim, and methylene blue).

[0295] Suitable chemotherapeutic agents include but are not limited to 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, actinomycin, ramucirumab, cytarabine, cyclophosphamide (Cytoxan), cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, dacarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, asparaginase Erwinia chrysanthemi, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylatemesylate), carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alpha-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, methyl bis(chloroethyl)amine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronicacid), lenalidomide, rituximab, octreotide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, tioguanine, dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, 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, tafluposide, azathioprine, doxifluridine, vindesine and all-trans retinoic acid.

[0296] In the multiple embodiments where the pharmaceutical preparation contains, in addition to one or more of the polypeptides, polynucleotides, CRISPR-Cas complexes, carriers, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein, an auxiliary active agent, the amount of the auxiliary active agent, such as an effective amount, will vary depending on the auxiliary active agent. In some embodiments, the amount of the auxiliary active agent ranges from 0.001 micrograms to about 1 milligram. In other embodiments, the amount of the auxiliary active agent ranges from about 0.01 IU to about 1000 IU. In further embodiments, the amount of the auxiliary active agent ranges from 0.001 mL to about 1 mL. In still other embodiments, the amount of the auxiliary active agent ranges from about 1% w / w to about 50% w / w of the total pharmaceutical preparation. 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 preparation. In yet other embodiments, the amount of the auxiliary active agent ranges from about 1% w / v to about 50% w / v of the total pharmaceutical preparation.

[0297] Dosage form

[0298] In some embodiments, the pharmaceutical preparations described herein can be in dosage form. The dosage form can be suitable for administration by any suitable route. Suitable routes include, but are not limited to, rectal, epidural, intracranial, intraocular, inhalation, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intra-articular, intracavernous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intraventricular, and intradermal. Such preparations can be made by any method known in the art.

[0299] Dosage forms suitable for parenteral administration and / or suitable for any type of injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intra-articular, intracavernous, gingival, subgingival, intrathecal, intravitreal, intracerebral, and intraventricular) may include aqueous and / or non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, solutes that render the composition isotonic with the blood of the subject, and aqueous and non-aqueous sterile suspensions, which may include suspending and thickening agents. Dosage forms suitable for parenteral administration may be presented in single unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The dosage may be lyophilized and reconstituted in a sterile carrier prior to administration to reconstitute the dosage. In some embodiments, ready-to-use injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. See, e.g., 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.

[0300] Dosage forms can also be prepared to provide for extended or sustained release of any ingredient. In some embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be ingredients for which release is delayed. In other embodiments, release of optional excipients included is delayed. Suitable methods for delaying release of an ingredient include, but are not limited to, coating or embedding the ingredient in a material such as a polymer, wax, gel, and the like. Sustained release formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” Liberman et al., eds. (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 ed., Ansel et al., (Media, PA: Williams and Wilkins, 1995).

[0301] 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 formulated in an ointment, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be formulated with a paraffin or water - soluble ointment base. In some embodiments, the active ingredient can be formulated in 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 mouthwashes.

[0302] Dosage forms suitable for intranasal or inhaled administration include aerosols, solutions, suspension drops, gels, or dry powders. In some embodiments, one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein are included in a dosage form suitable for inhalation, which is in a reduced particle size form obtained or obtainable by micronization. In some embodiments, the particle size of the compound or salt or its solvate that has been size-reduced (e.g., micronized) is defined by a D50 value of from about 0.5 to about 10 microns, as measured by suitable methods known in the art. Dosage forms suitable for administration by inhalation also include particulate dusts or mists. Suitable dosage forms in which the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oily solutions / suspensions of the active ingredient (e.g., one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein and / or co-active agents), which can be generated by various types of metered pressurized aerosols, nebulizers, or insufflators.

[0303] 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 contain a solution or fine suspension of one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein and a pharmaceutically acceptable aqueous or non-aqueous solvent. The aerosol formulation can be presented in a sterile form in a sealed container in single or multiple dose amounts. For some of these embodiments, the sealed container is a single or multiple dose nasal container, or an aerosol dispenser equipped with a metering valve (e.g., a metered inhaler), designed for disposal once the contents of the container are exhausted.

[0304] Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure such as compressed air, carbon dioxide, or organic propellants, including but not limited to hydrofluorocarbons. The aerosol formulation in other embodiments is contained in a pump nebulizer. The pressurized aerosol formulation can also contain a solution or suspension of one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein. In further embodiments, the aerosol formulation can also contain co-solvents and / or modifiers incorporated to improve, for example, the stability and / or taste and / or fine particle mass characteristics (amount and / or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, e.g., 2, 3, 4, or 8 times daily, where 1, 2, or 3 doses are delivered each time.

[0305] For some dosage forms suitable and / or applicable for inhalation administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein, auxiliary active ingredients, and / or their pharmaceutically acceptable salts, this dosage form may also contain a powder matrix such as lactose, glucose, trehalose, mannitol, and / or starch. In some of these embodiments, one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein are in a reduced particle size form. In further embodiments, performance modifiers such as L-leucine or another amino acid, cellobiose octaacetate, and / or metal salts of stearic acid such as magnesium stearate or calcium stearate.

[0306] In some embodiments, an aerosol dosage form may be provided such that each metered aerosol contains a predetermined amount of an active ingredient, such as one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein.

[0307] Dosage forms suitable for ocular administration may include aqueous and / or non-aqueous sterile solutions, which may optionally be suitable for injection, and which may optionally contain antioxidants, buffers, bacteriostatic agents, solutes that render the composition isotonic with the fluids contained in or around the subject's eye, and aqueous and non-aqueous sterile suspensions that may include suspending and thickening agents.

[0308] For some embodiments, the dosage form contains a predetermined amount of one or more of the polypeptides, polynucleotides, carriers, cells, and combinations thereof described herein per unit dose. In some embodiments, such a predetermined amount of the unit dose may thus be administered once or more than once per day. Such pharmaceutical formulations may be prepared by any method well known in the art.

[0309] Effective amount

[0310] In some embodiments, the amount of the primary active agent and / or optional secondary agent may 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 primary and / or optional secondary agent included in the pharmaceutical formulation that achieves one or more therapeutic or desired effects. As used herein, "minimum effective", "minimum effective concentration", etc. refer to the lowest amount, concentration, etc. of the primary and / or optional secondary agent 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 primary and / or optional secondary agent included in the pharmaceutical formulation that achieves one or more therapeutic effects. In some embodiments, one or more therapeutic effects include transducing the CNS.

[0311] In some embodiments, an amount or effective amount, particularly in the case of delivering infectious particles (e.g., virus particles having a primary or secondary agent as a carrier), the effective amount of virus particles can be expressed as a titer (plaque-forming units per unit volume) or as an MOI (multiplicity of infection). In some embodiments, the effective amount can be from about 1X10 1 particles / pL, nL, μL, mL, or L to 1X10 20 particles / pL, nL, μL, mL, or L or more, such as 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 , 1x1018, 1x10 19 to / or about 1x10 20 particles / pL, nL, μL, mL, or L. In some embodiments, the effective titer can be from about 1X10 1 transforming units / pL, nL, μL, mL, or L to 1X10 20 transforming units / pL, nL, μL, mL, or L or more, such as 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 , 1x0 13 , 1x10 14 , 1x10 15 , 1x10 16 , 1x10 17 , 1x10 18 , 1x10 19 to / or about 1x1020 Convert the unit to pL, nL, μL, mL or L, or any value or sub-range within these ranges. In some embodiments, the MOI of the pharmaceutical formulation can be in the range of about 0.1 to 10 or more, such as 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 value or sub-range within these ranges.

[0312] 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, each of which can be in the range 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 any non-zero amount within any of these ranges or any value or sub-range within any of these ranges.

[0313] In some embodiments, the primary and / or optional secondary active agent present in the pharmaceutical formulation can be any non-zero amount within the range of from 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, 0.99, 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 any numerical value or sub-range within any of these ranges.

[0314] In some embodiments, based on the weight of the subject in need thereof or the average weight of the specific patient population to which the pharmaceutical formulation can be administered, the amount or effective amount of one or more of the active agents described herein contained in the pharmaceutical formulation can be in the range of about 1 pg / kg to about 10 mg / kg.

[0315] In embodiments where a secondary agent is included in the pharmaceutical formulation, the effective amount of the secondary active agent will vary depending on the secondary agent, the primary agent, the route of administration, the age of the subject, the disease, the disease stage, etc., which will be known to those of skill in the art.

[0316] When optionally present in the pharmaceutical formulation, the secondary active agent can be included in the pharmaceutical formulation or can be present as a separate compound or pharmaceutical formulation that can be administered simultaneously or sequentially with the compound, its derivatives, or its pharmaceutical formulation.

[0317] In some embodiments, when optionally present, the effective amount of the secondary active agent is any non-zero amount within the range of from 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 additional embodiments, the effective amount of the secondary active agent is any non-zero amount within the range of from 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 pharmaceutical formulation, or any numerical value or sub-range within these ranges.

[0318] Kits

[0319] The present invention also describes kits that contain one or more of the compositions, polypeptides, polynucleotides, vectors, cells, or other components and combinations thereof described herein, and one or more of the pharmaceutical formulations described herein. In some embodiments, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be presented as combination kits. As used herein, the terms "combination kit" or "kit of parts" refer to compounds or formulations, as well as additional components, that are used for packaging, screening, testing, marketing, commercializing, delivering, and / or administering a combination or single element, such as an active ingredient, contained therein. Such additional components include, but are not limited to, packaging, syringes, blister packs, bottles, etc. The combination kit can contain one or more components (e.g., one or more of polypeptides, polynucleotides, vectors, cells, and combinations thereof) or its formulations can be provided in a single formulation (e.g., liquid, lyophilized powder, etc.) or in separate formulations. The individual components or formulations can be contained in a single package within the kit or in separate packages. The kit can also include instructions in a tangible expression medium, which can contain information and / or guidance regarding the amounts of the components and / or formulations contained therein; safety information regarding the amounts of the components and / or formulations contained therein; information regarding amounts, dosages, use indications, screening methods, component design recommendations; and / or information regarding recommended treatment regimens for the components and / or formulations contained therein. As used herein, a "tangible expression medium" refers to a physically tangible and accessible medium, and not merely an abstract idea or unrecorded verbal discourse. "Tangible expression medium" includes, but is not limited to, words on a cellulose or plastic material, or data stored in a suitable computer-readable memory. The data can be stored on a unit device, such as a flash drive or CD-ROM, or stored on a server that a user can access via, for example, a network interface.

[0320] In one embodiment, the present invention provides a kit that contains one or more of the components described herein. In some embodiments, the kit contains a vector system and instructions for using the kit. In some embodiments, the vector system includes regulatory elements operably linked to one or more engineered polynucleotides (such as those engineered polynucleotides containing selective n-mers, as described elsewhere herein), and optionally includes a carrier molecule optionally operably linked to the regulatory element. In embodiments where a carrier molecule is contained within the kit, one or more engineered polynucleotides, such as those engineered polynucleotides containing selective n-mers as described elsewhere herein, can be included on the same or different vectors as the carrier molecule.

[0321] 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 direct repeat sequence and one or more insertion sites for inserting one or more guide sequences upstream or downstream (as applicable) of the direct repeat sequence, wherein upon expression, the guide sequence directs sequence-specific binding of a Cas9 CRISPR complex to a target sequence in a eukaryotic cell, wherein the Cas9 CRISPR complex comprises a Cas9 enzyme complexed with the guide sequence and the guide sequence hybridizes to the target sequence; and / or (b) a second regulatory element operably linked to an enzyme-encoding sequence that encodes the Cas9 enzyme comprising a nuclear localization sequence. If applicable, a tracr sequence may be provided. In some embodiments, the kit comprises components (a) and (b) located on the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein upon expression, each of the two or more guide sequences directs sequence-specific binding of a 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 drive accumulation of the CRISPR enzyme in detectable amounts in the nucleus of a eukaryotic cell. 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 MC20171, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella species SCADC, Acidaminococcus species 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 a DD or associated therewith), and may further include alterations or mutations of Cas9 and can 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 directs cleavage of one or both strands at the position of the target sequence. In some embodiments, the DD-CRISPR enzyme lacks or substantially lacks DNA strand cleavage activity (e.g., nuclease activity is no more than 5% compared to a wild-type enzyme or an enzyme with mutations or alterations that do not 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 length of the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16 - 30 nucleotides, between 16 - 25 nucleotides, or between 16 - 20 nucleotides.

[0322] Method of use for delivering a payload to the CNS

[0323] Compositions comprising one or more of a cell-selective targeting moiety, an engineered AAV capsid system polynucleotide, polypeptide, vector, engineered cell, engineered AAV capsid particle are generally useful for packaging one or more payloads and / or delivering them to neurons and glial cells of the CNS. In some embodiments, delivery is effected in a cell-selective manner based on the selectivity of the targeting moiety. In some embodiments, this is conferred by the tropism of the engineered AAV capsid, which may be influenced at least in part by including one or more of the monomeric or multimeric motifs described elsewhere herein. In some embodiments, compositions comprising one or more of the CNS targeting moieties, including engineered AAV capsid particles, wherein the capsid incorporates the targeting moiety, can be administered to a subject or to cells, tissues, and / or organs and promote payload transfer and / or integration into recipient cells. In other embodiments, engineered cells capable of producing compositions such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles), containing one or more of the targeting moieties can be generated from the polynucleotides, vectors, and vector systems, etc., described herein. This includes but is not limited to engineered AAV capsid system molecules (e.g., polynucleotides, vectors, and vector systems, etc.). In some embodiments, the polynucleotides, vectors, and vector systems described herein, capable of producing compositions such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles), containing one or more of the targeting moieties can be delivered to cells or tissues in vivo, ex vivo, or in vitro. In some embodiments, when delivered to a subject, the compositions can transform the subject's cells in vivo or ex vivo to produce engineered cells capable of preparing the compositions described herein, which contain one or more of the cell-selective targeting moieties described herein, including but not limited to engineered AAV capsid particles, which can be released from the engineered cells and deliver payload molecules in vivo to recipient cells or produce personalized engineered compositions (e.g., AAV capsid particles) for reintroduction into the subject from whom the recipient cells were obtained.

[0324] In some embodiments, the engineered cells can be delivered to a subject, where they can release the compositions of the invention produced (including but not limited to engineered AAV capsid particles), such that they can subsequently deliver a payload (e.g., a payload polynucleotide) to recipient cells. In bioproduction and various other applications, these general processes can be used in a variety of ways to treat and / or prevent diseases or their symptoms in a subject, generate model cells, generate modified organisms, provide cell selection and screening assays.

[0325] In some embodiments, compositions such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles) containing one or more of the targeting moieties can be delivered to nerve cells of the CNS. In another exemplary embodiment, compositions containing one or more targeting moieties can be delivered to glial cells of the CNS.

[0326] In some embodiments, engineered AAV capsid polynucleotides, vectors, and systems thereof can be used to generate a library of engineered AAV capsid variants, which can be mined for variants with desired cell selectivity. As demonstrated by the descriptions provided herein, supported by various examples, variants with desired cell selectivity as contemplated can be obtained using the present invention to obtain capsids with desired cell selectivity as described herein.

[0327] Therapeutic Methods

[0328] Provided herein are methods for treating a disease or disorder, the methods comprising administering a composition as disclosed herein to cells of the CNS in a subject in need thereof. In one aspect, the compositions used in the methods disclosed herein are capable of increasing transduction of neurons and / or glial cells of the CNS, allowing for direct delivery of a cargo and a therapeutic agent to such cell types. In various embodiments, a method is disclosed wherein the cargo is one or more polypeptides.

[0329] Disease or disorder

[0330] In various embodiments, a method is disclosed wherein the disease or disorder is cancer, a neurological disorder, or an infection.

[0331] In one embodiment, a method of treatment comprises administering to a subject in need thereof a composition as detailed herein. In an exemplary embodiment, the cancer is neuroepithelial cancer. In one embodiment, the cancer is a neuroepithelial tumor, such as an astrocytic tumor, such as diffuse astrocytoma (fibrillary, protoplasmic, gemistocytic, mixed), anaplastic (malignant) astrocytoma, glioblastoma (giant cell, gliosarcoma variant), pilocytic astrocytoma, pleomorphic xanthoastrocytoma, or subependymal giant cell astrocytoma; oligodendroglial tumors, such as oligodendroglioma, anaplastic (malignant) oligodendroglioma, ependymal tumors, ependymoma (cellular, papillary, clear cell, tanycytic type), anaplastic (malignant) ependymoma, myxopapillary ependymoma, subependymoma; mixed tumors, such as oligoastrocytoma or anaplastic (malignant) oligoastrocytoma; choroid plexus tumors, such as choroid plexus papilloma or choroid plexus carcinoma; neuronal and mixed neuronal-glial tumors, such as ganglioglioma, desmoplastic infantile astrocytoma / ganglioglioma, dysembryoplastic neuroepithelial tumor (DNET), dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos), desmoplastic infantile astrocytoma / ganglioglioma, central neurocytoma, anaplastic ganglioglioma, cerebellar liponeurocytoma, paraganglioma of the filum terminale; pineal tumors, such as pineocytoma, pineoblastoma, intermediate differentiated pineal parenchymal tumor; embryonal tumors, such as medulloblastoma (desmoplastic, large cell, melanotic, medullo-myoblastoma), medulloepithelioma, supratentorial primitive neuroectodermal tumor, PNET such as neuroblastoma, ganglioneuroblastoma, ependymoblastoma, or atypical teratoid / rhabdoid tumor; neuroblastoma tumors, such as olfactory (esthesioneuroblastoma), olfactory neuroepithelioma, neuroblastoma of the adrenal and sympathetic nervous systems; gliomas of unknown origin, such as astrocytoma, gliomatosis cerebri, chordoid glioma of the third ventricle.

[0332] In one embodiment, the cancer is a primary cancer that has metastasized to the brain or other regions of the central nervous system.

[0333] In one exemplary embodiment, the neurological disorder is caused by a neurodegenerative or 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. Examples of neurodevelopmental diseases include, but are not limited to, attention deficit / hyperactivity disorder (ADHD), autism, learning disorders, intellectual disability (also known as mental retardation), behavioral disorders, cerebral palsy, speech and language disorders, Tourette syndrome, schizophrenia, fragile X syndrome, and vision and hearing impairments.

[0334] Non-human transgenic animal

[0335] In one aspect, the present disclosure provides a method of creating a humanized transgenic non-human animal, comprising: delivering one or more cells of a non-human animal to a vector system or recombinant viral particle comprising a recombinant viral genome, wherein: the vector system or recombinant viral genome encodes a human transferrin polypeptide, wherein the encoded human transferrin polypeptide is under the control of a tissue-specific promoter or an miRNA binding element having selective activity within 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 endothelial cells of the CNS vasculature. In one exemplary embodiment, the recombinant viral particle, optionally an AAV viral particle, comprises a capsid polypeptide, optionally an AAV capsid polypeptide, wherein the capsid polypeptide comprises a CNS-specific n-mer motif. In one exemplary embodiment, the CNS-specific n-mer motif comprises X1-N-X3-X4-X5-X6-X7, wherein X5 is independently selected from K or R, and X1, X3, X4, X6, and X7 are independently selected from any amino acid, optionally wherein the total charge of the n-mer motif at neutral pH is between 0 and +2. In one exemplary embodiment, the CNS-specific n-mer motif comprises NNSTRGG (SEQ ID NO: 42429), GNSARNI (SEQ ID NO: 42430), and GNSVRDF (SEQ ID NO: 42431) or consists thereof. In one exemplary embodiment, the transgenic non-human animal is a rodent, optionally a mouse.

[0336] In one aspect, the present disclosure provides a humanized transgenic non-human animal comprising: one or more cells expressing a human transferrin polypeptide, optionally wherein the one or more cells are CNS cells. In an exemplary embodiment, the transgenic non-human animal is a rodent, optionally a mouse. In one aspect, the present disclosure provides a humanized transgenic non-human animal produced by the methods described herein. In an exemplary embodiment, the humanized non-human animal has an inhibited immune system.

[0337] It should be understood that in the methods of the present invention, in the case where 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, such as in cell culture, and in some cases not in vivo. In other embodiments, it can occur in vivo. In one aspect, the present invention provides a method of modifying an organism or non-human organism by manipulating a target sequence in a genomic locus of interest, comprising: delivering a non-naturally occurring or engineered composition, for example, via a particle or nanoparticle or vector (such as a viral vector, such as AAV, adenovirus, lentivirus).

[0338] A single cell or cell population can preferably be modified ex vivo and then reintroduced, for example, transplanted, to produce a transgenic organism expressing TFRC in certain cells. In some embodiments, the present invention encompasses a method of modifying a eukaryote, such as a transgenic eukaryote, comprising delivering a non-naturally occurring or engineered composition, for example, via a vector and / or particle and / or nanoparticle. The system can comprise one, two, three, or four different vectors; and the system can comprise a nanoparticle complex of one, two, three, or four different delivery system components. Thus, components I, II, III, and IV can be located on one, two, three, or four different vectors and can be delivered by one, two, three, or four different particles or nanoparticle complexes or AAVs, or components I, II, III, and IV can be located on the same or different vectors / particles / nanoparticles, and all combinations of positions are contemplated. A complex targeting the CNS or CNS tissue or CNS cells is advantageous.

[0339] In some embodiments, the vector is delivered to a eukaryotic cell in a transgenic eukaryote. In some embodiments, the modification occurs in the eukaryotic cell in cell culture. In one aspect, the present invention provides a method of producing a model eukaryotic cell or model transgenic eukaryotic organism comprising one or more human proteins.

[0340] 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 line obtained by crossing a transgenic mouse with another mouse line. In certain embodiments, the offspring (or multiple offspring) derived from the transgenic eukaryote (such as the mouse line) can successfully reproduce for at least five generations without exhibiting an increase in genomic instability or cytotoxicity levels. In one aspect, the present invention provides a method for simultaneously introducing multiple mutations in vitro or in vivo in a tissue, organ, or cell line (of the central nervous system). It is understood that using the novel targeting moiety tools disclosed herein, transgenic non-human eukaryotes can be generated, such as animal models that can be envisioned to have multiple mutations at any number of loci and are within the scope of the present invention. It should be understood that such transgenic non-human eukaryotes, such as animal models, provide valuable tools for research purposes, such as viral transduction, and open the door for the development and testing of novel therapeutic interventions targeting specific tissues involving mutations at multiple loci. Such uses are within the scope of the present invention.

[0341] The eukaryotic cell can comprise a constitutive promoter, or a tissue-specific promoter, or an inducible promoter; and, the eukaryotic cell can be part of a non-human transgenic eukaryote, such as a non-human mammal, primate, rodent, mouse, rat, rabbit, canine, dog, bovine, sheep, goat, pig, poultry, chicken, fish, insect, or arthropod; advantageously a mouse. The isolated eukaryotic cell or non-human transgenic eukaryote can express an additional protein or enzyme, such as TfR1; and, the expression of TfR1 can be driven by encoding it to be functionally or operably linked to a constitutive promoter, or a tissue-specific promoter, or an inducible promoter.

[0342] The eukaryotic cell can be a mammalian cell, such as a mouse cell, such as a mouse cell that is part of a transgenic mouse having cells that express TfR1.

[0343] In one aspect of the practice of the present invention, transgenic non-human eukaryotes, such as animals, are also provided. Preferred examples include animals comprising TfR1, in terms of the polynucleotide encoding TfR1 or the protein itself. In certain aspects, the present invention relates to constitutive or conditional or inducible TfR1 non-human eukaryotes, such as animals, for example, primates, rodents, such as mice, rats, and rabbits are preferred; and may include dogs or canines, livestock (cows / cattle, sheep / goats, goats, or pigs), fish, fowl or poultry, such as chickens, and insects or arthropods, where it is mentioned that it is advantageous if the animal is a model of a human or animal protein, cell, or tissue, since non-human eukaryotes, such as via induction of multiple, are preferably used in conditional modeling. To generate transgenic mice with constructs, as exemplified herein, pure linear DNA can be injected into the pronucleus of a fertilized egg from a pseudopregnant female (e.g., CB56 female). Then, founder mice can be identified, genotyped, and backcrossed to CB57 mice. Then the construct can be cloned and optionally verified, for example, by Sanger sequencing. Knock-ins are envisioned (alone or in combination).

[0344] Accordingly, the present invention relates to non-human eukaryotes, animals, mammals, primates, rodents, etc., or their cells or tissues that can be used as models. For example, the methods of the present invention can be used to create non-human eukaryotes, such as animals, mammals, primates, rodents, or cells, that contain modifications of one or more nucleic acid sequences related to or associated with such cells or tissues (such as the central nervous system). In the case of a multicellular organism, the cells can be in vivo or ex vivo. In the case of cultured cells, if appropriate culture conditions are met, and preferably if the cells are suitably adapted for this purpose (e.g., stem cells), a cell line can be established. Accordingly, cell lines are also envisioned.

[0345] In one aspect, the invention can relate to cells that have been transformed to contain TfR1, such as non-human eukaryotes, such as animals, such as mammals, such as primates, rodents, mice, rats, rabbits, etc., and even human cells, such as such cell carriers containing a nucleic acid molecule encoding TfR1 (e.g., a nucleic acid encoding a promoter and at least one NLS, advantageously two or more NLSs), or such cells with an altered genome, such as by a vector that is an integrating virus or by such cells being stem cells or cells that give rise to cell lines or living organisms (but where such organisms are advantageously non-human), and the cell line or living organism contains and expresses a nucleic acid molecule encoding TfR1. Such cells are then transplanted into or onto an animal suitable for expressing the cells or tissues. The cells proliferate on or in a non-human eukaryote (such as an animal model). Then, under the control of a promoter such as the U6 promoter and / or particles and / or nanoparticles, an RNA or a vector, such as an AAV, adenovirus, lentivirus containing or providing the RNA, is administered to a non-human eukaryote, such as an animal model, having proliferating xenografted TfR1-containing cells. The non-human eukaryote, such as an animal model, can then be used for testing, such as for potential therapies and / or putative treatments via a compound that may have pharmaceutical activity. The administration can be at or for or for in vivo delivery to the proliferating xenografted TfR1-containing cells, such as by direct injection at or near such proliferating xenografted TfR1-containing cells, or by injection in such a way that the RNA is delivered into the proliferating xenografted TfR2-containing cells, such as by injection into the blood or other administration, whereby the body function is transported into the proliferating xenografted TfR1-containing cells. In one aspect of the invention, the barcode labeling technique of WO / 2013 / 138585A1 can be applied to or integrated into the practice of the invention. WO / 2013 / 138585A1 provides methods for simultaneously determining the effect of test conditions on the viability or proliferation of each of a plurality of genetically heterogeneous cell types. The number of live cells in a sample after exposure to test conditions, as indicated compared to a reference cell number, indicates the effect of the test conditions on the viability or proliferation of each cell type. WO / 2013 / 138585A1 also provides methods for simultaneously determining the effect of test conditions on the viability or proliferation of each of a plurality of genetically heterogeneous cell types. The cited patent application is incorporated herein by reference.

[0346] Unless otherwise indicated, the practice of the invention employs conventional techniques to generate gene-modified mice. See Marten H. Hofker and Jan van Deursen, TRANSGENIC MOUSE METHODS AND PROTOCOLS, 2nd Edition (2011).

[0347] Screening and Cell Selection

[0348] In one aspect, provided herein is a method of screening for n-mer motifs that are capable of transducing central nervous system (CNS) tissue via binding to the transferrin receptor (TFRC) in a humanized transgenic non-human animal, comprising: introducing one or more compositions comprising a candidate n-mer motif into a humanized non-human transgenic animal as described in any one of claims 101 to 113; and detecting the binding of the composition to the transferrin receptor (TFRC) and / or detecting the transduction or uptake of one or more CNS cells of the humanized transgenic non-human animal. In an exemplary embodiment, the candidate n-mer 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, optionally wherein the total charge of the n-mer motif at neutral pH is between 0 and +2. In an exemplary embodiment, the composition is a viral particle comprising one or more capsid proteins, each of which comprises the candidate n-mer motif. In an exemplary embodiment, the viral particle is an AAV viral particle, and the one or more capsid proteins are AAV capsid proteins, optionally wherein the candidate n-mer motif is inserted between amino acids 588 and 589 of the AAV9 capsid polypeptide or at a similar position in the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74, or AAV rh.10. In an exemplary embodiment, at least one of the one or more compositions further comprises a cargo. In an exemplary embodiment, the cargo is or encodes a therapeutic nucleic acid or polypeptide, a selective label, or a control polypeptide or nucleic acid.

[0349] In one aspect, a method of screening for targeting moieties, including targeting moieties incorporated within AAVs, that are capable of transducing central nervous system (CNS) tissue via binding to the transferrin receptor (TFRC) in a humanized transgenic non-human animal, the method comprising: (a) introducing a plurality of vector systems into one or more humanized transgenic non-human animals that express human TFRC, and (b) detecting the targeting moiety that binds to the transferrin receptor (TFRC).

[0350] The engineered AAV capsid system vectors, engineered cells, and / or engineered AAV capsid particles described herein can be used in screening assays and / or cell selection assays. The engineered delivery system vectors, engineered cells, and / or engineered AAV capsid particles can be delivered to a subject and / or a cell. In some embodiments, the cell is a eukaryotic cell. The cell can be in vitro, ex vivo, in situ, or in vivo. The engineered AAV capsid system molecules, vectors, engineered cells, and / or engineered AAV capsid particles described herein can introduce exogenous molecules or compounds into the subject or cell to which they are delivered. The presence of the exogenous molecule or compound can be detected, which can permit the identification of the cell and / or its properties. In some embodiments, the delivered molecule or particle can confer a genetic or other nucleotide modification (e.g., a mutation, gene or polynucleotide insertion, and / or deletion, etc.). In some embodiments, the nucleotide modification can be detected in the cell by sequencing. In some embodiments, the nucleotide modification may result in a physiological and / or biological modification of the cell, which results in a detectable phenotypic change in the cell, which may permit the detection, identification, and / or selection of the cell. In some embodiments, the phenotypic change can be cell death, such as in embodiments where a CRISPR complex binds to a target polynucleotide resulting in cell death. Embodiments of the invention permit the selection of specific cells without a selection marker or a two-step process that may include a counter-selection system. The cell can be a prokaryotic cell or a eukaryotic cell.

[0351] In one embodiment, the present invention provides a method of selecting one or more cells by introducing one or more mutations into a gene in one or more cells, the method comprising: introducing one or more vectors into the cells, the one or more vectors optionally including one or more engineered delivery system molecules or vectors as described elsewhere herein, wherein the one or more vectors optionally include a CRISPR enzyme and / or drive the expression of one or more of: a guide sequence linked to a tracr pairing sequence, a tracr sequence, and an editing template; or other polynucleotides to be inserted into the cell and / or its genome; wherein, for example, the mutations that abrogate CRISPR enzyme cleavage are within and expressed in vivo by the CRISPR enzyme and / or the editing template when being expressed; allowing the editing template to undergo homologous recombination with the target polynucleotide in the cell to be selected; allowing the CRISPR complex to bind to the target polynucleotide to effect cleavage of the target polynucleotide within the gene, wherein the CRISPR complex comprises a CRISPR enzyme complexed with (1) a guide sequence that hybridizes to a target sequence within the target polynucleotide and (2) a tracr pairing sequence that hybridizes to the tracr sequence, wherein binding of the CRISPR complex to the target polynucleotide induces cell death, thereby allowing one or more cells into which one or more mutations have been introduced to be selected. In a preferred embodiment, the CRISPR enzyme is a Cas protein. In another embodiment of the present invention, the cells to be selected are eukaryotic cells.

[0352] Methods for screening engineered AAV capsid systems, molecules, vectors, engineered cells, and / or engineered AAV capsid particles, including but not limited to delivering one or more CRISPR-Cas system molecules to cells, can be used in detection methods such as fluorescence in situ hybridization (FISH). In some embodiments, one or more components of an engineered CRISPR-Cas system comprising a catalytically inactive Cas protein can be delivered to cells by the engineered AAV capsid system molecules, engineered cells, and / or engineered AAV capsid particles described elsewhere herein and used in FISH methods. The CRISPR-Cas system can include an inactivated Cas protein (dCas) (e.g., dCas9) that lacks the ability to generate DNA double-strand breaks, can be fused to a label such as a fluorescent protein such as enhanced green fluorescent protein (eEGFP), and co-expressed with a small guide RNA to target in vivo the regions flanking centromeres, the center, and telomeric repeats. The dCas system can be used to visualize both repetitive sequences and individual genes in the human genome. Such new applications of labeled dCas, dCas CRISPR-Cas systems, engineered AAV capsid system molecules, engineered cells, and / or engineered AAV capsid particles can be used to image cells and study functional nuclear architecture, especially in cases where small nuclear volumes or complex 3-D structures are used. (Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W,...

Claims

1. A composition comprising a targeting moiety that effectively increases transduction of central nervous system (CNS) tissue by binding to the transferrin receptor (TfR1), and optionally further comprising a cargo conjugated or otherwise associated with the targeting moiety.

2. The composition according to claim 1, wherein the targeting moiety binds to the extracellular domain of TFRC.

3. The composition according to claim 2, wherein the targeting moiety binds to one or more of the apical, helical, and / or protease-like domains of the extracellular domain.

4. The composition according to claim 3, wherein the targeting moiety binds to the apical domain.

5. The composition according to any one of the preceding claims, wherein the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises Y, M, F, and L; X2 comprises S, H, T, and A; X3 comprises K and R; X4 comprises A, G, I, L, M, N, Q, S, T, V, and H; X5 comprises N, G, A, L, M, Q, S, and T; X6 comprises A, T, H, N, F, I, P, L, Y, G, S, V, D, E, M, and Q; and X7 comprises D and N.

6. The composition according to claim 5, wherein: X1 comprises Y, M, and L; X2 comprises S, H, T, and A; X3 comprises K and R; X4 comprises A, G, I, L, M, N, Q, S, T, and V; X5 comprises N; X6 comprises A, T, H, N, F, I, P, L, and Y; and X7 comprises D and N; or X1 comprises Y, M, and L; X2 comprises S, H, T, and A; X3 comprises K and R; X4 comprises A, G, I, L, M, N, Q, S, T, and V; X5 comprises G; X6 comprises A, G, F, H, I, L, N, P, S, T, V, and Y; and X7 comprises D and N; or X1 comprises L and Y; X2 comprises A, H, and S; X3 comprises K and R; X4 comprises A, G, H, I, L, M, N, Q, S, T, and V; X5 comprises G; X6 comprises P; and X7 comprises D; or X1 comprises L and Y; X2 comprises A, H, and S; X3 comprises K and R; X4 comprises A, G, H, I, L, M, N, Q, S, T, and V; X5 comprises G; X6 comprises P; and X7 comprises N; or X1 comprises Y; X2 comprises S; X3 comprises K; X4 comprises A, I, L, M, N, Q, S, T, and V; X5 comprises G; X6 comprises X; and X7 comprises Y, P, T, Q, V, F, L, H, S, A, E, D, I, and M; or X1 comprises L and Y; X2 comprises S; X3 comprises R and K; X4 comprises V, I, T, L, and A; X5 comprises S and A; X6 comprises P, R, Y, F, H, I, K, and W; and X7 comprises D; or X1 comprises F, L, M, and Y; X2 comprises H; X3 comprises K and R; X4 comprises A, L, and M; X5 comprises A, G, L, M, N, Q, S, and T; X6 contains A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y; and X7 contains D and N; or X1 contains L, M, and Y; X2 contains H; X3 contains K and R; X4 contains A, L, and M; X5 contains A, G, L, M, N, Q, S, and T; X6 contains A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y; and X7 contains N; or X1 contains L, M, and Y; X2 contains H; X3 contains K and R; X4 contains A, L, and M; X5 contains A, G, L, M, N, Q, S, and T; X6 contains A, D, E, F, H, I, L, M, N, Q, P, S, T, V, and Y; and X7 contains D; or X1 contains L, M, and Y; X2 contains H; X3 contains K and R; X4 contains L; X5 contains S, Q, G, T, N, and L; X6 contains P, T, V, I, Q, L, and A; and X7 contains D; or X1 contains F; X2 contains S; X3 contains R; X4 contains L; X5 contains G; X6 contains A, H, N, L, V, S, P, and T; and X7 contains N; or X1 contains F; X2 contains A; X3 contains R; X4 contains T, S, and N; X5 contains G; X6 contains Y, F, H, P, and A; and X7 contains N; or X1 contains F; X2 contains H; X3 contains K and R; X4 contains L; X5 contains G; X6 contains I, P, and S; and X7 contains N and D.

7. The composition according to claim 6, wherein the n-mer 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).

8. The composition according to claim 7, wherein the n-mer motif is YSRIGPN (SEQ ID NO: 14632).

9. The composition according to claims 1 to 4, wherein the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 contains Y or L; X2 contains H; X3 contains A; X4 contains K, R, N and A; X5 contains G, Q, L and S; X6 contains P, L, I, N, D and T; and X7 contains N.

10. The composition according to any one of claims 1 to 4, wherein the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 contains A, F, H, I, L, N, P, R, S, T, V and Y; X2 contains A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, W and Y; X3 contains S; X4 contains S and T; X5 contains N; X6 contains G; and X7 contains I, R and V.

11. The composition according to claim 10, wherein: X1 contains F, L and Y; X2 contains D, E, H, N, Q, S and T; X3 contains S; X4 contains S and T; X5 contains N; X6 contains G; and X7 contains I and V; or X1 contains V, P, I, S, T, H and A; X2 contains A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T and Y; X3 contains S; X4 contains S and T; X5 contains N; X6 contains G; and X7 contains I and V; or X1 contains A, F, I, L, P, S, T, V and Y; X2 contains D, E, N, Q, S and T; X3 contains S; X4 contains T and S; X5 contains N; X6 contains G; and X7 contains R; or X1 contains R; X2 contains E, D, Q and T; X3 contains S; X4 contains S and T; X5 contains N; X6 contains G; and X7 contains I and V.

12. The composition according to claim 11, wherein the n-mer 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).

13. The composition according to any one of claims 1 to 4, wherein the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 contains R and T; X2 contains T, L, M, S, G, D, N, E, R, K, Y and W; X3 contains G, E, D, I, F, H, S, A, M, P, V, Y, W, Q and T; X4 contains D, T, E, H, N and G; X5 contains A, V, S, T and D; X6 contains Y, F, P and A; and X7 contains A and P.

14. The composition according to claim 13, wherein: X1 comprises R; X2 comprises T, M, L, and S; X3 comprises Y, S, A, M, I, F, and P; X4 comprises D; X5 comprises A, V, S, and T; X6 comprises Y and F; and X7 comprises P; or X1 comprises R; X2 comprises T, M, L, and S; X3 comprises Y, S, A, M, I, F, and P; X4 comprises D; X5 comprises A, V, S, and T; X6 comprises Y and F; and X7 comprises A; or X1 comprises R; X2 comprises G, T, D, S, N, and E; X3 comprises E, D, P, S, and G; X4 comprises D, T, E, H, and N; X5 comprises V, A, and T; X6 comprises Y and F; and X7 comprises P; or X1 comprises R; X2 comprises G, L, T, D, and S; X3 comprises D, P, S, and G; X4 comprises D, E, H, and N; X5 comprises V and T; X6 comprises Y and F; and X7 comprises P; or X1 comprises T; X2 comprises R, K, Y, and W; X3 comprises E, W, Y, Q, S, and T; X4 comprises G; X5 comprises D; X6 comprises P and A; and X7 comprises A and P.

15. The composition according to claim 14, wherein the n-mer 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).

16. The composition according to claims 1 to 4, wherein the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises L; X2 comprises C; X3 comprises K and R; X4 comprises P; X5 comprises C; X6 comprises L, S, D, A, N, Q, H, P, and V; and X7 comprises E, T, G, A, D, N, and S.

17. The composition according to claim 16, wherein the n-mer motif comprises LCKPCLD (SEQ ID NO: 36437) or LCKPCPT (SEQ ID NO: 36438).

18. The composition according to claims 1 to 4, wherein the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 comprises Y and F; X2 comprises W, F, and Y; X3 comprises S, T, H, A, and Q; X4 comprises G; X5 comprises I, T, V, Q, M, H, K, and R; X6 contains I, P, H, L, M, A, Q, T, V, K, and R; and X7 contains A, S, D, E, and N.

19. The composition according to claim 18, wherein: X1 contains Y and F; X2 contains W, F, and Y; X3 contains T and S; X4 contains G; X5 contains I, T, V, Q, M, and H; X6 contains I, P, H, L, M, A, Q, T, and V; and X7 contains A, S, D, and E; or X1 contains Y and F; X2 contains W, F, and Y; X3 contains T and S; X4 contains G; X5 contains I, T, V, Q, M, and H; X6 contains K and R; and X7 contains A, S, D, and E; or X1 contains Y and F; X2 contains W, F, and Y; X3 contains T and S; X4 contains G; X5 contains K and R; X6 contains I, P, H, L, M, A, Q, T, and V; and X7 contains A, S, D, and E; or X1 contains Y; X2 contains F; X3 contains T; X4 contains G; X5 contains K, R, Q, M, H, and I; X6 contains T, R, H, K, V, and L; and X7 contains E; or X1 contains Y; X2 contains F; X3 contains T, S, H, and A; X4 contains G; X5 contains K, R, and T; X6 contains I, P, H, L, M, A, Q, and T; and X7 contains D and N; or X1 contains Y; X2 contains W; X3 contains T; X4 contains G; X5 contains K, M, V, and T; X6 contains P, V, I, H, Q, T, M, and L; and X7 contains E and D; or X1 contains Y and F; X2 contains F; X3 contains S, H, A, and Q; X4 contains G; X5 contains K, Q, and R; X6 contains I, V, L, K, H, R, Q, and M; and X7 contains E.

20. The composition according to claims 1 to 4, wherein the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 contains K, R, S, G, N, T, M, Q, V, D, I, and E; X2 contains D, S, N, M, L, G, P, E, and A; X3 contains E, D, G, S, A, R, Q, T, P, and N; X4 contains F, Y, T, V, S, N, A, G, and H; X5 contains T, K, S, R, V, and H; X6 contains T, S, G, V, A, K, R, N, D, E, and H; and X7 contains F, W, and Y.

21. The composition according to claim 20, wherein: X1 contains K and R; X2 contains D, S, and N; X3 contains E; X4 contains F; X5 contains T, K, S, R, and V; X6 contains T, S, G, and V; and X7 contains F, W, and Y; or X1 contains K and R; X2 contains D; X3 contains D; X4 contains F and Y; X5 contains T, S, V, and H; X6 contains T, S, G, V, and A; and X7 contains F, W, and Y; or X1 contains S, R, G, N, T, M, and Q; X2 contains D; X3 contains G; X4 contains T, V, S, N, and Y; X5 contains S; X6 contains K and R; and X7 contains W; Or X1 contains R, V, D, I, Q, and K; X2 contains M, L, and G; X3 contains S, E, A, R, and Q; X4 contains D; X5 contains R; X6 contains T, A, S, G, K, and N; and X7 contains W; Or X1 contains D, I, E, Q, V, S, and K; X2 contains L, M, G, and P; X3 contains E, A, S, D, Q, and T; X4 contains S and A; X5 contains R; X6 contains D, S, E, T, G, and A; and X7 contains W; Or X1 contains G; X2 contains E, S, P, G, and A; X3 contains D, E, P, and N; X4 contains G, H, T, S, and N; X5 contains V; X6 contains R, K, and S; and X7 contains W and Y; Or X1 contains R; X2 contains E; X3 contains D, E, P, and N; X4 contains G, H, T, S, and N; X5 contains V; X6 contains R, K, and S; and X7 contains W and Y; Or X1 contains G; X2 contains G and S; X3 contains G, E, S, A, P, and D; X4 contains T, G, and S; X5 contains S; X6 contains S, T, H, K, R, A, and N; and X7 contains W.

22. The composition according to claim 21, wherein the n-mer 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).

23. The composition according to any one of claims 1 to 4, wherein the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 contains V, I, R, N, and D; X2 contains A, G, and S; X3 contains L, T, S, H, and G; X4 contains K, R, and E; X5 contains G; X6 contains W, R, A, and I; and X7 contains D and G.

24. The composition according to claim 23, wherein the n-mer 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).

25. The composition according to any one of claims 1 to 4, wherein the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 contains L, M, and W; X2 contains F, R, W, K, T, and Y; X3 contains D and S; X4 contains G; X5 contains T; X6 contains P, G, S, N, A and R; and X7 contains A, P, S and Y.

26. The composition according to any one of claims 1 to 4, wherein the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence of X1-X2-X3-X4-X5-X6-X7, wherein: X1 contains P, N and K; X2 contains Y and F; X3 contains A; X4 contains R and K; X5 contains S; X6 contains P, V, A, R, I, L, S, E; and X7 contains E, D, M and L.

27. The composition according to any one of claims 1 to 4, wherein the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence of Z1-X1-Z2-X2-X3-X4-X5, wherein Z1 is selected from the group consisting of Y, F and L, Z2 is selected from the group consisting of S, R and K, and X1 to X5 are independently selected amino acids.

28. The composition according to claim 27, wherein X1 contains A, S or H.

29. The composition according to claim 27, wherein X2 contains S, T, L or I.

30. The composition according to claim 27, wherein X3 contains N or G.

31. The composition according to claim 27, wherein X4 contains G.

32. The composition according to claim 27, wherein X5 contains N, D, I, V or R.

33. The composition according to any one of claims 1 to 4, wherein the n-mer 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).

34. The composition according to any one of claims 1 to 4, wherein the targeting moiety comprises an n-mer motif, the n-mer motif comprising or consisting of the amino acid sequence of X1-H-X2-L-X3-X4-X5, wherein X1 to X5 are independently selected amino acids.

35. The composition according to claim 34, wherein the n-mer motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608).

36. The composition according to any one of claims 1 to 4, wherein the n-mer motif comprises 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).

37. The composition according to any one of claims 1 to 27, wherein the n-mer is selected from any one of the amino acid sequences listed in Tables 1 to 22, or any combination thereof.

38. The composition according to any one of claims 1 to 27, wherein the n-mer motif is selected from the amino acid sequences of SEQ ID NOs: 10952 - 20481 and 36241 - 42428.

39. The composition according to any one of the preceding claims, wherein the targeting moiety is a part of a viral capsid protein.

40. The composition according to any one of the preceding claims, wherein the targeting moiety is inserted into or replaces Loop IV, Loop VIII or both of the AAV capsid protein.

41. The composition according to claim 40, wherein the targeting 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).

42. The composition according to any one of the preceding claims, wherein the targeting moiety is inserted between two amino acids of one or more capsid proteins such that the targeting moiety is on the outside of the AAV capsid.

43. The composition according to any one of the preceding claims, wherein the viral capsid protein is an AAV viral capsid protein.

44. The composition according to any one of the preceding claims, wherein the targeting moiety is inserted between amino acids 588 and 589 of the capsid protein of AAV9, or at a similar position in the capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74 or AAV rh.

10.

45. The composition according to any one of the preceding claims, wherein the targeting moiety is inserted between two consecutive amino acids within amino acids 451 to 460 of the capsid protein of AAV9, or at a similar position in the capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74 or AAV rh.

10.

46. The composition according to any one of the preceding claims, wherein the capsid protein is VP1, VP2, VP3 or a combination thereof.

47. The composition according to any one of the preceding claims, wherein the cargo is a polynucleotide, a recombinant AAV genome comprising a transgene, one or more polypeptides, a ribonucleoprotein complex.

48. The composition according to claim 47, wherein the polynucleotide encodes one or more polypeptides and / or RNAi oligonucleotides.

49. The composition according to claim 48, wherein the polynucleotide encodes one or more polypeptides.

50. The composition according to claim 49, wherein the one or more polypeptides comprise an enzyme or an antibody.

51. The composition according to any one of the preceding claims, wherein the polynucleotide encodes a CRISPR-Cas system.

52. The composition according to any one of claims 47 to 51, wherein the polynucleotide is operably linked to a regulatory sequence that promotes expression in the CNS.

53. A viral capsid or viral particle comprising the composition according to any one of claims 1 to 52.

54. The viral capsid or viral particle according to claim 53, further comprising a recombinant viral genome, wherein 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.

55. The viral capsid or viral particle according to claim 54, wherein the therapeutic protein or nucleic acid, the control polypeptide or nucleic acid, and / or the selectable marker polypeptide or nucleic acid is operably linked to a regulatory sequence that promotes expression in the CNS.

56. The viral capsid or viral particle according to any one of claims 53 to 55, wherein the viral capsid or viral particle is an AAV viral capsid or an AAV viral particle.

57. The viral capsid or viral particle according to claim 54, wherein the recombinant viral genome is a recombinant AAV viral genome.

58. A viral capsid or viral particle according to any one of claims 53 to 57, wherein the targeting moiety is inserted between amino acids 588 and 589 of the capsid protein of AAV9, or at a similar position in the capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74 or AAV rh.

10.

59. A vector system comprising one or more vectors, wherein at least one of the one or more vectors encodes a targeting moiety that effectively increases transduction of central nervous system tissue (CNS) by binding to the transferrin receptor (TFRC), and optionally wherein at least one of the one or more vectors encodes a recombinant AAV genome comprising a transgene encoding a protein or polypeptide.

60. The vector system according to claim 59, wherein the targeting moiety binds to the extracellular domain of TFRC.

61. The vector system according to claim 60, wherein the targeting moiety binds to at least one of the apical, helical and / or protease-like domains of the extracellular domain.

62. The vector system according to claim 61, wherein the targeting moiety binds to the apical domain.

63. The vector system according to claim 59 or 62, wherein the targeting moiety comprises any n-mer motif as described in claims 5 to 26.

64. The vector system according to claim 59 or 62, wherein the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence Z1-X1-Z2-X2-X3-X4-X5, wherein Z1 is Y, F or L, Z2 is S, R or K, and X1 to X5 are independently selected amino acids.

65. The vector according to claim 64, wherein X1 comprises A, S or H.

66. The vector according to claim 64, wherein X2 comprises S, T, L or I.

67. The vector according to claim 64, wherein X3 comprises N or G.

68. The vector according to claim 64, wherein X4 comprises G.

69. The vector according to claim 64, wherein X5 comprises N, D, I, V or R.

70. The vector system according to claim 65, wherein the n-mer 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).

71. The vector system according to claim 59 or 60, wherein the targeting moiety comprises an n-mer motif comprising or consisting of the amino acid sequence X1-H-X2-L-X3-X4-X5, wherein X1 to X5 are independently selected amino acids.

72. The vector system according to claim 71, wherein the n-mer motif is VHRLQDK (SEQ ID NO: 16602) or LHALSHN (SEQ ID NO: 16608).

73. The vector system according to claim 59 or 62, wherein the n-mer motif comprises 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).

74. The vector system according to claim 59 or 60, wherein the n-mer is selected from any one of those listed in Tables 1 to 22, or any combination thereof.

75. The vector system according to claim 59 or 60, wherein the n-mer motif is selected from SEQ ID NOs: 10952 - 20481 and 36241 - 42428.

76. The vector system according to any one of claims 59 or 75, wherein the targeting moiety is part of a viral capsid protein.

77. The vector system according to any one of claims 59 or 76, wherein the targeting moiety is inserted or substituted in Loop IV and / or Loop VIII.

78. The vector system according to claim 77, wherein the targeting 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).

79. The vector system according to claim 76, wherein the targeting moiety is inserted between two amino acids of one or more capsid proteins such that the targeting moiety is on the outside of the AAV capsid.

80. The vector system according to claim 79, wherein the viral capsid protein is an AAV viral capsid protein.

81. The vector system according to claim 79, wherein the targeting moiety is inserted between amino acids 588 and 589 of the capsid protein of AAV9, or at a similar position in the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74 or AAV rh.

10.

82. The vector system according to claim 79, wherein the targeting moiety is inserted between amino acids 451 to 460 of the capsid protein of AAV9, or at a similar position in the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74 or AAV rh.

10.

83. The vector system according to claim 76, wherein the capsid protein is VP1, VP2, VP3 or a combination thereof.

84. The vector system according to any one of claims 59 or 83, wherein the cargo is a polynucleotide, a recombinant AAV genome comprising a transgene, one or more polypeptides, a ribonucleoprotein complex.

85. The vector system according to claim 84, wherein the polynucleotide encodes one or more polypeptides and / or RNAi oligonucleotides.

86. The vector system according to claim 85, wherein the polynucleotide encodes one or more polypeptides.

87. The vector system according to claim 86, wherein the one or more polypeptides comprise an enzyme or an antibody.

88. The vector system according to any one of claims 59 or 87, wherein the polynucleotide encodes a CRISPR-Cas system.

89. The vector system according to any one of claims 84 to 88, wherein the polynucleotide is operably linked to a regulatory sequence that promotes expression in the CNS.

90. A polypeptide encoded or produced by the vector system according to any one of claims 59 or 88.

91. The polypeptide according to claim 90, wherein the polypeptide is a capsid protein, optionally an AAV capsid polypeptide.

92. A particle produced by the vector system according to any one of claims 59 or 89.

93. The particle according to claim 92, wherein the particle is a viral particle, optionally an AAV particle.

94. A cell comprising the composition, vector, polypeptide or particle according to any one of the preceding claims.

95. A method of delivering one or more cargos to the CNS, comprising: administering in vivo or in vitro the capsid and / or viral particle according to any one of claims 1 to 58.

96. The method according to claim 95, wherein the vehicle is a recombinant AAV genome comprising a transgene, a polynucleotide encoding an RNAi oligonucleotide, a polynucleotide encoding a polypeptide, or a polypeptide.

97. The method according to claim 96, wherein the polypeptide comprises an enzyme or an antibody.

98. The method according to claim 95, wherein the vehicle is a Cas polypeptide, a guide molecule, or both.

99. The method according to claim 95, wherein the vehicle encodes a nuclease or a nucleic acid component of an RNA-guided nuclease.

100. The method according to claim 95, wherein the vehicle is one or more polynucleotides encoding a nuclease or a nucleic acid component of the RNA-guided nuclease.

101. A method of creating a humanized transgenic non-human animal, comprising: delivering one or more cells of a non-human animal into a vector system or a recombinant viral particle comprising a recombinant viral genome, wherein: the vector system or the recombinant viral genome encodes a human transferrin polypeptide, wherein the encoded human transferrin polypeptide is under the control of a tissue-specific promoter or a miRNA binding element having selective activity within a desired cell, tissue, or organ.

102. The method according to claim 101, wherein the one or more cells are endothelial cells.

103. The method according to any one of claims 101 or 102, wherein the one or more cells are CNS cells.

104. The method according to claim 102, wherein the one or more cells are cells of the CNS vasculature, lung, kidney, liver, or any combination thereof.

105. The method according to claim 104, wherein the endothelial cells are endothelial cells of the CNS vasculature.

106. The method according to any one of claims 101 to 105, wherein the recombinant viral particle, optionally an AAV viral particle, comprises a capsid polypeptide, optionally an AAV capsid polypeptide, wherein the capsid polypeptide comprises a CNS-specific n-mer motif.

107. The method according to claim 106, wherein the CNS-specific n-mer motif comprises X1-N-X3-X4-X5-X6-X7, wherein X5 is independently selected from K or R, and X1, X3, X4, X6, and X7 are independently selected from any amino acid, optionally wherein the total charge of the n-mer motif at neutral pH is between 0 and +2.

108. The method according to claim 106, wherein the CNS-specific n-mer motif comprises NNSTRGG (SEQ ID NO: 42429), GNSARNI (SEQ ID NO: 42430), and GNSVRDF (SEQ ID NO: 42431) or consists thereof.

109. The method according to any one of claims 101 to 108, wherein the transgenic non-human animal is a rodent, optionally a mouse.

110. A humanized transgenic non-human animal, comprising: One or more cells expressing a human transferrin polypeptide, optionally wherein the one or more cells are CNS cells.

111. The humanized transgenic non-human animal according to claim 110, wherein the transgenic non-human animal is a rodent, optionally a mouse.

112. A humanized transgenic non-human animal produced by the method according to any one of claims 101 to 109.

113. The humanized transgenic non-human animal according to any one of claims 101 to 112, wherein the humanized non-human animal has an inhibited immune system.

114. A method for screening an n-mer motif capable of transducing central nervous system (CNS) tissue via binding to transferrin receptor (TFRC) in a humanized transgenic non-human animal, comprising: introducing one or more compositions comprising a candidate n-mer motif into the humanized non-human transgenic animal according to any one of claims 101 to 113; and detecting the binding of the composition to transferrin receptor (TFRC) and / or detecting the transduction or uptake of one or more CNS cells of the humanized transgenic non-human animal.

115. The method according to claim 114, wherein the candidate n-mer motif comprises or consists of X1-N-X3-X4-X5-X6-X7, wherein X5 is independently selected from K or R, and X1, X3, X4, X6 and X7 are independently selected from any amino acid, optionally wherein the total charge of the n-mer motif at neutral pH is between 0 and +2.

116. The method according to any one of claims 114 or 115, wherein the composition is a viral particle comprising one or more capsid proteins, each of the capsid proteins comprising the candidate n-mer motif.

117. The method according to claim 116, wherein the viral particle is an AAV viral particle, and the one or more capsid proteins are AAV capsid proteins, optionally wherein the candidate n-mer motif is inserted between amino acids 588 and 589 of the AAV9 capsid polypeptide or at a similar position in the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh.74 or AAV rh.

10.

118. The method according to claims 114 to 117, wherein at least one of the one or more compositions further comprises a cargo.

119. The method according to claim 118, wherein the cargo is or encodes a therapeutic nucleic acid or polypeptide, a selectable marker, or a control polypeptide or nucleic acid.

120. A recombinant AAV particle comprising (1) a recombinant capsid protein having an n-mer motif peptide targeting TFR1, the n-mer motif peptide being inserted within the capsid protein such that when incorporated into the AAV particle, the peptide is displayed on the surface; and (2) a recombinant AAV genome comprising a transgene encoding a therapeutic polypeptide or polynucleotide operably linked to one or more regulatory sequences that promote expression of the transgene in CNS cells, the transgene flanked by AAV ITR sequences, wherein the AAV particle has enhanced binding, transduction, or transgene expression in CNS cells relative to an AAV particle having a reference capsid lacking the targeting n-mer motif.

121. The recombinant AAV particle of claim 120, wherein the targeting n-mer motif peptide is a heptamer peptide having an amino acid sequence as recited in any one of claims 5 to 26.

122. The recombinant AAV particle of claim 120, wherein the targeting n-mer motif is a heptamer peptide having the amino acid sequences of SEQ ID NOs: 10952-20481 and 36241-42428.

123. The recombinant AAV particle of any one of claims 120 to 122, wherein the peptide has an amino acid sequence as recited in claim 7, 12, 15, 17, 22, or 24.

124. The recombinant AAV particle of any one of claims 120 to 123, wherein the recombinant capsid protein is an AAV9 capsid protein (SEQ ID NO: 20506), an AAV9K449R capsid protein (SEQ ID NO: 20507), or a capsid protein having at least 90%, 95%, or 99% sequence identity to the AAV9 capsid protein and forms a capsid that transduces CNS cells.

125. The recombinant AAV particle of any one of claims 120 to 124, wherein the peptide is inserted between amino acids 588 and 589 of the AAV9 capsid protein or at the corresponding position in another AAV capsid protein.

126. The recombinant AAV particle of any one of claims 120 to 125, wherein the peptide is inserted between two consecutive amino acids within amino acids 451 to 460 of the AAV9 capsid protein or at the corresponding position within another AAV capsid protein.

127. A method of delivering a therapeutic polypeptide or polynucleotide to the CNS of a subject in need thereof, the method comprising administering to the subject a recombinant AAV particle of any one of claims 120 to 126.

128. A recombinant engineered AAV capsid protein having an n-mer motif peptide targeting TFR1, the targeting TFR1 n-mer motif peptide being inserted within the capsid protein such that when incorporated into an AAV particle, the peptide is displayed on the surface.

129. The recombinant engineered AAV capsid protein according to claim 128, wherein the targeting n-mer motif peptide is a heptamer peptide having the amino acid sequence as described in any one of claims 5 to 26.

130. The recombinant engineered AAV capsid protein according to claims 128 to 129, wherein the targeting n-mer motif is a heptamer peptide having the amino acid sequences of SEQ ID NOs: 10952-20481 and 36241-42428.

131. The recombinant engineered AAV capsid protein according to any one of claims 128 to 130, wherein the peptide has the amino acid sequence as described in claim 7, 12, 15, 17, 22 or 24.

132. The recombinant engineered AAV capsid protein according to any one of claims 128 to 131, wherein the recombinant capsid protein is an AAV9 capsid protein (SEQ ID NO: 20506), an AAV9 K449R capsid protein (SEQ ID NO: 20507) or a capsid protein having at least 90%, 95% or 99% sequence identity with the AAV9 capsid protein, and forms a capsid that transduces CNS cells.

133. The recombinant engineered AAV capsid protein according to any one of claims 128 to 132, wherein the peptide is inserted between amino acids 588 and 589 of the AAV9 capsid protein or at the corresponding position of another AAV capsid protein.

134. A host cell for producing the recombinant AAV particles according to any one of claims 120 to 127, the host cell comprising a first construct and a second construct, the first construct comprising a nucleic acid encoding the recombinant AAV capsid protein, and the second construct comprising a nucleic acid encoding the recombinant AAV genome.

135. A method for producing the recombinant AAV particles according to any one of claims 120 to 127, the method comprising culturing the host cell under conditions sufficient to produce the recombinant AAV particles and harvesting the recombinant AAV particles.

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